ROLES OF FTSN AND DEDD IN INITIATING E. COLI CELL CONSTRICTION BING LIU

Transcription

1 ROLES OF FTSN AND DEDD IN INITIATING E. COLI CELL CONSTRICTION by BING LIU Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January, 2015

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Bing Liu candidate for the degree of PhD*. Committee Chair Arne Rietsch Committee Member Piet de Boer Committee Member Robert Bonomo Committee Member Pieter de Haseth Date of Defense August 21, 2014 *We also certify that written approval has been obtained for any proprietary material contained therein

3 Table of Contents List of Tables... iv List of Figures... v ABSTRACT... vii INTRODUCTION... 9 Chapter 1. Self-enhanced FtsN activity during the initiation of E. coli cell constriction INTRODUCTION RESULTS DISCUSSION Chapter 2. Roles for FtsA and the FtsBLQ subcomplex in FtsN-mediated triggering of cell constriction INTRODUCTION RESULTS DISCUSSION Chapter 3. DedD assists in initiating cell constriction through an FtsNindependent pathway INTRODUCTION RESULTS DISCUSSION SUMMARY AND FUTURE DIRECTIONS MATERIALS AND METHODS FIGURES TABLES BIBLIOGRAPHY iii

4 List of Tables Table 1 Extragenic suppressors enable growth of cells producing otherwise nonfunctional FtsN mutant variants Table 2 FtsL D93G or FtsB D59H bypasses the requirement for E FtsN Table 3 Viability of ΔftsN cells harboring (combinations of) suppressing mutations in ftsa, B and/or L Table 4 Requirement for N FtsN in ftsb or ftsl mutant cells lacking E FtsN Table 5 Suppressing mutations reduce average cell size Table 6 FtsB E56A promotes early septal murein synthesis Table 7 Localization of GFP-DedD fusions in BL40 cells Table 8 Localization of GFP-DedD mutant variants in BL40 cells Table 9 Plasmids used in this study Table 10 E.coli strains used in this study iv

5 List of Figures FIG. 1 Schematic overview of SR assembly in E. coli FIG. 2 Further definition of the essential domain of FtsN FIG. 3 Localization of TT GFP- S FtsN at constriction sites depends on E FtsN and PBP3 activities FIG. 4 Localization of TT GFP- S FtsN and TT GFP in murein amidase mutants FIG. 5 Western analyses of TT GFP- S FtsN FIG. 6 Model for self-enhanced FtsN activity during the initiation of cell constriction FIG. 7 Western analyses of periplasmic FtsN fusions FIG. 8 Western analyses of FtsN fusions with internal deletions FIG. 9 Genetic screens for extragenic suppressors of nonfunction ftsn alleles FIG. 10 Suppressing mutations rescue division of cells producing otherwise nonfunctional FtsN variants FIG. 11 Conserved residues in N FtsN are important for its function and localization FIG. 12 Western analyses of FtsN 1-81 fusions FIG. 13 Viability of ΔftsN cells due to (combinations of) compensating mutations in ftsa, ftsb, and/or ftsl FIG. 14 FtsB E56A promotes septal murein synthesis in ftsn + cells FIG. 15 Cell shape and lysis phenotypes of various strains in LB ΔNaCl medium at 42 C, and suppression by ΔftsN FIG. 16 Lethality of Δ E FtsN-suppressing mutations in ftsb and/or ftsl on LB ΔNaCl medium at 42 C in the presence of E FtsN FIG. 17 The model for the SR to initiate septal murein synthesis upon initiation of cell constriction FIG. 18 Domain analysis of DedD FIG. 19 Septal localization of GFP-DedD depends on E FtsN and FtsI v

6 FIG. 20 Conserved residues in TM DedD are important for DedD s function in cell division FIG. 21 Western analyses of GFP-DedD fusions in BL40 cells FIG. 22 Western analyses of GFP-DedD mutants in BL40 cells FIG. 23 More E FtsN activity is required for cell division in the absence of DedD FIG. 24 Western analyses of DedD and FtsN fusions in ΔdedD ftsn slm117 cells FIG. 25 Mutations in TM DedD affect interactions between N DedD and FtsL FIG. 26 BATCH analyses for DedD and FtsN FIG. 27 Combining ΔdedD with ΔponB leads to massive cell lysis FIG. 28 Model for E FtsN and N DedD in the regulation of spg synthesis FIG. 29 Massive cell lysis in ΔdedD ΔponB cells is suppressed by PBP1B or DedD FIG. 30 Toxicity of excess FtsQ, B, or L molecules in ΔdedD cells FIG. 31 Suppressing the toxicity of excess FtsQ in ΔdedD cells FIG. 32 Overexpression of FtsQ inhibits cell division in the absence of DedD. 174 FIG. 33 Z-ring formation is not affected by overexpression of GFP-FtsQ in the absence of DedD FIG. 34 Overexpression of certain septal ring proteins is toxic to ΔdedD cells. 177 FIG. 35 FtsB E56A suppresses the division defects in ΔdedD cells FIG. 36 The model for E FtsN and N DedD to initiate septal murein synthesis upon initiation of cell constriction vi

7 Roles of FtsN and DedD in Initiating E. Coli Cell Constriction Abstract by BING LIU Upon onset of cell constriction, E. coli cells begin to synthesize new peptidoglycan perpendicularly to the lateral cell wall at the division site, which requires the activity of several essential Septal Ring (SR) components, including FtsQ, FtsB, FtsL, and FtsN, in addition to the murein synthases. As the last of the essential cell division proteins to accumulate significantly at the SR, FtsN initiates cell constriction and septal murein (spg) synthesis through its essential domain ( E FtsN) in the periplasm. In this study, we demonstrated that the cytoplasmic domain of FtsN ( N FtsN) is a weak septal targeting determinant that helps concentrate FtsN molecules at the division site through interactions with FtsA. The C-terminal SPOR domain of FtsN ( S FtsN), on the other hand, is a strong septal targeting determinant that depends on initiation of spg synthesis for septal localization and thus, on the essential activity of E FtsN. These results suggest that FtsN joins the SR and triggers spg synthesis in a self-enhancing fashion. In order to identify the targets for E FtsN, we set up genetic screens for extragenic suppressors of non-functional ftsn mutants with substitutions in one of the three critical residues in E FtsN, W83, Y85, and E90. The suppressing vii

8 mutations identified in ftsl or ftsb are able to direct E FtsN-independent cell constriction and induce early spg synthesis in cells carrying native ftsn. Our results suggest that the FtsBLQ subcomplex plays important roles in the regulation of spg synthesis and cell constriction. We also found that another SR component with a C-terminal SPOR domain, DedD, helps to stimulate spg synthesis through its critical functional domain in the N-terminal conserved region ( N DedD). N DedD may also interact with the FtsBLQ subcomplex and activate the murein synthases through an E FtsN-independent pathway. Our results suggest a model for FtsN and DedD in the initiation of cell constriction in E. coli. FtsN and DedD induce conformational changes in the FtsBLQ subcomplex through E FtsN and N DedD, leading to the release of an inhibitory activity of the FtsBLQ subcomplex on the murein synthases and, thus, initiation of spg synthesis and cell constriction. viii

9 INTRODUCTION The cell envelope of Gram-negative bacteria like Escherichia coli consists of three layers, the inner membrane (IM), the outer membrane (OM), and the peptidoglycan (PG, murein) layer in between them. Cytokinesis in E. coli is a complicated process involving coordinated invagination of all the three cell envelope layers, a task that is accomplished by a ring-shaped apparatus referred to as the Septal Ring (SR) or divisome (1-3). In addition to constricting the two membrane layers, the SR needs to synthesize new septal peptidoglycan (spg) perpendicular to the lateral PG and split the newly synthesized spg in order to separate the daughter cells and to create space for the invagination of the OM. The peptidoglycan layer in E. coli is a giant molecule composed of glycan strands of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues crosslinked by short peptides (4, 5). The last step of peptidoglycan synthesis is the incorporation of bactoprenol-bound precursor MurNAc(pentapeptide)-GlcNAc (Lipid II) into the existing peptidoglycan layer in the periplasm, a task that is accomplished by murein synthases (6, 7). There are three classes of known murein synthases in E. coli: bifunctional tansglycosylases /transpeptidase (PBP1A, PBP1B, and PBP1C), monofunctional transpeptidases (PBP2 and PBP3), and monofunctional transglycosylases (MtgA) (7). Among them, PBP1A and PBP1B are the two major bifunctional murein synthases with PBP1B more specifically involved in synthesizing septal murein, although PBP1A can take over its function when PBP1B is absent (8-11). The two monofunctional transpeptidases, PBP2 and PBP3 (FtsI), are essential for cell elongation and cell 9

10 division, respectively (12). The newly synthesized spg is processed by murein hydrolases, especially the three murein amidases AmiA, AmiB, and AmiC, to split the spg into two daughter layers (13-15). According to a current model, murein synthesis in E. coli is performed by multiprotein machineries composed of murein synthases and hydrolases (4, 16, 17). Two different types of machineries exist in E. coli for inserting new peptidoglycan at the lateral cell wall or at the division site, respectively, although they share some similar enzymes. Upon initiation of cell constriction, new peptidoglycan synthesis is redirected from the lateral cell wall to the division site. The Z-ring is shown to be able to direct insertion of some peptidoglycan along the lateral cell wall at midcell before cell constriction (16, 18-20). But synthesis of spg perpendicular to the lateral cell wall requires a mature SR containing all the essential components. The Septal Ring. The SR is a complicated molecular machine composed of more than two dozen proteins (Fig. 1). Ten SR proteins are essential to the cell division process, including FtsA, B, I, K, L, N, Q, W, Z, and ZipA, which are considered the core components of the SR. Cells lacking any of the essential SR components cannot divide but keep elongating to long multi-nucleoid filaments before they eventually die. In addition to the core components of the SR, a growing pool of more than twenty proteins have been discovered as non-essential components of the SR, with the recently identified members including ZapB (21), ZapC (22, 23), ZapD (24), ZapE (25), YmgF (26), Blr (27), and the SPOR-domain proteins DedD, 10

11 DamX, and RlpA (28, 29). Although nonessential, at least some of these additional SR components play redundant but important roles in cell division. The SR is assembled at midcell before cell constriction. Construction of a constriction-competent SR involves assembly of the SR components following a specific order as illustrated in Fig. 1 (3, 30). The first known event is polymerization of FtsZ molecules at the prospective division site (31). The tubulin-like FtsZ is the key SR component in E. coli and most other prokaryotes (32). Besides leading SR assembly, it is believed to provide at least part of the driving force for inner membrane invagination (33-36). FtsZ polymerization is regulated both temporally and spatially (31, 37, 38). At least two negative regulatory systems, the minicell system (Min) and nucleoid occlusion (NO), restrict FtsZ polymerization to midcell in between segregated nucleoids (39-42). FtsZ-polymers and FtsZ binding proteins, including ZipA, FtsA, and the Zap proteins, constitute the Z-ring on the cytoplasmic side of inner-membrane. The Z- polymers are tethered to the membrane by the two essential FtsZ-binding proteins, FtsA and ZipA. While ZipA is a type Ib bitopic integral membrane protein (N-out), FtsA is a peripheral membrane protein that associates with the phospholipid layer via a C-terminal amphiphatic helix. Though both proteins are normally essential for cell fission and viability, FtsA is evolutionarily better conserved, and certain FtsA mutant derivatives allow E. coli cells to divide almost normally in the complete absence of ZipA (43-48). FtsA belongs to the actin family of proteins but with a distinct subdomain arrangement. In FtsA, a IC domain was inserted to the opposite side of the molecule compared to the 11

12 original IB domain in actin (49). Recent structural evidence indicates that FtsA can indeed form actin-like polymers along the cell membrane (50). In addition to Z-ring assembly, FtsA also plays important roles in recruitment of additional SR components (51-55), as well as in regulating constriction of the Z-ring or maybe the entire SR (28, 36, 56-58). Right before constriction, the remaining essential SR components are recruited to the Z-ring following a mostly linear assembly pathway, leading to maturation of a constriction-competent SR (30). spg synthesis perpendicular to the lateral cell wall requires the activity of the late essential SR components. FtsI (PBP3) is the transpeptidase specialized in synthesizing spg (12). It interacts with FtsW (59), which is believed to be a Lipid II flippase that transports the lipidbound precursor from the cytoplasmic to the periplasmic side of the inner membrane (60). The essential functions of the remaining five core SR proteins are still poorly defined. The large C-terminal cytoplasmic domain of FtsK coordinates chromosome segregation with cell constriction by assisting in the resolution of chromosome dimers and in moving DNA away from the closing septal pore, but those functions of FtsK are not essential (61, 62). The three bitopic inner membrane proteins, FtsB, FtsL, and FtsQ, form a trimeric protein subcomplex that serves as a scaffold for recruiting downstream SR components during SR assembly (63-71). The subcomplex may also have a regulatory function as discussed later in this study. As the last essential cell division protein to accumulate sharply at the SR, FtsN has been proposed to trigger the cell 12

13 constriction phase upon its arrival at the SR (3, 28, 30, 72, 73). However, how FtsN might do this still remains unclear. During the constriction phase, additional non-essential SR components join the SR and play important roles in different aspects of cell constriction (1-3, 16, 74). SR activities during cell constriction. The SR has several complicated tasks to accomplish in order to achieve cell constriction. Firstly, the IM needs to be constricted, which is believed to be driven at least partially by contraction of the Z-ring, and maybe the inward growth of spg (33-36). Secondly, spg needs to be inserted at the septum perpendicular to the long axis of cell, which requires the Lipid II flippase FtsW, the transpeptidase PBP3, and at least one of the murein synthases with transglycosylase activity, PBP1B, PBP1A, or MtgA (8, 10, 12, 60). The FtsBLQ subcomplex, FtsN, and maybe FtsK are also required for spg synthesis, as E. coli cells cannot initiate spg synthesis without these essential SR components (52, 75-78). Next, the newly synthesized spg is simultaneously and carefully processed by murein hydrolysases, most prominently the murein amidases AmiA, AmiB, and AmiC, from the OM side to separate spg and make room for OM invagination (13, 14, 79-81). Lastly, the OM invagination involves both active and passive processes. The Tol-Pal complex is a non-core subsystem of the SR consisting of the IM proteins TolA, TolQ, and TolR, periplasmic TolB, and the OM lipoprotein Pal (82, 83). The system utilizes proton motive force to establish 13

14 transient connections of the OM with the PG layer (via Pal-PG interactions), as well as with the IM (via Pal-TolA interactions), causing the OM to be drawn inwards (84-86). In addition, generally distributed and abundant PG-binding OM proteins such as OmpA and Lpp help ensure that the OM remains closely connected to the underlying PG layer (87-89). In the past decades, great efforts have been made to better understand the cell division process in bacteria. But many questions remain to be answered, including the mechanism for the SR to initiate spg synthesis and cell constriction and the essential functions of the five core SR components, FtsK, FtsB, FtsL, FtsQ, and FtsN. In this study, we tried to find answers to some of these questions. Roles of FtsN in cell constriction. As the last of the essential SR components to accumulate sharply at the SR before/at the onset of cell constriction, FtsN is proposed to trigger the constriction phase upon its arrival at the SR (3, 28, 30, 72, 73). As illustrated in Fig. 2A, FtsN is a type II bitopic inner membrane protein with a small cytoplasmic domain, a transmembrane domain, and a large periplasmic domain (90). The periplasmic domain of FtsN is largely unstructured except for three short helices in the juxtamembrane region and the C-terminal SPOR domain ( S FtsN), which was demonstrated to bind peptidoglycan in vitro (91-94). The N-terminal cytoplasmic domain of FtsN ( N FtsN), on the other hand, could directly interact with the IC domain of FtsA (54, 55, 95). With N FtsN interacting with the Z-ring and 14

15 S FtsN binding the peptidoglycan layer, FtsN has the potential to coordinate Z-ring contraction with spg synthesis in the periplasm. The discovery of ftsa mutant alleles with substitutions in the IC domain of FtsA, such as ftsa E124A, which could bypass the absolute requirement for FtsN suggested that the interaction with FtsA might be essential for its function (28, 57). But in the absence of FtsN, cells could barely divide even with overproduced mutant FtsA variants (28), suggesting that cell division required some other parts of FtsN under normal conditions. Indeed, previous results proved that the essential activities of FtsN relied on a periplasmic portion of the protein (90, 94). The cytoplasmic domain, the transmembrane domain, and the C-terminal S FtsN are dispensable for its essential function. Study in our lab narrowed down the minimal essential domain of FtsN ( E FtsN) to a small peptide centered around helix H2 in the periplasm (28). Production of E FtsN could restore normal cell division in ΔftsN cells when it was transported to the periplasm, but E FtsN itself did not contribute much to septal localization of FtsN. Meanwhile, the C-terminal S FtsN localized sharply to the division site by itself and it was able to concentrate FtsN molecules to the division site. Thus, S FtsN is a strong septal targeting determinant and contributes to the efficiency of cell constriction by helping to concentrate E FtsN at the division site (28). But what is the essential function of FtsN ( E FtsN)? FtsN was shown to directly interact with the bifunctional murein synthase PBP1B and stimulate both the transglycosylase and transpeptidase activities of PBP1B in vitro (96). Bacterial two-hybrid and FRET assays also indicated interactions between FtsN 15

16 and several other murein synthases, including PBP3 and MtgA (97-100). These findings supported a hypothesis that FtsN could trigger cell constriction through stimulating the activities of murein synthases at the septum. However, activating PBP1B is probably not the essential function of FtsN, as PBP1B is not essential in E. coli and stimulating the PBP1B activities in vitro requires the N-terminal nonessential part of FtsN (8, 10, 96). In addition, FtsN was also proposed to function in stabilizing the SR through interactions with multiple SR components (95, ). Our study, as shown below, helps to understand the essential function of FtsN in cell constriction. In Chapter 1, we demonstrate that the strong septal targeting determinant, S FtsN, depends on the newly synthesized and processed spg for septal localization, which normally requires the essential activity of E FtsN, FtsI (PBP3), and one of the murein amidases, AmiA, AmiB, or AmiC. On the other hand, the cytoplasmic domain of FtsN ( N FtsN) is a weak septal targeting determinant that helps to concentrate FtsN molecules at the septum in an S FtsN-independent fashion through interaction with FtsA. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of FtsN in triggering the constriction phase of the division process. In Chapter 2, we used genetic screens to identify suppressing mutations in three essential division genes, ftsa, ftsb, and ftsl, which restored cell division in cells producing normally non-functional variants of ftsn. FtsA I143L that carries a substitution in the Ic domain of FtsA was able to bypass the absolute requirement 16

17 for ftsn upon overproduction. The suppressing mutations in ftsl or ftsb, on the other hand, could restore cell constriction independent of E FtsN, but some mutations required the cytoplasmic N FtsN-FtsA interaction. Our results demonstrated for the first time that the FtsBLQ subcomplex actively participates in regulating septal murein synthesis and cell constriction and suggest a model for the SR to initiate cell constriction. In the model, the FtsBLQ subcomplex acts as a switch for septal murein synthesis. Upon its arrival at the SR, E FtsN induces conformational changes in the FtsBLQ subcomplex, which then triggers septal murein synthesis and cell constriction. Roles of DedD and other SPOR-domain proteins in cell division. In addition to FtsN, there are another three proteins in E. coli containing a SPOR domain, DedD, DamX, and RlpA. DedD and DamX are both bitopic innermembrane proteins with a similar topology as FtsN, while RlpA is an outermembrane lipoprotein ( ). All three proteins were found to localize to the SR during cell division but none of them were essential for cell division (28, 29). Thus, they were qualified as non-essential components of the SR (Fig. 1). Cells lacking DedD displayed a distinct cell chaining phenotype and DedD was essential for cell division in ftsn slm117 cells where the essential activity of FtsN is limited due to the truncation of S FtsN by a transposon insertion, suggesting that DedD is required for efficient cell division in E. coli (28). Cells lacking DamX showed no obvious division phenotype, but deleting damx in ΔdedD or ftsn slm117 cells greatly aggravated the defects in cell division (28). The absence of DamX 17

18 was also found to suppress a ts allele in ftsq (29). Thus, DamX is a nonessential division protein with functions seemingly redundant to that of DedD or FtsN. The absence of rlpa did not lead to any division phenotype in either WT cells or in combination with other mutations in E. coli (28). But a recent study indicated that RlpA functions as a lytic transglycosylase in Pseudomonas aeruginosa that helps to separate spg during cell constriction (106). In Chapter 3, we identified the critical functional domain of DedD ( N DedD) in its N-terminal conserved region. N DedD s function in cell constriction is partially redundant to that of FtsN. Massive cell lysis occurred primarily at the constriction site when the activity of E FtsN or N DedD was diminished in cells lacking PBP1B, suggesting that both E FtsN and N DedD are ultimately required for stimulating spg synthesis. N DedD is also a weak septal targeting determinant and the interaction between N DedD and the FtsBLQ complex is important for both the function and septal localization of N DedD. Other than their functions in cell constriction, both DedD and FtsN are important for the stability of the SR when the numbers of certain SR components are out of balance. 18

19 Chapter 1. Self-enhanced FtsN activity during the initiation of E. coli cell constriction. INTRODUCTION FtsN is an essential cell division protein that joins the SR before the onset of cell constriction (75, ). As with most SR proteins, it is unclear what the essential role of FtsN is. The ftsn gene was first identified as a multicopy suppressor of a ts allele in essential division gene ftsa (75). Elevated levels of FtsN were subsequently found to also suppress some ts alleles in ftsi, ftsk and ftsq (75, 111), and even to allow the propagation of cells with a complete lack of FtsK (101, 112), or of FtsEX (113). Depletion of FtsN allows assembly of all the other known essential components into non-constricting SR s, but the number of ring structures per unit of cell length in FtsN - filaments is 2 to 3 fold lower than in wildtype cells (109). Bacterial two hybrid studies suggest that FtsN interacts with several other SR proteins, including FtsA, FtsI (PBP3), FtsQ, FtsW, and MtgA (54, 97-99). Moreover, it was recently shown that the requirement for FtsN itself can be bypassed in cells producing certain mutant forms of FtsA that are thought to stabilize the SR to a greater degree than native FtsA (57). These observations are all compatible with a general role of FtsN in stabilizing the ring structure. In addition, it was recently found that FtsN interacts directly with PBP1B, one of the major bifunctional murein synthases in E.coli, and that it can stimulate both its transglycosylase and transpeptidase activities in vitro (96). Thus, in addition to stabilizing the SR, FtsN may have a more specific role in modulating septal murein synthesis. Lastly, based on the fact that FtsN is the last known essential 19

20 protein to join the SR, it is attractive to speculate the protein plays a role in triggering the constriction phase (3, 54). To what degree any of these proposed functions contribute to the essentiality of FtsN remains unclear. FtsN is a typeii bitopic transmembrane species of 319 residues with a small cytoplasmic domain (residues 1-30), a single transmembrane domain (residues 31-54), and a large periplasmic domain (residues ) (90) (FIG. 2). The periplasmic domain comprises three short regions with a-helical character that are centered around residues (H1), (H2), and (H3), an unstructured glutamine-rich linker (residues ), and a C-terminal globular SPOR domain (residues ) that has affinity for peptidoglycan (92, 94). Previous results obtained in our lab show that the essential function of FtsN can be performed by a surprisingly small periplasmic peptide of at most 35 residues that is centered around helix H2, but that this essential domain ( E FtsN) itself is unlikely to contribute much, if anything, to the accumulation of FtsN at constriction sites (28). On the other hand, the non-essential periplasmic SPOR domain ( S FtsN) localized sharply to these sites by itself, while SPOR-less FtsN derivatives localized poorly, suggesting that S FtsN is a strong septal targeting determinant. FtsN S contributes to the efficiency of cell division by helping concentrate E FtsN at the division site (28). In this study, we showed that septal localization of S FtsN depended on coproduction of E FtsN, in-cis or in-trans, unless cells were provided with the FtsA E124A protein (57) to allow constriction to ensue in the complete absence of E FtsN. Localization of S FtsN also depended on the activity of FtsI (PBP3) and the 20

21 presence of at least one of the periplasmic murein amidases AmiA, B, or C. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of FtsN in triggering the constriction phase of the division process. In addition, the results taken together with earlier biochemical work (91, 92, 96) suggest that S FtsN is recruited to some form of septal murein that accumulates only transiently at sites of active constriction. RESULTS Further definition of the essential domain of FtsN ( E FtsN). Starting at its N-terminus, FtsN consists of a small cytoplasmic (FtsN 1-30, N FtsN), a trans-membrane (FtsN 31-54, TM FtsN), and a large periplasmic (FtsN , C FtsN) domain. The latter further consists of juxta-membrane α-helices H1 (~FtsN 62-67, H1 FtsN), H2 (~FtsN 80-93, H2 FtsN) and H3 (~FtsN , H3 FtsN), a long linker peptide (FtsN , L FtsN), and a C-terminal globular SPOR domain (FtsN , S FtsN) (FIG. 2A). To define the essential domain of FtsN ( E FtsN), we previously used complementation assays with various portions of FtsN fused to the C-terminus of either cytoplasmic GFP or Tat-targeted periplasmic TT GFP (28). These showed that ΔftsN cells can be rescued by trans-membrane GFP-FtsN 1-90 or periplasmic TT GFP-FtsN , but not by GFP-FtsN 1-81 or TT GFP-FtsN (FIG. 2A). Rescue by the former two fusions suggested that E FtsN resides in the T71-E90 interval; although the failure of the fully periplasmic TT GFP-FtsN fusion to support cell 21

22 division in ΔftsN cells raised the possibility that C-terminal truncation of FtsN near/at the boundary (e.g. in functional GFP-FtsN 1-90 ) imposes a requirement for additional N-terminal residues in the 1-70 interval. We tested additional fusions to define the boundaries of E FtsN more precisely for this study (FIG. 2). Periplasmic fusions TT GFP-FtsN and TT GFP-FtsN were as effective as TT GFP-FtsN in stimulating cell division, and ΔftsN cells producing any one of these fusions propagated readily on both rich (LB) and minimal (M9) medium. In contrast, a TT GFP-FtsN fusion was not functional, suggesting E FtsN starts between L75-P79. Further trimming of TT GFP-FtsN at the C-terminal end yielded TT GFP-FtsN 75-93, the smallest periplasmic and functional fusion we identified. Though TT GFP-FtsN could still rescue ΔftsN cells, it was significantly less effective in doing so than the other functional fusions. Consequently, cells of strain CH34/pLP218 [ΔftsN/P lac :: tt gfp-ftsn ] could grow on M9 in the presence of IPTG, but suffered a lethal division defect on LB, even at high concentrations of inducer (FIG. 2B). Western analyses ruled out the possibility that TT GFP-FtsN was rendered unstable due to the truncation of residues P94-P99 (FIG. 7). Thus, E FtsN does not extend past Q93, but residues in the P94-P99 interval promote the activity of the essential domain, when produced as a fully periplasmic fusion to TT GFP. As alluded to above, the C-terminal boundary of E FtsN is slightly different when the domain is expressed as a trans-membrane fusion. The finding that fusion GFP-FtsN 1-90 appears fully functional while TT GFP-FtsN is not suggested some additional requirement for the first 70 residues in E FtsN function, 22

23 when the latter is truncated at E90. Additional fusions were studied to further explore this possibility. Like TT GFP-FtsN 71-90, a larger periplasmic fusion containing all periplasmic residues up to E90 ( TT GFP-FtsN ) was still incapable of rescuing ΔftsN cells. This implied that the presence of residues in the interval are not sufficient to explain the functionality of GFP-FtsN 1-90, and raised the possibility that specific residues in the cytoplasmic ( C FtsN) or transmembrane ( TM FtsN) domains might be important instead. This was tested with a variant of a functional trans-membrane RFP-FtsN 1-90 fusion in which both C FtsN and TM FtsN were replaced with corresponding domains ( C MalF and TM1 MalF, respectively) of the MalF protein. As summarized in FIG. 2A, the RFP- MalF FtsN variant was still capable of supporting cell division in ΔftsN cells. Altogether, the complementation results indicate that the functionality of transmembrane XFP-FtsN 1-90 fusions on one hand, and the non-functionality of periplasmic TT GFP-FtsN or TT GFP-FtsN fusions on the other, is not determined by any specific FtsN residues in the 1-71 interval. Rather, being membrane tethered per se may help to position E FtsN at an optimal distance from the membrane and/or hold it in an active conformation. We also explored the importance of residues between TM FtsN and E FtsN by studying derivatives of a full length GFP-FtsN fusion (encoded by pch201) in which 15 of these (FtsN 59-73, including H1) are replaced with unrelated peptides of 5 (pbl205), 15 (pbl211) or 95 (pbl210) residues, with the latter two mostly derived from the unstructured P/Q-rich domain of the ZipA protein (114). As summarized in FIG. 2A, all three substitution derivatives supported viability of 23

24 ΔftsN cells. Notably, however, substitution of FtsN with only 5 residues (a deficit of 10 residues) severely compromised the ability of the protein to support cell division, while substitution with an equal number, or with 80 additional, residues had little effect (FIG. 2C). None of the substitutions affected the stability of the fusion proteins (FIG. 8). We infer that restriction of the maximal distance of E FtsN to the outer leaflet of the inner membrane is far more detrimental to function than expansion of this distance. Hence, one, and possibly the only, role of the residues between TM FtsN and E FtsN is to simply serve as a linker that allows E FtsN to reach a sufficient distance from the IM. E FtsN-dependent septal localization of S FtsN. In a previous study, we showed that S FtsN is a septal targeting determinant that helps concentrate FtsN at the SR during cell division (28). E FtsN, on the other hand, does not contribute much to septal localization of FtsN (28). But septal localization of S FtsN seemed to be dependent on the essential activity of E FtsN. In the E FtsN-depletion strain CH34/pMG20/pMG4 [ΔftsN/P BAD :: TT bfp-ftsn /P lac :: TT gfp-ftsn ], production of a fusion of the essential domain to Tat-targeted Blue fluorescent protein ( TT BFP- E FtsN) can be modulated with arabinose and that of TT GFP- S FtsN with IPTG. In the presence of both inducers, cells appeared to divide normally and TT GFP- S FtsN accumulated at each constriction site (FIG. 3B). In the absence of arabinose ( E FtsN - ), however, TT GFP- S FtsN did not form regularly spaced rings, as might be expected if S FtsN could target SR s independently of E FtsN, but accumulated in rings only at rare constrictions still remaining in some of the filaments (FIG. 3C, and not 24

25 shown). So S FtsN targeting to constriction sites normally indeed depends on the presence of E FtsN. E FtsN-independent septal localization of S FtsN. Two scenarios might explain the E FtsN-dependent accumulation of S FtsN at SRs. The first is that S FtsN can interact directly with E FtsN, and that the latter acts as a pilot for the former. We considered this unlikely because all GFP fusions that lacked S FtsN, especially TT GFP- E FtsN, localized poorly, if at all (28). The second is that, even though E FtsN by itself fails to localize, it can still stimulate cell constriction when its concentration in the periplasm is sufficiently high, and that S FtsN is attracted to some other feature of the constricting SR. The recent discovery that a mutant FtsA protein (FtsA E124A ) allows cells to grow and divide in the complete absence of FtsN (57) provided us with means to conclusively rule out the first scenario. To this end, we first replaced ftsa on the chromosome of strain TB28 with ftsa E124A, and then introduced the ΔftsN (ftsn<>aph) knock-out lesion by P1- mediated transduction. While cells of the BL18 [ftsa E124A ] recipient strain divided at a normal frequency as expected (57), the resultant ftsa E124A ΔftsN transductant cells required additional copies of the ftsa E124A allele, such as on plasmid pbl12, for cell division. Even then, division was not completely normal as cells of the resulting strain BL20/pBL12 [ΔftsN, ftsa E124A )/ftsa E124A ] displayed a chaining phenotype (FIG. 3D&E). 25

26 We next compared the localization patterns of periplasmic TT GFP and TT GFP- S FtsN in BL20/pBL12 derivatives that carry a single copy of pbl5 [P lac :: TT gfp] or pbl6 [P lac :: TT gfp-ftsn ] integrated at atthk022. Expression of unfused TT GFP in strain BL20(iBL5)/pBL12 resulted in an homogenous fluorescent halo around each cell without obvious ring-like accumulations at constriction sites (FIG. 3E), suggesting that chaining was due to slow constriction rather than to any defect that would cause a grossly expanded inter-membrane distance at sites of constriction. In contrast, TT GFP- S FtsN accumulated at each constriction site of BL20(iBL6)/pBL12 cells (FIG. 3D). We conclude that S FtsN is readily able to recognize these sites in the complete absence of any other domain of FtsN, including E FtsN. Septal localization of FtsN S is dependent on PBP3(FtsI) activity. Given that recruitment of S FtsN to division sites appeared to depend on the onset of the constriction phase of the fission process (FIG. 3C), and that the SPOR domains of both E.coli FtsN (92) and B.subtilis CwlC (91) have affinity for murein, it is reasonable to suppose that the feature of constriction sites that is recognized by S FtsN includes some form of septal murein. To test this idea further we monitored localization of TT GFP- S FtsN after treatment of cells with cephalexin, a β-lactam that specifically interferes with the murein transpeptidase activity of penicillin-binding protein 3 (PBP3, FtsI), which is specifically required for the synthesis of septal murein during cell constriction (115). As illustrated in FIG. 3F, TT GFP- S FtsN localized throughout the periplasm 26

27 in cephalexin-treated filaments of TB28(iBL50) [WT(P lac :: TT gfp-ftsn )], save for some very weak accumulations at some of the remaining constrictions. In contrast, and as expected (116, 117), a ZipA-GFP fusion accumulated in regularly-spaced rings in cephalexin-treated TB28(iBL48) [WT(P lac ::zipa-gfp)] filaments (FIG. 3G). Septal localization of FtsN S is dependent on murein amidase activity. The purified periplasmic domain of FtsN (FtsN ) was previously found to preferentially bind the longest (> 25 disaccharide units) glycan strands that remain upon amidase treatment of purified sacculi, and to bind poorly to sacculli from cells that are deficient in murein amidase activities (92). This suggested that the septal localization of S FtsN might be impaired in amidase-deficient cells as well, and we tested this using a set of isogenic strains lacking (combinations of) the amia, B, and C genes. TT GFP- S FtsN still accumulated sharply at constriction sites in cells of strains BL25 [ΔamiA], BL7 [ΔamiB], and BL9 [ΔamiC], as well as in the (relatively short) cell chains formed by strains BL10 [ΔamiB, ΔamiC] and BL11 [ΔamiA, ΔamiC] (FIG. 4A, and data not shown) indicating that neither of the amidases is specifically required for accumulation of S FtsN at septa. In the long cell chains formed by the triple knock-out strain BL12 [ΔamiA, ΔamiB, ΔamiC], however, the localization pattern of TT GFP- S FtsN (FIG.4 C) closely resembled that of unfused TT GFP (FIG. 4D). Thus fluorescence appeared throughout the periplasm though some weak septal accumulations along the cell chains were apparent as well. 27

28 As the latter were similarly apparent with the TT GFP control, we assume these reflected an increased inter-membrane distance at such sites. The results indicated that efficient targeting of S FtsN to sites of constriction indeed requires the activity of at least one of the three periplasmic murein amidases in E.coli (FIG. 5). Model for self-enhanced FtsN activity at onset of cell constriction. Substantial accumulation of FtsN at the division site does not occur until the onset of cell constriction. Under normal conditions, initiation of cell constriction requires the essential activity of E FtsN. Taken together with existing biochemical evidence (91, 92, 96), our results suggest that FtsN joins the SR in a self-enhancing manner, which is a property that is consistent with a role in triggering the constriction phase of the division process. As illustrated in FIG. 6, our results suggest the following order of events at the onset of cell constriction: i) Once the SR matures to the point where the murein transpeptidase PBP3 (FtsI) has joined the assembly, the SR is poised to initiate the constriction process. ii) However, cell constriction requires a sufficient level of E FtsN activity at the SR and the majority of FtsN is spread along the membrane prior to synthesis of any septal murein. iii) Even so, a fraction of the FtsN molecules in the membrane interact with the poised SR structure, likely aided by weak specific interactions between the N-terminal portion of FtsN with other SR components (101), and their associated E FtsN domains stimulate a small amount of constriction and septal murein synthesis. iv) This murein is 28

29 processed by multiple activities, including murein amidases, leading to the generation of a sharply localized and transient form of septal murein that also constitutes a high-affinity binding substrate for S FtsN. v) Accumulation of this substrate at the SR recruits additional FtsN molecules from elsewhere in the membrane by binding their S FtsN moieties. vi) This, in turn, leads to an increased local concentration of E FtsN at the SR, and an enhanced progression of steps iiivi, and so on. DISCUSSION The minimal essential domain of FtsN ( E FtsN) required for cell division was identified as a small peptide in the periplasmic part of FtsN. The C-terminal SPOR domain ( S FtsN) functions as a strong septal targeting determinant that contributes to efficient cell constriction by concentrating E FtsN at the SR (28). Our results have shown that septal targeting of S FtsN actually depends on the essential activity of E FtsN, although this dependency could be bypassed by the FtsA E124A mutant protein. Septal localization of S FtsN was also proved to be dependent on the activities of PBP3 (FtsI) and the three amidases, which are involved in synthesis and processing of septal murein, respectively. Given that SPOR domains from FtsN and CwlC were found to be able to bind peptitoglycan in vitro (91, 92, 96), all these results suggested a self-enhancing model for FtsN at the division site, which is compatible with its function in triggering cell constriction. 29

30 Our work provides useful information for identifying the substrate that S FtsN recognizes at the division site. Though alternative mechanisms cannot be excluded, it seems likely that S FtsN and other SPOR domains are attracted to constriction sites by the local accumulation of some form of murein. As GFP- S FtsN did not linger at newly created poles, this material must only be transiently available for binding at the constricting SR. The failure of S FtsN to accumulate at constriction sites in cells lacking the three murein amidases AmiA, B, and C, correlates well with the biochemical evidence that the periplasmic domain of FtsN (including S FtsN) bound poorly to sacculi from a similar mutant, and that both this domain, as well as the isolated SPOR domain of CwlC ( S CwlC), still interacted with material that remained upon digestion of murein sacculi with purified amidases (91, 92). The simplest interpretation of these results is that S FtsN specifically recognizes glycan strands that have been, at least partially, denuded of peptide side-chains (presumably both free and cross-linked) by amidase action (92). Such denuded strands are likely subject to (partial) detachment from the sacculus and/or to subsequent processing by lytic transglycosylases, which could be responsible for the transient nature of the SPOR-binding substrate. Another possibility is that SPOR domains recognize a murein geometry that is uniquely present at the SR and inherently transient in normally dividing cells, such as the junction between newly split and as yet unsplit septal murein. Our results also suggest that the ability of S FtsN to recognize its binding substrate contributes to FtsN function by helping to concentrate E FtsN at the SR as soon as constriction begins. Helping to increase E FtsN activity at the SR may 30

31 be the only function of the SPOR domain, but it is attractive to speculate on additional roles. For example, FtsN seems well placed to also serve a quality control function during the constriction process. Thus, any problem in septal murein assembly or processing that reduces the availability or accessibility of the S FtsN substrate might well lead to a reduction in the concentration of E FtsN at the SR, and a commensurate reduction in the constriction rate. The existence and importance of such a function may only become apparent under special circumstances, and further work will be needed to explore this possibility further. Now, to understand why FtsN is normally essential for cell division, it is pertinent to understand what E FtsN does. This question if further investigated in the next chapter. 31

32 Chapter 2. Roles for FtsA and the FtsBLQ subcomplex in FtsNmediated triggering of cell constriction. INTRODUCTION In the previous chapter, we showed that FtsN triggers cell constriction through its essential domain ( E FtsN) in the periplasm. To understand how FtsN initiates cell constriction, we now need to find out how E FtsN triggers septal murein synthesis and cell constriction. As a small peptide of at most 14 residues (FtsN ), the action of E FtsN at the SR is more likely to be allosteric rather than catalytic. In that case, E FtsN needs to interact with a protein target(s) at the SR in order to trigger cell constriction. As the interaction involving E FtsN is essential for cell division, the target(s) for E FtsN must be either an essential protein or a group of proteins that collaborate to play an essential role in cell division. Many proteins involved in cell division or murein synthesis, including several essential SR components, were demonstrated to interact with FtsN using bacterial two-hybrid analyses or in vitro biochemical assays (95-102). But none of these approaches were able to distinguish the interactions specifically involving E FtsN. We attempted to find the target(s) for E FtsN by comparing an FtsN fusion carrying a non-functional substitution in E FtsN ( E FtsN - ) with an E FtsN + FtsN fusion for interactions with various proteins involved in cell division and murein synthesis using the bacterial two-hybrid (BACTH) assay (118)(data not shown). But no difference was detected between the mutant and native FtsN fusions. It is possible that E FtsN itself does not contribute much, if any, to the binding affinity with the target, which is supported by the finding that isolated E FtsN was unable 32

33 to localize to the septa by itself (28). These properties of E FtsN render most biochemical assays ineffective for identifying the target for E FtsN. We thus turned to genetic approaches and developed genetic screens for extragenic suppressors of non-functional ftsn mutants with substitutions in one of the three critical residues in E FtsN, W83, Y85, and E90. From the screens, we identified one E FtsN*-suppressing mutation in ftsa (ftsa I143L ), one in ftsb (ftsb D59H ), and another one in ftsl (ftsl D93G ). Similar to the previous identified ftsa E124A allele, ftsa I143L bypassed the absolute requirement for ftsn only when the mutant protein was overproduced. ftsb D59H and ftsl D93G, on the other hand, restored cell division in the absence of E FtsN, but they instead depended on the cytoplasmic domain of FtsN ( N FtsN) and more specifically, on the N FtsN-FtsA interaction. Further screens using randomly mutagenized libraries identified additional E FtsN*-suppressing mutations in ftsb and ftsl. The ftsb alleles with substitutions in residue E56 allowed cell division in complete absence of ftsn. Further investigations on these suppressing mutations indicate that they accelerate the cell division process through promoting septal murein synthesis. Our results indicate that the FtsBLQ protein subcomplex actively participates in the regulation of septal murein synthesis and cell constriction. Based on the results, we propose a model for FtsN in the initiation of cell constriction. In the model, E FtsN promotes conformational changes in the FtsBLQ subcomplex upon its arrival at the SR, leading to the release of an inhibitory activity of the FtsBLQ subcomplex on the murein synthases and, thus initiation of spg synthesis and cell constriction. 33

34 RESULTS Identification of critical residues in E FtsN. To identify individual residues critical to FtsN function, each residue in the FtsN interval (~ E FtsN, see chapter 1) was subjected to site-scanning (residues 80-93) and/or site-directed mutagenesis, and resulting mutants were tested for their ability to rescue growth of the FtsN-depletion strain CH31 [P BAD ::ftsn] in the absence of arabinose. We analyzed 114 ftsn alleles with mutations of a single codon, yielding a total of 57 distinct residue substitutions within the FtsN interval. A summary of the latter is illustrated in FIG. 9A. 26 out of the 57 distinct substitutions had no effect on the ability of GFP-FtsN to rescue FtsN-depleted cells, and such permissible substitutions were identified for each of the 19 residues in the targeted interval. The remaining 31 substitutions abrogated the essential function of FtsN. Interestingly, all affected one of only three residues; W83, Y85, or L89. With the exception of Y85G, permissible substitutions in these three residues generally conserved their aromatic and/or hydrophobic character, though most substitutions were not tolerated. For example, Phe was permissible at each one of the three positions, but Ala was not and Y85 could also not be substituted with I, L, V, or W. The fact that site-scanning mutagenesis did not yield nonpermissible substitutions in any other residue within the interval is notable, and indicates that W83, Y85, and L89 are the only FtsN residues that are truly critical for division and viability. 34

35 Screen for extragenic suppressors of non-functional ftsn alleles. Given that E FtsN is essential, periplasmic, and small, we hypothesize that it stimulates cell constriction via allosteric regulation of a periplasmic domain of one of the other essential SR proteins. Its immediate target could be directly involved in spg synthesis (e.g. FtsI=PBP3, or FtsW), or its allosteric 'signal' could be relayed by additional SR components via more circuitous routes before reaching the spg synthases. To help elucidate the function of E FtsN, we performed screens for extragenic suppressors of specific lethal mutations in one of each of the three essential residues of FtsN identified above. Forcing cells to survive with a non-functional FtsN(Y85W) variant, for example, would ideally select for compensatory mutations in the gene for the immediate target of E FtsN that restore a productive E FtsN-target interaction. Strain BL86 [ΔftsN ΔrecA ΔlacIZYA leu::tn10] carrying plasmid pbl200 [aada repa ts P syn135 ::ftsn I-SceI ci857 PλR::i-sceI] was constructed to serve as host in these screens (FIG. 9B). At 30 C, BL86/pBL200 cells express WT FtsN exclusively from a constitutive promotor on the plasmid. At elevated temperatures (above ~35 C), however, ftsn expression ceases because pbl200 both fails to replicate, and self-destructs due to derepression of the plasmidborne gene for the I-SceI meganuclease, which cuts the plasmid at an I-SceI site that is unique in the strain. Incubation of BL86/pBL200 in LB at C caused severe cell filamentation and reduced cfu >10 8 -fold after 24 hr, indicating effective elimination of pbl200. For screens, the strain was transformed with an 35

36 unstably inherited mini-f plasmid [bla laci P lac ::gfp-ftsn*::lacz] encoding a mutant GFP-FtsN* variant as well as LacZ under control of the lac promotor, and cells were grown at C for 24-48hr in liquid LB with IPTG. In case cultures showed an appreciable density, survivors were colony-purified on solid medium with IPTG, and then subjected to several tests to ensure that they indeed propagated without native FtsN (Spec S ) but still required FtsN* (retention of gfpftsn* allele on mini-f plasmid, and IPTG-dependent cell division). Most essential SR proteins (save FtsB, FtsK, FtsN itself, and ZipA) are encoded by a gene cluster at 2' on the chromosome, near the leu::tn10 marker in strain BL86. To map a suppressor mutation, therefore, we first determined if it was co-transducible with leu::tn10 (see methods). If so, the nucleotide sequence of (parts of) the 2' cluster of the suppressed strain was determined by conventional means. If not, the entire chromosome of the suppressed strain was subjected to deep sequencing, and compared to that of unsuppressed BL86. Screens were performed with BL86/pBL200 producing non-functional GFP- FtsN variants with one of seven distinct substitutions in one of the three critical residues of E FtsN (W83L, W83M, W83T, Y85S, Y85W, L89H or L89S). E FtsN*- suppressors were identified in screens with GFP-FtsN W83L, GFP-FtsN Y85S, and GFP-FtsN Y85W (FIG. 9C). But only screens with GFP-FtsN W83L and GFP-FtsN Y85W yielded spontaneous suppressor mutations in cell division genes, and these are discussed below. Suppression of ftsn Y85W by ftsa I143L. 36

37 Screening for survivors of strain BL86/pBL200 carrying pbl215 [P lac ::gfpftsn Y85W ::lacz] yielded two promising clones, BL86-KK1/pBL215 and BL86- AK1/pBL215, that had lost pbl200 and relied on pbl215 for survival. Cotransduction experiments indicated that both harbored leu::tn10-linked suppressing mutations. The co-transduction frequency for BL86-KK1/pBL215, was ~58%, and nucleotide sequencing of the chromosomal ftsqaz region revealed a transversion in codon 143 of ftsa (ATC>CTC), causing substitution of I with L (I143L) in the 1C domain of the FtsA protein (FtsA I143L ). Notably, this same allele was previously isolated as a suppressor of a hypomorphic variant of the FtsQ protein (FtsQ V92D ) (119). To verify that the ftsa I143L mutation is sufficient for survival of cells producing GFP-FtsN Y85W as the sole source of FtsN, we first converted WT strain TB28 to ftsa I143L by gene replacement (120). The resulting strain, BL114 [ftsa I143L ], was then transformed with pbl215 [P lac ::gfpftsn Y85W ::lacz] or a vector control (prc7 [P lac ::lacz]), and transformants were tested for their ability to tolerate the introduction of ΔftsN<>aph via P1-mediated transduction in the presence of IPTG. Attempts to transduce ΔftsN<>aph to strain BL114 [ftsa I143L ] carrying the vector control yielded no viable transductants, indicating that a chromosomal ftsa I143L allele is not sufficient to bypass the need for FtsN altogether (Table 1). In contrast, BL114/pBL215 cells readily tolerated removal of chromosomal ftsn yielding strain BL120/pBL215 [ftsa I143L ΔftsN/ P lac ::gfp-ftsn Y85W ] (Table 1). BL120/pBL215 cells managed to grow in the presence of IPTG but displayed a cell chaining phenotype, indicating that FtsAI 143L was unable to restore wildtype division frequency with FtsN Y85W (FIG. 37

38 10A). Consistent with the transduction results, BL120/pBL215 cells still required FtsN Y85W for cell division, as they became long filaments in LB medium without IPTG (FIG. 10A). The ΔftsN<>aph lesion could also be readily introduced in BL114 [ftsa I143L ] cells carrying extra copies of ftsa I143L on plasmid pbl236 [repa ts ftsa I143L ]. However, cells of the resulting strain BL120/pBL236 [ftsa I143L ΔftsN/ repa ts ftsa I143L ] were highly filamentous at 30 C, and failed to propagate when replication of pbl236 ceased at 42 C (FIG. 10B and data not shown). Thus, an elevated level of FtsA I143L can bypass the absolute essentiality of FtsN, but compensates for its absence only incompletely. These properties of FtsA I143L are similar to those of the FtsA E124A variant described previously (28, 57). It is interesting to note that both variants carry substitutions in the 1C domain of FtsA, though they affect residues that reside at opposite ends of this domain (57, 119). Suppression of ftsn Y85W by ftsl D93G. The co-transduction frequency of the suppressing mutation in clone BL86- AK1/pBL215 with leu::tn10 was ~75%, and nucleotide sequencing of the chromosomal ftsl-z region revealed a transition in codon 93 of ftsl (GAC>GGC), causing a D93G substitution in the periplasmic domain of the FtsL protein (FtsL D93G ) (FIG. 13A). To test if this substitution is indeed sufficient for survival of cells producing GFP-FtsN Y85W as the sole source of FtsN, we first recombineered the ftsl D93G mutation onto the chromosome of a leu::tn10 derivative of TB28. Strain BL154 [leu::tn10 ftsl D93G ] was then transformed with pbl215 [P lac ::gfp- 38

39 ftsn Y85W ::lacz], and tested for its ability to tolerate the introduction of ΔftsN<>aph via transduction in the presence of IPTG. BL154 carrying a vector control could not be transduced with ΔftsN<>aph, indicating that the ftsl D93G mutation did not compensate for the complete absence of FtsN (Table 1). The presence of pbl215, by contrast, readily allowed the creation of strain BL157/pBL215 [leu::tn10 ftsl D93G ΔftsN/ P lac ::gfp-ftsn Y85W ], implying that the FtsL D93G protein indeed imparted functionality to FtsN Y85W again (Table 1). Suppression of ftsn W83L by ftsb D59H. Screening for survivors of strain BL86/pBL200 carrying pbl216 [P lac ::gfpftsn W83L ::lacz] yielded clones BL86-AK11/pBL216 and BL86-AK12/pBL216 that had lost pbl200 and relied on pbl216 for survival. The suppressing mutation in neither clone was co-transducible with leu::tn10, indicating they mapped outside the 2' cluster. Genomic sequencing revealed the same transversion (GAT>CAT) in both clones, suggesting they were siblings. The mutation maps to ftsb, and leads to a D59H substitution in the periplasmic domain of the protein (FtsB D59H ) (FIG. 13A). To verify that this mutation is sufficient for survival of cells producing GFP-FtsN W83L as the sole source of FtsN, we first again used gene replacement (120) to convert the ftsb gene of WT strain TB28 to ftsb D59H. The resulting strain, BL140 [ftsb D59H ], was then transformed with pbl216 [P lac ::gfp-ftsn W83L ::lacz], and tested for its ability to tolerate the introduction of ΔftsN<>aph via transduction in the presence of IPTG. Indeed, transductants were readily obtained (Table 1) and 39

40 the resulting strain, BL141/pBL216 [ftsb D59H ΔftsN/ P lac ::gfp-ftsn W83L ], required IPTG for growth (FIG. 10F). Thus, FtsB D59H renders FtsN W83L functional again. ftsb D59H or ftsl D93G cells no longer require E FtsN for viability, but depend on N FtsN in its absence. Additional experiments indicated that neither suppression of ftsn W83L by ftsb D59H, nor of ftsn Y85W by ftsl D93G, occurred in an allele-specific fashion. Production of a periplasmic fusion to the WT E FtsN peptide ( TT GFP-FtsN ) was still sufficient to compensate for the absence of full length FtsN in ΔftsN ftsb D59H or ΔftsN ftsl D93G cells (Table 2). Thus, neither FtsB D59H nor FtsL D93G prevented the wildtype E FtsN peptide from executing its essential activity. In addition, either ftsb D59H or ftsl D93G was sufficient to rescue ΔftsN cells producing any of the tested GFP-FtsN* variants with otherwise lethal substitutions of W83, Y85, or L89 in their E FtsN domain (Table 1, FIG. 10E and F). Because neither ftsb D59H nor ftsl D93G was sufficient to bypass the need for FtsN altogether (Tables 1 and 2), this lack of allele specificity was unexpected. It also raised the possibility that the E FtsN domain might no longer be essential in ftsb D59H or ftsl D93G cells, but that they become critically dependent on another part of the FtsN protein when E FtsN is non-functional or absent. This possibility was confirmed by the observation that production of GFP-FtsN Δ(64-101), an otherwise non-functional fusion in which the E FtsN domain and flanking residues are replaced with an arbitrary peptide, was sufficient to rescue ΔftsN ftsb D59H and ΔftsN ftsl D93G cells (Table 2). 40

41 To elucidate which of the non- E FtsN domains of FtsN gained a critical function in ftsb D59H or ftsl D93G cells, GFP-FtsN derivatives that lacked E FtsN and one or more additional domains were tested for their ability to support division of ΔftsN ftsb D59H and/or ΔftsN ftsl D93G cells. As summarized in Table 2, fusions GFP- FtsN 1-81 and GFP-FtsN 1-71 could rescue these cells, implying that most of periplasmic FtsN, including its SPOR domain, was dispensable for this function. A variant of GFP-FtsN 1-81 in which the transmembrane domain of FtsN is replaced with that of MalF (GFP-FtsN MalF FtsN ) also rescued, showing that TM FtsN was also not specifically required. In contrast, a variant of GFP-FtsN 1-81 in which the cytoplasmic domain of FtsN is replaced with cytoplasmic MalF residues (GFP-MalF FtsN ) failed to correct a ΔftsN lesion in ftsb D59H or ftsl D93G cells. Hence, unlike WT cells, ftsb D59H or ftsl D93G cells can survive without the essential periplasmic domain of FtsN, provided that the cytoplasmic domain of FtsN is still present (Table 2). We also tested whether the two E FtsN*-suppressing mutations in the IC domain of FtsA, FtsA I143L and FtsA E124A, behaved like FtsB D59H or FtsL D93G. Cells carrying chromosomal ftsa I143L or ftsa E124A allowed ftsn deletion when they produced any of the non-functional GFP-FtsN* variants or TT GFP-FtsN containing WT E FtsN (Tables 1 and 2). So similar to FtsB D59H and FtsL D93G, neither of the FtsA mutants enabled cell division in an allele-specific fashion, although ftsa I143L or ftsa E124A cells seemed to grow better with GFP-FtsN* variants carrying substitutions in residue Y85 than in W83 or L89 (FIG. 10C and D). But different from FtsB D59H or FtsL D93G, neither FtsA I143L nor FtsA E124A, when 41

42 expressed at normal levels, was able to rescue cell division with FtsN fusions lacking E FtsN, such as GFP-FtsN Δ(64-101) or GFP-FtsN 1-81 (Table 2). Thus, the ftsa mutations rescue cell division in a fashion that is different from the ftsb or ftsl mutations, and independent of the cytoplasmic domain of FtsN. N FtsN residues required for viability of ftsb D59H or ftsl D93G cells lacking E FtsN. The N-terminal cytoplasmic domain of E.coli FtsN ( N FtsN) consists of only ~30 residues, and sequence comparisons indicate that the most N-terminal ones (FtsN 1-7 ) are the best conserved (FIG. 11A). To test if this region is important for viability of ftsb D59H or ftsl D93G cells that lack E FtsN, we compared the complementing properties of GFP-FtsN and GFP-FtsN 1-81 fusions with those of derivatives bearing a double substitution (DY>SA) in the two conserved adjacent FtsN residues D5 (D5S) and Y6 (Y6A). Consistent with the notion that ftsb D59H or ftsl D93G cells gained the ability to survive in the presence of either the N FtsN or E FtsN domain (see above), production of GFP-FtsN ( N FtsN +, E FtsN + ), GFP- FtsN DY>SA ( E FtsN + ), or GFP-FtsN 1-81 ( N FtsN + ) supported cell division and viability of ΔftsN ftsb D59H and ΔftsN ftsl D93G cells. In contrast, the GFP-FtsN 1-81,DY>SA fusion failed to rescue ΔftsN ftsb D59H or ΔftsN ftsl D93G cells (Table 2). Western analyses ruled out the possibility that the double substitution affected the stability of GFP-FtsN 1-81,DY>SA (FIG. 12). Thus, residue D5 and/or Y6 in the N-terminal cytoplasmic domain of FtsN play a critical role in the E FtsN-independent division process that can occur in ftsb D59H or ftsl D93G cells. 42

43 The extreme N-terminus of FtsN promotes localization to the SR, and interaction with FtsA in BACTH assays. The N FtsN domain has been implicated in direct interaction with the FtsA protein (95), raising the possibility that this interaction is important for E FtsNindependent division of ftsb D59H or ftsl D93G cells. Support for this idea came from BACTH assays in which we used truncated (SPOR-less) versions of FtsN to help increase the specificity of the assay by reducing the incidence of spurious interactions between division proteins due to their accumulation and high concentration at the SR. Notably, T18-FtsA interacted robustly with T25-FtsN in this assay, but failed to do so with a version bearing the DY>SA substitution. In contrast, both T25-FtsN and T25-FtsN 1-105,DY>SA interacted weakly, but comparably, with a T18-FtsQ fusion (FIG. 11B). Even though accumulation of FtsN at SR's is primarily determined by its periplasmic SPOR domain, we previously noted that SPOR-less versions of GFP-FtsN still weakly accumulate at SR's (28). The interaction of N FtsN with FtsA suggested an involvement of N FtsN in this weak accumulation. Accordingly, fusions GFP-FtsN 1-81 and GFP-FtsN MalF FtsN (both N FtsN + ) showed weak accumulation at constriction sites of WT cells, while GFP-MalF FtsN ( N FtsN - ) did not. Moreover, the GFP-FtsN 1-81,DY>SA fusion also failed to localize, emphasizing the importance of residues D5 and/or Y6 in N FtsN function (FIG. 11C). 43

44 These results support a model in which a fraction of FtsN molecules in the cell can be recruited to the SR in a SPOR-independent fashion via an interaction between N FtsN and FtsA. Moreover, the results suggest that the same cytoplasmic N FtsN-FtsA interaction is critical for the survival of ΔftsN ftsb D59H and ΔftsN ftsl D93G cells that lack the periplasmic E FtsN domain. Synergy of FtsA I143L with FtsB D59H or FtsL D93G in compensating for the absence of FtsN. Why the E124A or I143L substitutions in the IC domain of FtsA promote survival of cells with compromised FtsN function is unclear, but one straightforward scenario is that N FtsN promotes a conformational state of FtsA that is also favored by the residue substitutions. Because ftsb D59H or ftsl D93G cells can survive with N FtsN as the sole portion of FtsN in the cell, this scenario predicted that introduction of the ftsa I143 (or ftsa E124A ) allele in ftsb D59H or ftsl D93G cells might allow their survival in the complete absence of FtsN. Hence, we created strains BL149 [ftsa I143L ftsb D59H ], BL151 [ftsa E124A ftsb D59H ], and BL164 [ftsa I143L ftsl D93G ], and tested their ability to survive complete removal of ftsn by transduction of the ΔftsN<>aph allele. Indeed, while parent strains bearing only one of the mutant ftsa, B or L genes failed to yield viable ΔftsN transductants, the three new strains readily tolerated removal of ftsn (Table 3). The resulting strains, BL150 [ΔftsN ftsa I143L ftsb D59H ], BL152 [ΔftsN ftsa E124A ftsb D59H ], and BL165 [ΔftsN ftsa I143L ftsl D93G ], grow in LB media but displayed obvious division defects (FIG. 13B). 44

45 Synergy of FtsB D59H with FtsL D93G in compensating for the absence of FtsN. Why D59H or D93G substitutions in the periplasmic domains of FtsB or L, respectively, can compensate for absence of the otherwise essential domain of FtsN ( E FtsN) is also unclear. Similar to the proposed role of N FtsN in directly promoting a conformational change of FtsA that promotes constriction of the SR, one parsimonious possibility is that E FtsN directly or indirectly promotes a conformational change of the FtsBLQ subcomplex that is also promoted by the FtsB D59H and FtsL D93G substitutions. However, the FtsB D59H or FtsL D93G substitutions do not fully compensate for absence of E FtsN, as ftsb D59H or ftsl D93G cells still require N FtsN in lieu of E FtsN for survival (see above). We constructed strain BL159 [ftsb D59H ftsl D93G ] to test if the mutations in the two genes act synergistically in compensating for a lack of FtsN activity. Interestingly, the ΔftsN<>aph allele could be readily introduced into BL159, yielding strain BL163 [ftsb D59H ftsl D93G ΔftsN] that managed to divide in complete absence of ftsn (Table 3 and FIG. 13B). Thus, combining the two E FtsN*-suppressing mutations in ftsb and ftsl could compensate for the complete absence of ftsn. Random mutagenesis identifies regions in FtsB and FtsL that determine essentiality of E FtsN. Plasmids pbl304 [P syn135 ::ftsb D59H ] and pbl305 [P syn135 ::ftsl D93G ] are low copy-number plasmids that direct constitutive production of FtsB D59H and FtsL D93G, respectively. Strain TB28 [wt] carrying either one of these plasmids 45

46 readily tolerated removal of chromosomal ftsn, provided that it also harbored a second plasmid directing production of either N FtsN, or E FtsN, or both (not shown). Thus, ftsb D59H and ftsl D93G are both dominant over WT ftsb and ftsl, respectively, in allowing viability in the absence of E FtsN. We took advantage of this in plasmid-based screens for additional ftsb and ftsl alleles that overcome the lethality that is normally associated with loss of E FtsN function. Error-prone PCR was used to prepare libraries of randomly mutated ftsb and ftsl in the context of plasmids pbl336 [P syn135 ::ftsb] and pjh2 [P syn135 ::ftsl], respectively. Libraries were then introduced into strain JH1 [ΔftsN ΔrecA ΔlacIZYA] already harboring pbl200 [aada repa ts P syn135 ::ftsn I-SceI ci857 PλR::i-sceI], and one of several mini-f derivatives encoding variants of GFP-FtsN that lack functional E FtsN (see Methods). Transformants were then plated at 37 o C (or 42 o C) to induce loss of pbl200 and, hence, select for plasmid-borne ftsb or ftsl alleles that allow survival of E FtsN cells. These screens yielded a total of 16 clones from the pbl336 [P syn135 ::ftsb] library (~7000 screened), and 13 from the pjh2 [P syn135 ::ftsl] one (~9000 screened). Subsequent sequence analyses and subcloning, to resolve clones with multiple silent and/or missense mutations in ftsb (9/16) or ftsl (9/13), then allowed us to identify a total of 6 and 5 relevant single residue substitutions in FtsB and FtsL, respectively. Interestingly, substitutions in both proteins were limited to 3 (FtsB A55, E56, and D59) or 4 (FtsL E88, N89, D93, and H94) neighboring residues (FIG. 13A). The mutations in ftsb and ftsl were subcloned to plasmid pbl336 [P syn135 ::ftsb] or pjh2 [P syn135 ::ftsl], respectively, yielding a group of plasmids that 46

47 direct constitutive production of FtsB or FtsL variants (Table 4). Strain TB28 [wt] carrying the ftsb or ftsl plasmids, respectively, survived deletion of ftsb or ftsl on the chromosome in each case, showing that all identified FtsB and FtsL mutant proteins were functional in supporting cell division in the presence of WT FtsN. The resulting BL155 [ΔftsB] and BL156 [ΔftsL] strains producing plasmid encoded FtsB or FtsL mutant variants were transformed with either an empty vector (pmlb1113δh3), or with plasmid pmg13 [P lac ::gfp-ftsn 1-81 ] encoding an N FtsN + E FtsN - fusion, and then tested for their ability to survive introduction of an ΔftsN<>cat allele by transduction. All four FtsL variants (FtsL E88V, FtsL E88K, FtsL N89S, FtsL H94Y ) and two FtsB ones (FtsB A55T and FtsB D59V ) performed similarly to the two previously characterized mutants, FtsL D93G and FtsB D59H. Thus, they supported cell division in the absence of E FtsN but all required the presence of N FtsN to do so (Table 4). Interestingly, however, the four FtsB variants with substitutions in residue E56 (FtsB E56V, FtsB E56A, FtsB E56K, FtsB E56G ) were all able to compensate for a complete absence of ftsn (Table 4). To verify this result, we replaced the chromosomal ftsb gene in wildtype strain TB28 with one of three ftsb E56 alleles, ftsb E56A, ftsb E56K, or ftsb E56V, using gene replacement (120) or Gene Doctoring methods (121). The resulting strains, BL167 [ftsb E56A ], BL172 [ftsb E56K ] and BL171 [ftsb E56V ], readily survived introduction of the chromosomal ΔftsN<>aph allele, confirming that a chromosomal copy of these ftsb super alleles was sufficient to rescue cell division in the complete absence of ftsn (Table 3). In LB medium, none of the ΔftsN strains, BL173 [ΔftsN ftsb E56A ], BL175 [ΔftsN ftsb E56K ] 47

48 and BL174 [ΔftsN ftsb E56V ], divided like wildtype cells, but all three propagated without gross defects in cell viability (FIG. 13C and 16A). The ftsb E56A mutation promotes septal murein synthesis. The E FtsN*-suppressing mutations allow cell division in the absence of E FtsN, but they can still work with wildtype E FtsN and restore nearly wildtype division phenotypes in ftsn + cells (FIG. 14A). In fact, ftsn + cells carrying an E FtsN*- suppressing mutation were shorter than wildtype cells. We measured around 350 cells from each strain carrying either one or a combination of two suppressing mutations on the chromosome, and found that mutant cells were on average at least 10% smaller than wildtype cells when grown in LB medium at 30 C (Table 5). The ftsa I143L mutation appeared to have the most pronounced effects on cell size. On average, ftsa I143L cells were only two thirds the size of wildtype cells. One of the super ftsb allele, ftsb E56A, also reduced cell size by nearly 30%. Combining ftsa I143L with either ftsb D59H or ftsl D93G did not further reduce the average cell size, suggesting that the suppressing mutations did not affect cell size in a cumulative fashion. In fact, cells carrying both ftsb D59H and ftsl D93G were somewhat larger than cells carrying only ftsb D59H or ftsl D93G, though still smaller than wildtype cells. How do these E FtsN*-suppressing mutations reduce cell size? The most straightforward scenario is that the mutations reduce the growth rate of the mutants. We measured the growth rates of all the strains listed in Table 5 in LB medium at 30 C. All strains showed nearly identical growth curves and the 48

49 mutants had similar doubling times as wildtype cells during exponential growth. Thus, the E FtsN*-suppressing mutations do not affect cell size by simply reducing the growth rates of the mutants. It is more likely, therefore, that the mutations accelerate the cell division process. As a multistage process, cell division could be affected by the suppressing mutations at various time points. In order to investigate which step(s) of the cell division process is affected by the suppressing mutations, we monitored and compared localization of two different markers in addition to cell constriction in wildtype and ftsb E56A cells. To monitor Z-ring assembly, we used a cytoplasmic GFP-ZapA. To monitor septal murein synthesis/processing, we utilized a RFP fusion to the SPOR domain of FtsN ( SS DsbA-RFP- S FtsN) that is directed to the periplasm by an N-terminal Sec-dependent signal sequence from DsbA (122, 123). As the SPOR domain is likely to recognize a processed form of newly synthesized septal murein (Chapter 1), accumulation of periplamic RFP- S FtsN at the division site is expected to follow the initiation of septal murein synthesis. As summarized in Table 6, 20% more ftsb E56A cells were scored with an RFP- S FtsN ring than wildtype cells (84% vs 64%). The increase was mostly due to the appearance of RFP- S FtsN rings in cells that did not yet show any visible sign of constriction. More than 30% of RFP- S FtsN rings appeared in ftsb E56A cells without visible cell constriction, which was more than double of that in wildtype cells (FIG. 14B). We calculated the average age for wildtype or ftsb E56A cells to start showing an RFP- S FtsN ring according to the formulas developed by den Blaauwen and colleagues (38). As is shown in FIG. 14C, RFP- S FtsN rings 49

50 appeared 5 minutes earlier in ftsb E56A cells than in wildtype cells with a similar doubling time of 43 minutes. ftsb E56A also seemed to slightly accelerate Z-ring assembly, but its primary effect in accelerating cell division is to promote premature septal murein synthesis. This finding is compatible with the previous results above that FtsB and FtsL mutant variants promote septal murein synthesis and cell constriction independently of E FtsN. These results indicate that the FtsBLQ protein subcomplex does not merely serve as a scaffold for SR assembly, but actively participates in the regulation of cell constriction initiation. Super ftsb alleles caused morphological changes and cell lysis in ftsn + cells at 42 C. One of the suppressing mutations identified here (ftsl E88V, Table 4) was previously identified as a ts mutation that caused cell lysis at 42 C in LB medium containing no salt (124, 125). We tested whether this ts property also applied to other E FtsN*-suppressing mutations that we identified in this study. We first attempted to reproduce the cell lysis phenotype of ftsl E88V mutants using strain BL156 [ΔftsL] strains harboring either pjh2 (P syn135 ::ftsl) or pbl333 (P syn135 ::ftsl E88V ). For the ts test, we utilized a special medium (LB ΔNaCl) that contains half the contents of regular LB medium and no salt (0.5% Tryptone, 0.25% Yeast extract). In regular LB with 0.5% NaCl at 30 C, cells producing FtsL E88V grew like wildtype cells except that they were slightly shorter than cells producing wildtype FtsL (FIG. 15A). When both strains were grown in LB ΔNaCl at 42 C, cells producing wildtype FtsL appeared normal, but ftsl E88V cells lost the 50

51 rod shape and became shorter and roundish. A few dead cells were found among ftsl E88V cells, but no massive cell lysis was observed. It is possible that the multicopy plasmid produces more FtsL E88V protein than a chromosomal ftsl E88V allele in the previous study, and affected the ts phenotype of the mutants. Thus, we decided to test the mutant strains carrying suppressing mutations on the chromosome. As is shown in FIG. 16A, neither ftsb D59H nor ftsl D93G alone affected cell viability on LB ΔNaCl plate at 42 C as assessed by spot titer assays. But similar to cells overproducing FtsL E88V, cell carrying either mutation became roundish and displayed a mild cell lysis phenotype in LB ΔNaCl medium at 42 C (FIG. 15 B and data not shown). Combination of ftsb D59H with ftsl D93G, on the other hand, reduced cell viability by nearly 10 5 fold on an LB ΔNaCl plate at 42 C. In liquid LB ΔNaCl medium at 42 C, most of ftsb D59H ftsl D93G cells became nearly spherical and the culture was filled with dead cells and cell debris that was generated from lysed cells (FIG. 15C). The three strains carrying one of the super ftsb alleles, BL167 [ftsb E56A ], BL172 [ftsb E56K ] and BL171 [ftsb E56V ], also displayed severe ts phenotypes. All three ftsb alleles reduced cell viability by at least 10 5 fold and they induced cell morphological conversion and cell lysis in LB ΔNaCl at 42 C (FIG. 15D and 16A). The two suppressing mutations in ftsa, ftsa I143L and ftsa E124A, were also tested. But unlike suppressing mutations in ftsb and ftsl, neither mutation in ftsa caused a severe change in cell shape or cell lysis at 42 C (FIG. 15E and data not shown). Thus, the ts phenotype is a unique feature of the suppressing mutations in ftsb and ftsl. Also, the severity of the ts 51

52 phenotype correlates with the ability of the suppressing mutation to rescue cell division in cells lacking E FtsN. Mutations that depend on N FtsN to rescue cell division in the absence of E FtsN caused mild morphologic changes and cell lysis, without affecting cell viability. Mutations that can compensate for complete absence of ftsn, including the ftsb super alleles and combination of ftsb D59H with ftsl D93G, caused the most severe cell shape changes and cell lysis in LB ΔNaCl at 42 C (FIG. 15 and 16). Why did the suppressor mutations cause morphological changes and cell lysis at 42 C? The fact that the super alleles induced more severe ts phenotypes raised the possibility that the ts phenotypes might be a result from overstimulating septal murein synthesis. In favor of this hypothesis, simply deleting ftsn in cells carrying the super alleles could restore normal cell viability and rod shape (FIG. 15 and 16). Spot titer analyses in FIG. 16B confirmed that defects in cell viability resulted from having both the essential activity of E FtsN and a super ftsb allele. In LB ΔNaCl medium at 42 C, overproducing E FtsN + FtsN fusion proteins in wildtype cells caused similar morphological changes as ftsb D59H or ftsl D93G cells (FIG. 15F). Thus, with regards to the ts phenotypes, harboring an E FtsN*-suppressing mutation in ftsb or ftsl is equivalent to having more E FtsN activity in the periplasm. Assuming the suppressing mutations can stimulate septal murein synthesis independent of E FtsN, the ts phenotypes might result from overstimulating septal murein synthesis in the periplasm. But why cells only display these phenotypes at elevated temperature in LB ΔNaCl medium is still unclear. Further studies answering this question can provide more 52

53 information about the regulation of septal murein synthesis and the function of FtsN and the FtsBLQ complex. DISCUSSION In this study, we identified suppressing mutations in three essential cell division genes, ftsa, ftsb, and ftsl, which can direct cell constriction in the absence of E FtsN. Our results suggest that the mutant alleles promote premature spg synthesis, and that FtsA and the FtsBLQ subcomplex play important roles in the regulation of spg synthesis. The results also indicate that the cytoplasmic domain of FtsN, in addition to E FtsN, is involved in controlling cell constriction, probably by communicating with the cytoplasmic Z-ring through the IC domain of FtsA. The essential domain of FtsN, on the other hand, regulates spg synthesis and cell constriction through direct or indirect interactions with the FtsBLQ subcomplex in the periplasm. Our results provide the first evidence that the FtsBLQ subcomplex plays important roles in regulating spg synthesis and cell constriction. As essential components of the SR, FtsB, FtsL, and FtsQ, form a relatively conserved subcomplex as part of the SR, which serves as a scaffold for recruiting downstream SR components during SR assembly (63-70). There are also sudies suggesting that the FtsBLQ subcomplex is involved in other aspects of cell division, like PG synthesis, but without any direct evidence ( ). The mutant alleles in ftsb and ftsl that we identified can restore spg synthesis in the absence of the normal activation signal, E FtsN, although incompletely. Plus, in 53

54 cells with normal E FtsN activity, having an additional E FtsN*-suppressing mutation in ftsb or ftsl induced early spg synthesis and caused cell morphological changes or even cell lysis at elevated temperature in a special medium containing no salt. All these results suggest that, the FtsBLQ subcomplex, at least FtsB and FtsL, are actively involved in regulating spg synthesis. The properties of the suppressing mutations in ftsb and ftsl provide valuable information about how the FtsBLQ subcomplex functions in regulating spg synthesis. Clustering of these mutations is notable, and suggests that the affected residues comprise small periplasmic subdomains in FtsB and FtsL that are important in determining the essentiality of E FtsN (FIG. 13A). In FtsB, the affected residues reside in the fourth (A55 and E56) or fifth (D59) complete heptad repeat of the periplasmic coiled-coil domain of the protein for which a partial crystal structure is available (130). In FtsL, the affected residues reside just C-terminal to the third complete heptad repeat of the predicted periplasmic coiled-coil domain of the protein. Whether this region forms a fourth heptad or a turn in the protein structure is presently not clear. But it is worth emphasizing that the two small periplasmic subdomains are positioned at almost similar distances away from the inner membrane, right at the border of the regions in each protein involved in FtsB-FtsL interaction (63, 64). Interestingly, E FtsN is placed about the same distance away from the outer leaflet of the inner membrane. Reducing the distance between E FtsN and the inner membrane compromises the activity of E FtsN (FIG. 2). These findings are compatible with a hypothesis that E FtsN 54

55 interacts with the FtsBLQ subcomplex, perhaps with the two periplasmic subdomains of FtsB and FtsL. The mutations in FtsB and FtsL might mimic the conformation of the FtsBLQ subcomplex as if E FtsN is engaged. The biochemical properties of the substitutions in FtsB and FtsL suggest that the FtsBLQ subcomplex functions like a switch, which controls the activity of the murein synthases until E FtsN switches it. 9 out of the 12 mutations were substitutions of Aspartic acid or Glutamic acid residues, which converted the negatively charged side chains to positively charged, hydrophobic, or uncharged side chains. In the four ftsb super alleles, for instance, the E56 residue of FtsB was either converted to a positively charged Lysine residue, an hydrophobic Alanine or Valine residue, or an uncharged glycine residue. Also, residue E56 resides at the g position in the heptad repeats (FIG. 13A) that usually plays important roles in determining homo- or hetero-dimerization specificity of coiled coils ( ). The rest of the affected residues were mostly at the c or f positions on the hydrophilic surface of coiled-coil structures that are important for both the stability of the coiled coils and interactions with other proteins (135, 136). Substitutions to these residues can affect the specificity and stability of the coiled coil structures formed by FtsB and FtsL and, possibly the interactions with their target. Given the fact that the mutations in ftsb or ftsl promote spg synthesis, we thus propose that the FtsBLQ subcomplex regulates cell constriction by enforcing a checkpoint in the cell division process that prevents spg synthesis before the arrival of the activation signal, E FtsN (FIG. 17A). 55

56 Our results also provide valuable information about the function of FtsA and the normally nonessential cytoplasmic domain of FtsN ( N FtsN). Our results and recent studies demonstrated that N FtsN helps accumulate some of the FtsN molecules to the division site before S FtsN-dependent septal localization of FtsN, suggesting a role of N FtsN for concentrating E FtsN activities at the SR for initiating spg synthesis before visible constriction (28, 55). This role of N FtsN fits well with our model for self-enhanced FtsN action in the initiation of cell constriction (Chapter 1). But we also found that some ftsb or ftsl mutant alleles rely on N FtsN to allow cell constriction in the absence of E FtsN, suggesting that N FtsN also plays important roles in initiating cell constriction besides septal localization. N FtsN interacts directly with the IC domain of FtsA (54, 95), and we proved that the two ftsa mutant alleles with substitutions in the IC domain could bypass the requirement for the N FtsN-FtsA interaction for FtsB D59H or FtsL D93G to restore cell division in the absence of E FtsN. It raised the possibility that the cytoplasmic Z-ring also needs to be activated by FtsN for initiating cell constriction (FIG. 17A). With its cytoplasmic N FtsN interacting with the Z-ring and its SPOR domain binding the peptitoglycan layer, FtsN is an ideal candidate for coordinating inner membrane invagination with spg synthesis in the periplasm. Of course, we cannot rule out the possibility that other SR components, like the FtsBLQ subcomplex or FtsI (PBP3), actually mediate communication between the cytoplasmic and periplasmic portions of the SR. Indeed, the suppressing mutation in ftsa, ftsa I143L, was previously identified as a suppressor for a localization-deficient FtsQ variant (119), suggesting a role of FtsA outside the Z- 56

57 ring and communications between FtsA and the FtsBLQ subcomplex. The identification of two ftsa alleles that can restore cell division in the absence of ftsn (Ref. 57 and above) also suggests that the activation signal for spg synthesis could originate from the cytoplasmic Z-ring. There is a possibility that the activation signal is transferred from the Z-ring to FtsN, the FtsBLQ subcomplex, or directly to PBP3, to activate spg synthesis in the periplasm (FIG. 17D). Given the present results, we propose a model for the SR to trigger spg synthesis and cell constriction in E. coli. As illustrated in FIG. 17, the ultimate target for the SR to activate upon initiation of cell constriction is the multiprotein machinery for synthesizing septal murein, which contains PBP3(FtsI) and other murein synthases and hydrolases. PBP3 could be activated in the periplasm (FIG. 17B). The FtsBLQ subcomplex functions as a switch for PBP3 activity and suppresses spg synthesis when FtsN has not yet accumulated at the SR (FIG. 17A). The primary activation signal, E FtsN, induces conformational changes in the FtsBLQ subcomplex upon its arrival at the SR and converts the FtsBLQ subcomplex to its on state that can now stimulate spg synthesis (FIG. 17, green arrows). Of course, we cannot rule out the possibility that the FtsBLQ subcomplex functions as a co-activator that forms a transient protein complex with E FtsN to activate PBP3. In that case, E FtsN might also directly interact with PBP3 or the target for initiating spg synthesis (FIG. 17B, cyan arrow). In either case, the mutations in ftsb or ftsl promote the on state of FtsBLQ as if E FtsN is 57

58 engaged and induce septal murein synthesis even when E FtsN is absent or nonfunctional. The Z-ring also needs to be activated for the initiation of cell constriction. Activation signal from E FtsN is transmitted to the Z-ring through the N FtsN-FtsA interaction and/or maybe commonications between the FtsBLQ subcomplex and FtsA (FIG. 17C, blue arrows), converting FtsA to its on state that stimulates Z- ring contraction coordinated with spg synthesis. Alternatively, FtsA activated by N FtsN might stimulate the on state of the FtsBLQ subcomplex or PBP3 activity can be stimulated by the contraction of the Z-ring via another route (FIG. 17D, orange arrows). The mutations in the IC domain of FtsA mimic the on state as if FtsA is activated by N FtsN and induce cell division in the absence of both E FtsN and N FtsN. Of course we cannot rule out the possibility that PBP3 can be activated in either the periplasm or the cytoplasm, or it needs to be activated in both. Either FtsN or the FtsBLQ subcomplex has the potential to mediate the communications between the periplasmic and cytoplasmic parts of the SR and coordinate spg synthesis with Z-ring contraction. Future studies, especially biochemical evidences that help clarify the interactions among FtsN, FtsBLQ, FtsI and maybe other SR components will help us refine the current model. 58

59 Chapter 3. DedD assists in initiating cell constriction through an FtsN-independent pathway. INTRODUCTION Proteins containing a SPOR domain are widespread among bacteria. To date, more than 7,500 proteins from over 2,000 species are predicted to carry a SPOR domain (PF05036, pfam), including four proteins in E. coli, FtsN, DedD, DamX, and RlpA (137). SPOR domain proteins from a wide range of bacterial species were found to specifically accumulate at the division site during cell constriction, due to a common feature of the SPOR domains to recognize the constriction site (28, 29, 138). The four SPOR domain proteins in E. coli are also targeted to the constriction site, raising the question of whether they participate in the cell division process (28, 29, 107). In fact, FtsN is an essential SR protein involving in septal murein synthesis and initiating cell constriction (3, 28, 72, 73, 96). DedD and DamX, on the other hand, belong to the nonessential components of the SR that contribute to the efficiency of cell constriction (28, 29). Similar to most SR proteins, how these SPOR domain proteins function in cell division remains unclear. In this study, we investigate the function of DedD in E. coli cell constriction. As a nonessential component of the SR, DedD was shown to participate in the cell constriction process (28). E. coli cells lacking DedD display a cell chaining phenotype that resembles the phenotype of ftsn slm117 cells. The division defects of ftsn slm117 cells are caused by a transposon insertion in the ftsn gene, which lead to a reduction of the essential activity of FtsN at the SR due to C- 59

60 terminal truncation of FtsN, including S FtsN (28). The similar phenotype of ΔdedD cells suggested that DedD contributes to the efficiency of cell constriction. More interestingly, DedD is essential for cell constriction in ftsn slm117 cells. ftsn slm117 cells depleted of DedD form nonseptate filaments, suggesting that DedD has an important role in cell constriction, which becomes essential when the essential activity of FtsN is limited. DedD is a typeii inner-membrane protein with a similar topology to FtsN (FIG. 18A). DedD has a very small cytoplasmic domain ( Cyto DedD, residues 1 to 8), a transmembrane domain ( TM DedD, residues 9 to 26), and a large periplasmic part (residues 27 to 220) containing a C-terminal SPOR domain ( S DedD, residues 140 to 216). Similar to S FtsN, S DedD accumulates sharply to the constriction site and helps targeting DedD to the SR during cell constriction (28, 29). In this study, we identified the critical functional domain of DedD ( N DedD). N DedD corresponds to the N-terminal conserved region of DedD, and includes TM DedD. N DedD s function in cell constriction is partially redundant to that of E FtsN. Sufficient E FtsN and N DedD activities are required for maintaining the integrity of the peptidoglycan layer during cell constriction when septal murein synthesis is compromised. Massive cell lysis occurred primarily at the constriction site when the activity of E FtsN or N DedD was diminished in cells lacking PBP1B, suggesting that both E FtsN and N DedD are ultimately required for stimulating septal murein synthesis, presumably through modulating the activity of PBP3. N DedD also includes a septal localization determinant and the 60

61 interaction between N DedD and the FtsBLQ subcomplex is important for both the function and septal localization of N DedD. Other than their functions in cell constriction, both DedD and FtsN are important for the stability of the SR when the numbers of certain SR components are out of balance. RESULTS Septal localization of S DedD depends on E FtsN and PBP3. In a previous study, we showed that septal localization of S DedD depends on the activities of the three murein amidases, AmiA, AmiB, and AmiC, but it appeared to be less sensitive to depletion of E FtsN or inhibition of PBP3 (FtsI) activity, compared to S FtsN (28). Even though there is a possibility that S DedD might recognize a different target at the septum, a more reasonable interpretation is that S DedD has higher affinity for septal murein than S FtsN and the remaining activities of E FtsN or PBP3 after treatment were able to produce enough targets for S DedD (28). We used more stringent depletion conditions and showed that septal localization of GFP-DedD was indeed dependent on E FtsN and PBP3 (FIG. 19). The results suggest that S DedD recognizes certain form of septal murein and helps targeting DedD to the constriction site. The critical domain for DedD s function in cell division corresponds to in its N-terminal conserved region. A large portion of DedD is evolutionary not well conserved, except for the C-terminal SPOR domain and an N-terminal region of about 50 residues, 61

62 including Cyto DedD, TM DedD, and a small juxtamembrane region in the periplasm (FIG. 20A). Previous studies showed that most of the FtsN protein was dispensable for its essential function, including the SPOR domain (28, 90, 92). We speculated that the critical functional domain(s) of DedD required for its function in cell constriction might reside in its N-terminal conserved region. We investigated which part of DedD was required for its function in cell constriction by complementation analyses using plasmids expressing GFP fusions to various portions of DedD. The fusion proteins were expressed upon induction with IPTG and tested for their abilities to correct the division defects of BL40 (ΔdedD ΔdamX) cells. We used strain BL40 instead of a ΔdedD strain because ΔdedD ΔdamX cells exhibited more pronounced division defects than ΔdedD cells (28). Also, ectopically expressed functional DedD fusions should restore a wild-type division phenotype in BL40 cells, as cells lacking damx alone have no division-related phenotype (28, 29). As summarized in FIG. 18A, GFP fusions to full length DedD expressed from plasmid pfb236 (P lac ::gfp-dedd) (FIG. 18C) or to SPOR-less DedD expressed from ppc1 (P lac ::gfp-dedd ) (FIG. 18F) were able to correct the division defects of BL40 cells, although BL40 cells harboring ppc1 needed higher concentrations of IPTG (50 µm) to resemble wild-type cells than the ones with pfb236 (<5 µm) (data not shown). In addition to the SPOR domain, a large portion of the periplasmic part of DedD was dispensable for its function, as we could truncate DedD from the C-terminus to residue 54 without abolishing its 62

63 function. Production of GFP-DedD 1-54 could restore BL40/pBL95 (P lac ::gfp-dedd 1-54 ) cells to a nearly wild-type division phenotype as illustrated in FIG. 18D. GFP-DedD 1-35 expressed from plasmid pbl37 (P lac ::gfp-dedd 1-35 ) failed to compensate for the absence of DedD in BL40 cells with any concentration of IPTG (FIG. 18A and G). Western analyses indicated that GFP-DedD 1-35 was produced at a slightly lower level than other fusions in the presence of 50 µm IPTG (FIG. 21). But this was probably not the reason for its non-functionality as GFP-DedD 1-35 also failed to have an effect on the division phenotype, even at a much higher inducer level (250 µm IPTG, data not shown). Another possibility was that GFP-DedD 1-35 was defective in membrane insertion with only a few residues on the periplasmic side of the membrane. It was supported by the observation that fusion GFP-DedD 1-35 was found dispersed evenly in the cytoplasm rather than accumulating along the cell membrane (FIG. 18G ). We improved the stability of GFP-DedD 1-35 in the membrane by attaching an RFP molecule to its C-terminus. Production of GFP-DedD RFP from plasmid pbl101 (P lac ::gfp-dedd ecrfp) could partially suppress the cell chaining phenotype of BL40 cells (FIG. 18H). BL40/pBL101 cells were shorter than BL40/pMLB1113ΔH3 (FIG. 18B) cells with 50 µm IPTG, but they never resembled wild-type cells at any inducer level. We concluded that the N-terminal conserved region of DedD, with at most the first 54 residues, was sufficient for performing its function in cell constriction. The importance of the N-terminal region of DedD was confirmed by complementation analyses with fusion GFP-MalF DedD expressed from 63

64 plasmid pbl33 (P lac ::gfp-malf dedd ). In this construct, the cytoplasmic and transmembrane domains of DedD are replaced by the corresponding domains of MalF (TM1) (139). Production of GFP-MalF DedD was unable to correct the division defects in BL40 cells, although the fusion localized sharply to the constriction sites (FIG. 18D&D ). The results above demonstrated that the critical functional domain of DedD ( N DedD) required for its function in cell constriction resides in its N-terminal conserved region. N DedD consists of the first 54 residues at most, including the cytoplasmic domain, the transmembrane domain, and some residues in the juxta-membrane region in the periplasm. The rest of the periplasmic part of DedD, including the SPOR domain, is dispensable for its function in cell constriction. N DedD is a septal targeting determinant. It is well accepted that SPOR domains are efficient septal targeting determinants in E. coli cells, which can concentrate SPOR-domain containing proteins at sites of cell constriction (28, 29). Previous studies also showed that the N-terminal part of FtsN contained a weak septal localization determinant that can drive a small portion of FtsN molecules to the division sites (ref. 28 and Chapter 2). We investigated whether N DedD also possessed affinity for the SR using GFP fusions to SPOR-less DedD parts. Functional DedD fusions without S DedD, such as GFP-DedD and GFP-DedD 1-54, did not seem to localize well in otherwise wild-type cells (data not 64

65 shown). However, ring-like accumulation of the fusion proteins at the constriction site was witnessed in cells lacking native DedD, such as MG14 (ΔdedD) or BL40(ΔdedD ΔdamX) cells. The rings formed by GFP-DedD or GFP-DedD 1-54 did not appear to be as sharp as the ones formed by GFP-DedD, but they were easily distinguishable from the peripherally distributed fluorescent signal along the cell membrane (FIG. 18). As summarized in Table 7, a GFP-DedD ring was observed in nearly half (48%) the BL40/pPC1 (ΔdedD ΔdamX/P lac ::gfpdedd ) cells in an exponentially growing culture. Without the SPOR domain, GFP-DedD showed defects in septal localization compared to GFP-DedD, which formed sharp rings in 62% of the BL40/pFB236 (ΔdedD ΔdamX/P lac ::gfpdedd) cells. The differences between the two fusions in septal localization were particularly obvious in deeply constricted cells. A GFP-DedD ring was detected in most of (96%) the deeply constricted BL40/pFB236 cells, while only 36% of deeply constricted BL40/pPC1 cells were scored with a GFP-DedD ring. But in cells at early stages of constriction, GFP-DedD localized to the SR almost as efficiently as GFP-DedD. Nearly all the BL40/pPC1 cells (96%) with a shallow constriction site were observed with a GFP-DedD ring. Also, a fraction (24%) of GFP-DedD rings were detected in cells that did not yet show a visible constriction site under DIC. These results suggest that the N-terminal part of DedD contains a weak localization determinant that can help DedD join the SR early in the division process. We then tested if the minimal critical functional domain ( N DedD) that we identified possessed affinity for the SR. GFP-DedD 1-54 showed a similar 65

66 localization pattern as GFP-DedD (Table 7 and FIG. 18). A GFP-DedD 1-54 ring appeared in most (94%) of ΔdedD ΔdamX cells with shallow constrictions, but the ring was absent in majority of the deeply constricted cells. We also noticed that BL40/pBL95 cells producing GFP-DedD 1-54 displayed a relatively high fluorescent background in the cytoplasm. Western analyses indicated that GFP-DedD 1-54 was not completely stable (FIG. 21), suggesting that the elevated fluorescent background might be due to the release of intact GFP moieties from the fusion. However, we could not rule out the possibility that the fusion GFP- DedD 1-54 was deficient in membrane insertion. Nevertheless, the results proved that the first 54 residues of DedD are sufficient for determining septal localization of N DedD. From the results above, we conclude that N DedD possesses affinity for the SR. Septal localization of N DedD fusions only became clearly visible when the fusions were the only source of DedD in the cell, suggesting N DedD only has a relatively weak affinity for the SR. Ring-like accumulation of N DedD mostly coincided with shallow constriction sites, but the rings were not readily visible in the majority of cells entering late stages of constriction. Accumulation of N DedD also occurred in some cells that had not yet show any visible constriction, suggesting that N DedD might join the SR relatively early in the cell division process. Several conserved residues in TM DedD are important for N DedD s function in cell constriction. 66

67 Sequence alignment indicated that N DedD is well conserved among Gammaproteobacteria (FIG. 20A). To better define the functional domain of DedD, we mutated several conserved residues in N DedD and tested whether the mutations affect DedD s function. For this study, GFP fusions to either wild-type or mutant DedD were expressed from single-copy CRIM plasmids (140) integrated in the chromosome of BL40 cells. We used GFP fusions to DedD instead of DedD 1-54 to reduce the fluorescent background in the cytoplasm when studying in vivo localization of the fusion proteins. As is shown in FIG. 20B, BL40(iBL345) [ΔdedD ΔdamX (P lac ::gfp-dedd )] cells resembled wild-type cells when they were grown in M9- maltose medium containing 250 µm IPTG. Ring-like accumulation of GFP-DedD at midcell was observed in a proportion of the BL40(iBL345) cells (25%) (FIG. 20B and Table 8). Fewer cells were scored with a GFP-DedD ring here than previous observed in BL40 cells carrying multicopy plasmids in LB medium. That was expected because fewer cells undergo active cell constriction in M9-maltose medium as also indicated by the observation that only 33% of BL40(iBL345) cells showed a visible constriction site. When a highly conserved residue, Glycine 11 in DedD TM, was mutated to Alanine or Cysteine, fusions GFP-DedD 1-118, G11A (FIG. 20C) or GFP-DedD 1-118, G11C (data not shown) failed to compensate for the absence of DedD in ΔdedD ΔdamX cells. Interestingly, the substitutions also affected septal localization of the mutant proteins. Accumulation of GFP-DedD 1-118, G11A at the constriction site was only scored in 9% of the BL40(iBL345)(G11A) [ΔdedD ΔdamX (P lac ::gfp- 67

68 dedd 1-118, G11A )] cells. In rest of the cells, the fusion protein was distributed homogenously along the cell membrane, although 80% of the cells/chains had at least one constriction site. Only 6% of the constriction sites in BL40(iBL345)(G11A) cells were scored with a GFP-DedD 1-118, G11A ring (Table 8), which is comparable to membrane proteins with no localization specificity, such as ZipA GFP (28). Western analyses indicated that GFP-DedD 1-118, G11A was as stable as GFP-DedD (FIG. 22). We thus conclude that point mutations in residue G11 affect both the function and septal localization of N DedD. We also mutated another conserved residue in DedD TM, Proline 24, to Alanine. The mutant protein GFP-DedD 1-118, P24A also failed to correct the division defects in BL40(iBL345)(P24A) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, P24A )] cells (FIG. 20D). But different from the mutations in residue G11, mutation P24A did not completely eliminate the mutant protein s affinity for the division site. In BL40(iBL345)(P24A) cells, 28% of the visible constriction sites were associated with a GFP-DedD 1-118, P24A ring, suggesting that P24A reduced the mutant s affinity for the septal ring. So residue P24 in DedD TM is also important for the function and septal localization of N DedD. A conserved residue in the periplasmic part of N DedD, Proline 42, was also substituted. Production of the mutant protein GFP-DedD 1-118, P42G restored a wild-type division phenotype in BL40(iBL345)(P42G) [ΔdedD ΔdamX (P lac ::gfpdedd 1-118, P42G )] cells (FIG. 20E), suggesting that the P42G substitution did not affect N DedD s function in cell constriction. Septal localization of N DedD was also not affected by the mutation. Ring-like accumulation of GFP-DedD 1-118, P42G was 68

69 observed in 26% of BL40(iBL345)(P42G) cells, similar to WT GFP-DedD in BL40(iBL345) cells (Table 8). These results confirm that the transmembrane domain of DedD ( TM DedD) is important for DedD s function in cell constriction, beyond its role as a membrane anchor. N DedD fusions with substitutions in one of two conserved residues in TM DedD were unable to compensate for the absence of DedD. TM DedD is also important for septal localization of N DedD. The point mutations in TM DedD could either eliminate or reduce N DedD s affinity for the SR. N DedD is required for cell division in ftsn slm117 cells. Previous results demonstrated that DedD became essential for cell constriction in ftsn slm117 cells, in which the activity of E FtsN at the SR is reduced due to a transposon insertion in ftsn (28). We investigated which domain(s) of DedD was required for cell constriction in ftsn slm117 cells via spot titer analyses using depletion strains derived from MG19 (ΔdedD ftsn slm117 ) carrying two different plasmids. One plasmid is pmg39 (P BAD ::dedd) that produces native DedD upon induction by arabinose. The other plasmid is one of the pmlb1113 derivatives that encode GFP fusions to various DedD portions under the control of the lac regulatory region. FIG. 23A indicated that all the strains grew equally well on LB agar containing 0.5% arabinose due to expression of DedD from plasmid pmg39. But on LB agar containing 0.2% glucose, none of the strains grew except for MG19/pMG39/pFB236 (ΔdedD ftsn slm117 /P BAD ::dedd/p lac ::gfp-dedd) (lane1). 69

70 Residual expression of GFP-DedD from plasmid pfb236 in the presence of 0.2% glucose appeared to be sufficient to support growth of ΔdedD ftsn slm117 cells. In contrast, the fusion GFP-MalF DedD , which lacks an intact N DedD domain failed to rescue strain MG19/pMG39/pBL33 (ΔdedD ftsn slm117 /P BAD ::dedd/p lac ::gfp-malf dedd ) even in the presence of IPTG (lane 2). It suggested that ftsn slm117 cells required the activity of N DedD for cell constriction. We then assessed DedD constructs with truncations from the C-terminus. On LB agar containing 50 µm IPTG, strain MG19/pMG39/pBL95 (ΔdedD ftsn slm117 /P BAD ::dedd/p lac ::gfp-dedd 1-54 ) managed to grow at places spotted with cultures diluted to OD 600 of , suggesting that N DedD was able to support cell division in ΔdedD ftsn slm117 cells (lane 4). Surprisingly, a longer DedD fusion with C-terminal truncation, GFP-DedD 1-118, failed to rescue ΔdedD ftsn slm117 cells at any IPTG concentrations (lane 3 and data not shown). The result was unexpected as the fusion GFP-DedD contains intact N DedD and is fully capable of compensating for the absence of DedD in BL40 (ΔdedD ΔdamX) cells (FIG. 18F). Western analyses indicated that fusion GFP-DedD was not any less stable than GFP-DedD 1-54 in ΔdedD ftsn slm117 cells (FIG. 24). One possible explanation would be that residues 55 to 118 can inhibit the activity of N DedD before cell constriction. When DedD joins the SR during constriction, the residues then disengage from N DedD as S DedD binds septal murein and stretches out the periplasmic part of DedD. If this were the case, the selfinhibitory activity also appears to depend on the condition of FtsN. It emerges 70

71 when the activity of E FtsN is limited as in ftsn slm117 cells. Nonetheless, the results above prove that N DedD is sufficient to support cell constriction in ΔdedD ftsn slm117 cells. DedD s function in cell constriction is independent of FtsN. Cells with full-length FtsN can divide without DedD. But when the essential activity of FtsN is reduced, like in ftsn slm117 cells, DedD is required for cell constriction (28). It appears that DedD can either partially substitute or enhance the activity of E FtsN. To assess DedD s effect on FtsN s function more directly, we compared the levels of ectopically-encoded FtsN required for viability of ΔftsN or ΔftsN ΔdedD cells using spot titer analyses. To better control the production levels of the fusion proteins, various FtsN fusions were expressed from singlecopy CRIM constructs incorporated in the chromosome. As is shown in FIG. 23B, residual production of GFP-FtsN supported growth of the ΔftsN strain CH34(iMG59) [ΔftsN(P lac ::gfp-ftsn)] on LB agar without any inducer (lane 1). In contrast, BL73(iMG59) [ΔftsN ΔdedD(P lac ::gfp-ftsn)] cells, which lack DedD, required minimally 500 µm IPTG to grow to a comparible extent (lane 4). The differences became even more promounced with FtsN fusions lacking S FtsN. For example, CH34(iMG62) [ΔftsN(P lac ::gfp-ftsn )] (lane 3) cells grew nearly as well as CH34(iMG59), but BL73(iMG62) [ΔftsN ΔdedD(P lac ::gfp-ftsn )] cells barely survived, even in the presence of 500 µm IPTG (lane 6). So, E. coli cells need more E FtsN molecules in the absence of DedD. 71

72 One possibility is that DedD helps stabilize the FtsN fusions. But western analyses indicated that the FtsN fusions were not any less stable in the absence of DedD (FIG. 24B). Another possibility is that DedD enhances the activity of E FtsN. If so, overexpression of DedD should be able to suppress the division defects in ftsn slm117 cells. However, as is shown in FIG. 23C, ftsn slm117 cells overexpressing GFP-DedD still showed the cell chaining phenotype. Conversely, overexpression of GFP-FtsN was similarly unable to suppress the division defects in ΔdedD ΔdamX cells (FIG. 23D). These results suggest that DedD does not function through simply enhancing the activity of FtsN. DedD s function in cell constriction is partially redundant with that of FtsN, but it works in a pathway that is independent of FtsN. DedD interacts with the FtsBLQ protein subcomplex. In order for DedD to localize to the SR and function in cell constriction, it is likely to interact with components of the SR and/or the peptidoglycan synthesis machinery. We utilized bacterial two-hybrid (BACTH) analyses to test for interactions between DedD and proteins involved in cell division and peptidoglycan synthesis in E. coli. In the analyses, we used a DedD fusion without the SPOR domain (T18-DedD ) to try to reduce the number of falsepositive results caused by accumulation of these fusion proteins at the septa. But the results obtained using DedD looked quite similar to the results obtained with full-length DedD, except for some minor differences in the intensity of several interactions (FIG. 25A and 26A). The results shown in FIG. 25A 72

73 suggested strong interactions between DedD and FtsA, ZipA, FtsK, FtsQ, FtsB, FtsL, FtsI, FtsN, DedD, PBP1A, PBP1B, and TolA. Interactions between DedD and FtsEX, FtsW, and PBP2 were also detected in the BACTH analyses. Most of the interactions detected here were also detected in the BACTH analyses using FtsN as the bait protein, except for the interactions with FtsL and FtsB. The BACTH analyses detected strong interactions between DedD and FtsL, while no interaction was detected between FtsN and FtsL (FIG. 26B). Interaction between DedD and FtsB also seemed to be stronger than the interaction involving FtsN and FtsB. Given the findings (FIG. 23D) that FtsN could not completely compensate for the loss of DedD function, the interactions between DedD and FtsL or FtsB might be important for DedD s function in cell division. FIG. 20 demonstrated that several point mutations in the transmembrane domain of DedD affected the function and septal localization of N DedD. We used BACTH assays to test if these mutations affected the interaction between DedD and other SR proteins. In FIG. 25B, T18-DedD fusions with mutations in N DedD were compared for interactions with FtsQ, FtsL, FtsB, FtsI, or FtsN. Interestingly, the two substitutions in residue G11 significantly reduced the interactions between N DedD and FtsL (FIG. 25B). Given the fact that the two mutations in G11 affected both the function and septal localization of N DedD, the results suggested that the interaction with FtsL might be important for N DedD s function in cell division. However, another point mutation in DedD TM, P24A, did not seem to affect the interaction between N DedD and FtsL, although GFP- 73

74 DedD 1-118, P24A lost the DedD function in cell division but could still localize to the septa. So the interactions with FtsL agreed well with the N DedD fusions ability to localize to the division site. But we still cannot rule out the idea that the interaction with FtsL is important for the function of N DedD, as it is possible that residue P24 does not contribute much to the binding energy with FtsL. Several mutations in N DedD also affected the interaction between N DedD and FtsB. Mutation P42G reduced the interaction between N DedD and FtsB in the BACTH assays, although P42G had no obvious influence on the function or septal localization of N DedD (FIG. 20). The two substitutions in residue G11 also slightly reduced the interaction between N DedD and FtsB, but mutation P24A did not seem to affect the interaction between them (FIG. 25B). As FtsB, FtsL and FtsQ form a protein subcomplex in the SR (69), the reductions detected between the two G11 mutants and FtsB might result from the mutations effect on the interaction with FtsL. This could also explain the reduction seen with the interaction between T18-DedD 1-118, P42G and FtsL. Mutation P42G affected the interaction between N DedD and FtsB and led to a slight reduction in its interaction with FtsL as well. Nonetheless, the BACTH analyses with the N DedD mutants together with the domain analyses and localization experiments (FIG. 18 and 20) indicate that DedD TM interacts with FtsL, and that this interaction is important for septal localization and maybe the function of N DedD. The juxtamembrane domain of N DedD in the periplasm may participate in an interaction with FtsB, but if this interaction is essential for its function in cell constriction is unclear. 74

75 DedD is required for septal murein synthesis in cells lacking PBP1Bs. The results above suggest that DedD s functions in cell constriction are partially redundant to those of FtsN. One of the functions proposed for FtsN, and probably its essential function in cell constriction, is to trigger septal murein synthesis through directly or indirectly modulating the activities of murein synthases (28, 96). It is possible that DedD also participates in regulating septal murein synthesis. This hypothesis was partially supported by bacterial two-hybrid analyses, which indicated interactions between DedD and several murein synthases in E. coli, including PBP1A, PBP1B and PBP3 (FIG. 25A). PBP1A and PBP1B are the two major bifunctional murein synthases in E. coli with both transglycosylase and transpeptidase activities; and PBP3 (FtsI) is a transpeptidase that is essential for cell division and septal murein synthesis (12, 141, 142). Abolishing the activities of different murein synthases by genetic modifications or β-lactam antibiotics treatment has various effects on E. coli cells. Cells might lose the rod shape, become filamentous, or undergo rapid cell lysis, depending on the specific murein synthases that were targeted (12, ). If DedD affects the function of certain murein synthases, blocking the activity of different murein synthases in cells lacking DedD might lead to different phenotypes, which can provide valuable information about the function of DedD. We first tested abolishing the activities of PBP1A or PBP1Bs in cells lacking DedD. Phage P1-mediated transduction of the pona (ΔponA<>kan) or ponb (ΔponB<>cat) null allele to strain MG14 (ΔdedD) was successful. But one 75

76 of the resulting strains, BL83 (ΔponB ΔdedD), was defective for growth, especially in liquid media. Overnight cultures of BL83 grown in LB medium typically had optical densities that were less than 30% of wild-type strains. Examination of the culture revealed that low cell density resulted from massive cell death among BL83 cells. Microscopy of overnight cultures revealed large numbers of dead cells and abundant cell debris in addition to remaining live cells with heterogeneous cell length (FIG. 27A). Some cells were captured in the process of cell lysis, with most of them prone to lyse at the division sites as indicated by spherical membrane bulges at midcell, suggesting that cell lysis in ΔponB ΔdedD cells is related to cell division. Cell lysis in ΔponB ΔdedD cells was also found to be growth phase dependent. After dilution of an overnight culture in fresh media, strain BL83 showed a similar growth curve to the wild-type strain (TB28) till late exponential phase, when the differences in cell density became obvious, coinciding with the onset of cell lysis in the BL83 culture (FIG. 27B and C). Production of PBP1B or DedD was able to prevent cell lysis in ΔponB ΔdedD cells (FIG. 29), suggesting that DedD becomes partially essential in cells lacking PBP1B, and is important for maintenance of cell wall integrity during cell division. Cells lacking PBP1Bs also required sufficient E FtsN activity. Phage P1- mediated transduction of the ponb (ΔponB<>cat) null allele to the ftsn slm117 strain TB77 was fruitless. We then combined the ponb null allele with ftsn slm117 using a CRIM plasmid to provide additional production of a GFP-FtsN fusion. The resulting strain BL84(iMG62) [ΔponB ftsn slm117 (P lac ::gfp-ftsn )] underwent rapid cell lysis at midcell when the extra FtsN fusion was depleted (FIG. 27D). 76

77 The cell lysis phenotypes above are reminiscent of that when PBP1B and PBP3 are blocked simultaneously, which also causes rapid cell lysis at division sites (12, ). We blocked the activity of PBP3 in ΔponB cells using the normally non-lytic β-lactam cephalexin. Most of the ΔponB cells in an exponentially growing culture lysed within one hour of cephalexin treatment. And cell lysis occurred primarily at midcell, although cells with leakages along the cell envelope were also witnessed (FIG. 29C). Given the fact that cells lacking PBP1A have no division phenotype, it is unlikely that PBP1A is the primary target for DedD or FtsN. The finding that diminishing the activities of DedD or FtsN in cells lacking PBP1B has similar effects as blocking PBP3 in such cells is compatible with the proposal that both DedD and FtsN are required for full PBP3 activity. N DedD works in a separate pathway from E FtsN, but both of them contribute to stimulating septal murein synthesis (FIG. 28). Toxicity of excess SR proteins in cells lacking DedD. In Chapter 2, we present evidence that the target for E FtsN is probably the FtsBLQ subcomplex. We also present evidence that the interaction with the FtsBLQ subcomplex is important for the function of DedD (FIG. 25). If the FtsBLQ subcomplex is also the target for N DedD, FtsL or FtsB might be able to suppress the division defects in ΔdedD cells when they are overproduced. Contrary to our expectations, expressing GFP fusions to FtsL, FtsB, or FtsQ actually aggravated the division defects in ΔdedD cells. As is shown in FIG. 30A, expression of GFP-FtsL from a multicopy plasmid induced by 10 µm IPTG 77

78 rendered ΔdedD cells slightly longer than the ones growing in LB medium without any inducer. When GFP-FtsL was highly overproduced with 100 µm IPTG, ΔdedD cells became filamentous, indicating that overproduction of GFP-FtsL inhibited cell division in ΔdedD cells. This was also the case for GFP-FtsB and GFP-FtsQ (data not shown). In the spot titer analyses shown in FIG. 30B, overproduction of GFP-FtsQ or GFP-FtsL inhibited colony formation of ΔdedD cells by at least fold compared with ΔdedD cells carrying an empty vector on LB agar with 500 µm IPTG. GFP-FtsB showed a weaker inhibitory activity and MG14/pMLB1113ΔH3 (ΔdedD/vector) cells grew nearly fold better than MG14/pBL193 (ΔdedD/P lac ::gfp-ftsb) cells in the presence of 500 µm IPTG. The inhibitory activities of FtsQ, B, and L in MG14 (ΔdedD) cells were due to the absence of N DedD. In the spot titer assays, overproduction of these fusion proteins had no detectable effect on the growth of TB28 (wt). And their inhibitory activities in ΔdedD cells could be suppressed by GFP fusions containing N DedD (FIG. 31 and data not shown). As cells lacking DedD had similar division defects as ftsn slm117 cell, we also tested whether overproduction of FtsQ, B, or L affected ftsn slm117 cells. The spot titer analyses in FIG. 30B showed that GFP-FtsQ and GFP-FtsL inhibited the growth of ftsn slm117 cells when overexpressed (at least and 10-fold respectively with 500 µm IPTG). But GFP-FtsB did not seem to have much effect on the growth of ftsn slm117 cells, although overproduction of GFP-FtsB slightly aggravated the division defects of ftsn slm117 cells (data not shown). So excessive 78

79 production of FtsQ, B, or L also inhibited cell division in ftsn slm117 cells with reduced E FtsN activities. DedD s function in cell division was shown to be partially overlapping with that of FtsN (Ref. 28 and FIG. 23). In addition to stimulating septal murein synthesis during cell constriction, FtsN may also stabilize the SR through interactions with multiple SR components (95, 97, 98, 102). The phenotype of ΔdedD cells with excess FtsQ, B, or L molecules suggested that DedD might also be important for the stability of the SR. When we observed ΔdedD cells overproducing the GFP fusion to FtsQ, B, or L, we noticed that the GFP fusions failed to accumulate at the SR in ΔdedD cells. In LB medium containing 100 µm IPTG, MG14/pCH196 (ΔdedD/ P lac ::gfp-ftsq) cells formed long filaments with only a few constriction sites (FIG. 32A). In the filamentous cells, GFP-FtsQ was mostly evenly distributed along the cell membrane, with rarely a few GFP-FtsQ rings. Under similar growth condition, cells of the otherwise wild-type strain (TB28/pCH196) overexpressing GFP-FtsQ were at most twice longer than normal cells and sharp GFP-FtsQ rings were present in most cells. The absence of FtsQ from the SR in ΔdedD cells leads to the absence of late SR components that depend on preassembly of FtsQ, including FtsW, FtsI, and FtsN (FIG. 32 and data not shown). In FIG. 32B, no ring-like accumulation of GFP-FtsN was detected in the filamentous ΔdedD cells overproducing FtsQ. Instead, GFP-FtsN formed bright patches and spots along the cell membrane, indicating that GFP- FtsN molecules formed abnormal aggregates outside the SR. Early SR components joining the SR before FtsQ, like ZipA, did not seem to be affected by 79

80 overproduction of FtsQ (FIG. 33). The absence of late SR components from the SR helps explain why cell division is blocked by excess FtsQ, B, or L molecules in ΔdedD cells. Toxicity of excess SR components in ΔdedD cells is not limited to FtsQ, B, and L. For the ten essential SR proteins, overproduction of FtsZ, FtsA, or ZipA, was significantly more toxic to ΔdedD cells than to wt cells (FIG. 34). In addition, while overproduction of FtsK, FtsQ, or FtsI had little effect on viability of wt cells, growth of ΔdedD cells was severely inhibited in each case (FIG. 34). Notably, overexpression of untagged FtsB or FtsL had much less of a detrimental effect on the growth and division phenotypes of ΔdedD cells than the GFP-tagged versions of the proteins (FIGs. 30B and 34, and data not shown). FtsB and FtsL rely on FtsQ and each other for stabilization in the cell (64, 76, 146). It is possible that attaching GFP to the N-terminus of FtsB or FtsL helped stabilizing them such that the GFP fusions to FtsB or FtsL showed more inhibitory activities in ΔdedD cells than the native proteins. We conclude that DedD is important for maintaining the integrity of the SR, especially in cases of fluctuations in the amount of certain SR components. DISCUSSION In this study, we narrowed down the minimal critical functional domain of DedD to its N-terminal conserved region ( N DedD) of at most 54 residues. The activity of N DedD is required for efficient cell constriction, especially in cells with reduced E FtsN activity at the SR, where N DedD becomes essential for initiating cell constriction. In cells lacking PBP1B, abolishing N DedD activity leads to 80

81 rupture of the peptidoglycan layer and cell lysis at the division site, suggesting that N DedD is ultimately required for stimulating septal murein synthesis. Our results also demonstrated that N DedD possesses affinity for the SR. N DedD is capable of accumulating at the constriction sites independent of S DedD, but not very efficiently. Given that septal localization of S DedD relies on synthesis and processing of septal murein (Ref. 28 and FIG. 19), our results suggest that DedD functions in cell constriction in a self-enhancing manner, similar to that of FtsN (28). (i) Before the onset of cell constriction, most of DedD molecules are not at the SR. But a small fraction of the DedD molecules are recruited to the SR via the N DedD domain, through interactions with preassembled SR components, presumably the FtsBLQ subcomplex. (ii) The DedD molecules at the SR help to activate the murein synthases at the division site, like PBP3 (FtsI), through an FtsN independent pathway, leading to some new sepal murein synthesis. (iii) The newly synthesized septal murein is then processed by multiple enzymes, including the amidases, generating a transient form of peptidoglycan that is recognized by the SPOR domains, including S DedD. (iv) S DedD accumulates at the constriction site and helps to recruit more DedD molecules, leading to increased N DedD activity at the constriction site. (v) The N DedD (and E FtsN) molecules at the SR simulate more septal murein synthesis, accelerating the previous steps and leading to sustained septal murein synthesis. The self-enhancing model for DedD differs from the FtsN model in at least one aspect. Septal targeting of S DedD normally does not absolutely require the 81

82 activity of N DedD. Instead, it relies on the essential activity of E FtsN. When sufficient E FtsN activity is present at the SR, cells can start cell constriction without N DedD. However, when the essential activity of FtsN is limited, like in ftsn slm117 cells, N DedD is required for initiating cell constriction. Even in cells with normal FtsN activity, the absence of DedD leads to slow cell constriction and this cannot be rescued by giving the cells more FtsN. Thus, N DedD stimulates septal murein synthesis and cell constriction through a pathway parallel to that of E FtsN. Both pathways converge at or before the ultimate target, the murein synthases specializing in septal murein synthesis. Based on the cell lysis phenotype in cells lacking N DedD or E FtsN in addition to PBP1B, PBP3 (FtsI) is an ideal target for N DedD and E FtsN. Other than PBP3, the FtsBLQ protein subcomplex is another potential target for N DedD and E FtsN. With the ability to interact with multiple SR proteins, the FtsBLQ subcomplex has long been proposed to function as a platform during SR assembly for recruiting downstream SR components (63-70). But there are also evidences supporting the idea that the FtsBLQ subcomplex participates in regulating septal murein synthesis (Refs and Chapter 2). We identified point mutations in the periplasmic part of FtsB and FtsL that enable septal murein synthesis and cell constriction in the absence of E FtsN. Cells carrying an ftsb or ftsl mutant allele in addition to native ftsn start septal murein synthesis earlier in the cell division process, reinforcing the idea that the FtsBLQ subcomplex is involved in regulating septal murein synthesis (Chapter 2). Interestingly, the E FtsN*-suppressing mutations in ftsb or ftsl are also capable of suppressing the 82

83 division defects of ΔdedD cells, suggesting that they affect both the N DedD and E FtsN pathways (FIG. 35 and data not shown). The bacterial two-hybrid analysis suggested direct interactions between N DedD and FtsBL, and the interaction with FtsL appeared to be important for the function of N DedD. With the proposed E FtsN interaction in the periplasm and the N DedD interaction involving their N- terminal regions close to the membrane, the FtsBLQ subcomplex has the ability to incorporate the activation signals from both the N DedD and E FtsN pathways. Given our results, we propose a model for FtsN and DedD in triggering septal murein synthesis during cell constriction (FIG. 36). The ultimate target for FtsN and DedD is the multiprotein machinery for synthesizing septal murein, with FtsI (PBP3) (and/or maybe FtsW) as their primary target. E FtsN and N DedD may activate PBP3 either directly or indirectly through other proteins, such as the FtsBLQ subcomplex. In the latter case (FIG. 36A), the FtsBLQ subcomplex functions as a switch for PBP3 activity and suppresses spg synthesis before interacting with E FtsN and N DedD. E FtsN and N DedD induce conformational changes in the FtsBLQ subcomplex upon their arrival at the SR and convert the FtsBLQ subcomplex to its on state that can now stimulate spg synthesis. Alternatively, the FtsBLQ subcomplex functions as a co-activator that forms a transient protein complex with E FtsN and maybe N DedD to activate PBP3 (FIG. 36B). Future studies, especially biochemical evidences that help clarify the interactions among FtsN, DedD, FtsBLQ, FtsI and maybe other SR components will help us refine the current model. 83

84 SUMMARY AND FUTURE DIRECTIONS Our results in this study suggest that the essential/critical functional domains of FtsN ( E FtsN) and DedD ( N DedD) stimulate septal murein synthesis and cell constriction by targeting at (or collaborating with) the FtsBLQ protein subcomplex during cell constriction. Both FtsN and DedD join the SR and function in cell constriction in self-enhancing fashions that increase E FtsN and N DedD molecules at the division site in coordination with spg synthesis and processing. In our model for cell constriction initiation, E FtsN interacts with a periplasmic domain of the FtsBLQ subcomplex, while N DedD talks to the FtsBLQ subcomplex at its juxta- and trans-membrane part, either directly or indirectly. The engagement of E FtsN and N DedD with the FtsBLQ subcomplex induces conformational changes in the FtsBLQ subcomplex, which probably leads to the release of its inhibitory activity on the murein synthases and switches on spg synthesis and cell constriction. In order to initiate spg synthesis, E FtsN and N DedD need to stimulate the murein synthases, especially PBP3 (FtsI), either directly or indirectly, and the FtsBLQ subcomplex plays an essential role in this process. The FtsBLQ subcomplex might work as a co-activator that forms a transient protein complex with E FtsN and N DedD that activates PBP3 through the FtsBLQ- E FtsN interface in the periplasm and the FtsBLQ- N DedD interface in the vicinity of the inner membrane. But the properties of the E FtsN*-suppressing mutations suggest that the FtsBLQ subcomplex is more likely to function as a switch for PBP3 activity. It suppresses PBP3 activity until the engagement with E FtsN and N DedD releases 84

85 its inhibitory activity. In that case, activation of PBP3 involves FtsBLQ-PBP3, FtsBLQ- E FtsN, and FtsBLQ- N DedD interactions, but E FtsN and N DedD may not directly interact with PBP3. Studies that can clarify the interactions between these proteins will help us refine our model for initiating spg synthesis. But as previously discussed, interactions involving specific (sub)domains in these proteins, especially E FtsN, are likely to be weak or transient so that they are not suitable for conventional biochemical approaches or two-hybrid assays. To find proteins that specifically interact with E FtsN, we used a site-directed in vivo crosslinking approach utilizing the photo-reactive amino acid analog Bpa that was incorporated into FtsN in place of various residues in E FtsN (147). From the crosslinking experiments, we identified a chaperon complex, YfgM-PpiD, which readily interacts with E FtsN (data not shown). But neither YfgM nor PpiD is essential and cells lacking YfgM and/or PpiD have no division phenotype, meaning that they are not the essential targets of E FtsN for triggering spg synthesis. One possibility is that several potential targets for E FtsN, such as FtsQ, FtsB, or FtsL, are present in E. coli cells in very small amounts compared to FtsN, which makes it challenging to detect the crosslinked protein complexes in cells producing these proteins at native levels (75, 77, 78, 92). Thus, it is worth trying to perform crosslinking in cells overproducing the proteins of interest, like FtsQ, FtsB, FtsL, FtsI, and maybe FtsN. It is also reasonable to crosslink with Bpa placed in the FtsB or FtsL subdomains in the periplasm that may interact with E FtsN and maybe PBP3. 85

86 The ts phenotypes of E FtsN + cells carrying additional suppressing mutations in ftsb or ftsl also deserve in-depth investigations. Figuring out the reason for the suppressing mutations to cause the ts phenotypes will provide useful information about the function of these essential proteins and the control mechanism for spg synthesis. The most straightforward scenario may be that the FtsBLQ subcomplex controls the activities of certain murein hydrolysases. Its inhibitory effect is released at elevated temperatures due to increased instability of the subcomplex and leads to hydrolysis of the peptidoglycan layer and, thus cell morphological changes and cell lysis. One argument against this hypothesis is that the changes in cell shape indicate that PG hydrolysis occurred along the lateral cell wall. It might be difficult for the FtsBLQ subcomplex accumulated at the septum to control the hydrolases activities along the lateral cell wall. Given that the suppressing mutations promote septal murein synthesis in addition to E FtsN, we favor another scenario that the ts phenotypes are caused by overstimulation of septal murein synthesis. This hypothesis is compatible with our model that the FtsBLQ subcomplex functions as a switch that inhibits spg synthesis until its engagement with the activation signal E FtsN. The suppressing mutations in ftsb or ftsl induce spg synthesis by promoting the conformational changes of the FtsBLQ subcomplex toward the on state. At elevated temperatures, the FtsBLQ subcomplex is more likely to disassociate from its targets, such as PBP3, and leads to overstimulation of the murein synthases at the septum. Overproduction of spg may cause depletion of the substrate for PG synthesis or maybe murein synthases along the lateral cell wall, which leads to 86

87 disruption of the balance between PG synthesis and hydrolysis and, thus cell morphological changes and cell lysis. We set up screens to identify multicopy suppressors for ftsb E56A cells in LB ΔNaCl at 42 C and identified three proteases (FtsH, DegP, and DegQ) in addition to native FtsB. Further studies that identify the targets for these proteases will help us better understand the regulation of spg synthesis. Our results suggest that N DedD may also target the FtsBLQ subcomplex, but at a different domain of the subcomplex around the inner membrane. Investigations on the interaction between N DedD and the FtsBLQ subcomplex, or other potential targets of N DedD, will help us to better understand the function of N DedD and FtsBLQ in initiating cell constriction. As the interaction between N DedD and FtsL involves the transmembrane domain of DedD, in vivo crosslinking with Bpa or cysteine residues placed in their transmembrane domains may help us identify the targets for N DedD. Another approach is to select for suppressing mutations in ftsblq that can compensate for the absence of dedd. Cells can live without dedd, but combining ΔdedD with ftsn slm117 or ΔponB leads to synthetic (sub)lethality. Suppressor screens in ΔdedD ΔponB cells using randomly mutagenized libraries of ftsb, ftsl, or ftsq may identify suppressing mutations in their trans- or juxta-membrane domains and support the former hypothesis. 87

88 MATERIALS AND METHODS Plasmids and Phages Plasmids used in this study are listed in Table 9. Relevant restriction sites and sequences for targeted recombination are indicated by underline in the primer sequences below. For pbl18 [P BAD ::rfp-ftsz], the 1930 bp XbaI-SalI fragment of pch313 [P lac ::rfp-ftsz] was used to replace the 1026 bp XbaI-SalI fragment of pmg20 [P BAD :: TT bfp-ftsn le]. For pbl33 [P lac ::gfp-malf dedd ], primers 5'- GCAGGGATCCGACGGGCAGAAAAAACATTATCAGGATGAG-3' and 5'- CCCAAGCTTTTAATTCGGCGTATAGCCCATTACCACGCC-3' were used to amplify a fragment of pfb236 [P lac ::gfp-dedd]. The product was digested with BamHI and HindIII, and the 588 bp fragment was used to replace the 330 bp BamHI-HindIII fragment of pch310 [P lac ::gfp-malf ftsn ]. To obtain pbl37 [P lac ::gfp-dedd le], primers 5'- CGCGGATCCGTGGCAAGTAAGTTTCAGAATCGGTTAGTGGGC-3' and 5'- GGCGCTCGAGCTGATAATGTTTTTTCTGCCCGTCC-3' were used to amplify a fragment of pfb236 [P lac ::gfp-dedd]. The product was digested with BamHI and XhoI, and the 111 bp fragment was used to replace the 330 bp BamHI-XhoI fragment of pmg12 [P lac ::gfp-ftsn le]. For pbl39 [P lac ::daca rfp], primers 5'- GCGTCTAGAAATAATTTTGTTTGAATTCAACTCAGGAGATATACC-3' and 5'- CGCTCGAGAACGGTTTCAAAGAAACGGAAGC-3' were used to amplify a fragment of 88

89 ptb12 [P lac ::daca]. After treatment with XbaI and XhoI, the 927 bp product was used to replace the 584 bp XbaI-XhoI fragment of pmg36 [P lac ::pal-rfp] (84). For pbl51 [P lac ::t18-dedd ], primers 5'- GTACGGCCATTACGGCCGTGGCAAGTAAGTTTCAGAATCG-3' and 5'- GATCGGCCGAGGCGGCCTTACTTTGGTTTAGGCGGCTCC-3' were used to amplify a fragment of pfb236 [P lac ::gfp-dedd]. The product was digested with SfiI, and the 370 bp fragment was used to replace the 1027 bp SfiI fragment of pch371 [P lac ::t18-rodz] (148). For mutant versions of pbl51 (G11A, P24A, P42G), the same primers were used to amplify fragments of the corresponding versions of pbl198. After treatment with SfiI, the 370 bp fragments were next used to replace the 2565 bp SfiI fragment of plp7 [P lac ::t18-pona] (149). For pbl52 [P lac ::t18-dedd], primers 5'- GTACGGCCATTACGGCCGTGGCAAGTAAGTTTCAGAATCG-3' and 5'- GATCGGCCGAGGCGGCCTTAATTCGGCGTATAGCCC-3' were used to amplify a fragment of pfb236 [P lac ::gfp-dedd]. The product was digested with SfiI, and the 676 bp fragment was used to replace the 1027 bp SfiI fragment of pch371 [P lac ::t18-rodz]. For mutant versions of pbl52 (G11A, P24A, P42G), the same primers were used to amplify fragments of the corresponding versions of pbl198. After treatment with SfiI, the 676 bp fragments were next used to replace the 2565 bp SfiI fragment of plp7 [P lac ::t18-pona]. 89

90 For pbl74 [P lac ::dsba ecrfp-cwlc ], the 229 bp BamHI-HindIII fragment of pbl34 was used to replace the 18 bp BamHI-HindIII fragment of ptu136. For pbl75 [P lac ::dsba ecrfp-cwlc ], the 1075 bp XbaI-HindIII fragment of pbl74 was used to replace the 1746 bp XbaI-HindIII fragment of pch201. pbl81 [aph P lac :: TT gfp-ftsn ] was obtained by replacing the 1817 bp XbaI-ApaI fragment of pbl50 [cat P lac :: TT gfp-ftsn ] with the 2046 bp XbaI- ApaI fragment of ptb14. To obtain pbl82 [bla P lac ::dsba rfp-dedd ], the 252 bp BamHI-HindIII fragment of pmg44 [P lac :: TT gfp-dedd ] was used to replace the 18 bp BamHI-HindIII fragment of ptu136 [P lac ::dsba rfp]. For pbl83 [aph P lac ::dsba rfp-dedd ], the 1064 bp AatII-NotI fragment of pbl82 was replaced with the 1415 bp AatII-NotI fragment of pbl81. For pbl87 [aada ci857 PλR::ftsI], the 1196 bp XbaI-SalI fragment of pdb346 (150) was replaced with the 1815 bp NheI-SalI fragment of pch197 [P T7 ::gfpftsi]. To obtain pbl91 [bla P lac ::dsba rfp-ftsn ], the 18 bp BamHI-HindIII fragment of ptu136 [bla P lac ::dsba rfp-] was replaced with the 244 bp BamHI- HindIII fragment of pbl6 [P lac :: TT gfp-ftsn ]. For pbl92, [aph P lac ::dsba rfp-ftsn ], the 1064 bp AatII-NotI fragment of pbl91 was replaced with the 1415 bp AatII-NotI fragment of pbl83. For pbl95 [P lac ::gfp-dedd le] and pbl96 [P lac ::gfp-dedd le], ppc1 was used as template to amplify fragments with forward primer 5'- 90

91 GCTGCTGGGATTACACATGGC-3' and reverse primer 5'- ACTACTCGAGATCAGGCTCATCACGATCGCCCGC-3' (pbl95) or 5'- CTGCCTCGAGCTCTTCCGCTGCGCCTTCCGGCGG-3' (pbl96). Products were digested with NheI (internal to fragments) and XhoI, and the resulting 201 bp (pbl95) or 261 bp (pbl96) fragment was used to replace the 360 bp NheI-XhoI fragment of pch310 [P lac ::gfp-malf ftsn ]. To construct pbl101 [P lac ::gfp-dedd rfp] and pbl102 [P lac ::gfp-dedd rfp], the 927 bp XbaI-XhoI fragment of pbl39 was replaced with the 884 bp and 941 bp XbaI-XhoI fragment of pbl37 and pbl95, respectively. For pbl115 [P T7 ::gfp-ftsn (1-63)-glqg-(68-101)-a-( ) ], one portion of pch201 was amplified with primers 5'- CAGCGAATTCCATATGGCACAACGAGATTATG-3' and 5'- CTGGCCTTGCAGGCCCTCGGACTCTTCTTTCTTGTGATG-3' and digested with EcoRI and SfiI, yielding a 205 bp fragment. A second portion was amplified with 5'- GTGGCCTGCAAGGCCAGAAAGTGACCGGAAACGG-3' and 5'- CAGGCCGAAGGGGCCTCTGTGGGCGCACGCACTCCCGG-3' and digested with SfiI, yielding a 113 bp fragment. The two fragments were then used in a three-way ligation with the 6771 bp EcoRI-SfiI fragment of pbl114. pbl115 encodes a fully functional version of GFP-FtsN in which an alanine residue is inserted between residues 101 and 102, and residues (TLQS) have been replaced with GLQG. In addition, pbl115 contains two unique SfiI sites flanking codons For pbl116 [P lac ::gfp-ftsn (1-63)-glqg-(68-101)-a-( ) ], the 976 bp BamHI-HindIII fragment of pbl115 was used to replace the 973 bp BamHI-HindIII fragment of 91

92 pch201. pbl116 encodes a fully functional version of GFP-FtsN in which an alanine residue is inserted between residues 101 and 102, and residues (TLQS) have been replaced with GLQG. In addition, pbl116 contains two unique SfiI sites flanking codons For pbl120 [P lac ::gfp-ftsi], the 1780 bp SfiI fragment of pch433 was used to replace the 16 bp SfiI fragment of plp13 (149). For pbl122 [P lac ::rfp-dedd le], the 2029 bp NheI-ClaI fragment of pch313 was replaced with the 1243 bp NheI-ClaI fragment of ppc1 [P lac ::gfp-dedd le]. For pbl128 [P lac ::gfp-dedd ftsn ], a portion of pch201 was amplified with primers 5'- GACTCGAGACGCATCACAAGAAAGAAGAGTCC-3' and 5'- CGAAGCTTATTCAAGAAGCTGACGTTGTTCTGG-3' and the 214 bp XhoI-HindIII fragment was used to replace the 23 bp XhoI-HindIII fragment of pbl95. For pbl131 [P BAD ::rfp-dedd le], the 1136 bp XbaI-SalI fragment of pbl122 was used to replace the 1930 bp XbaI-SalI fragment of pbl18 [P BAD ::rfpftsz]. For pbl136 [P lac ::gfp-ftsn Δ(64-101) ], oligo 5'- CGAGGGCCTGCAAGGCCAGAAAGTGACCGGAAACGGACTCGAGCCGGGAGTGCGTGCGC CCACAGAGGCCCCTTCGGCCGGTG-3' and its reverse complement were annealed and the 62 bp SfiI fragment of the product was used to replace the 113 bp SfiI fragment of pbl116. In pbl136, ftsn codons (TLQSQKVTGNGLPPKPEERWRYIKELESRQPGVRAPTE) are replaced by a SfiI fragment containing a unique XhoI site, and encoding an in-frame arbitrary 92

93 peptide of 22 residues (GLQGQKVTGNGLEPGVRAPTEA). The plasmid was used to facilitate directional cloning of 113 bp SfiI mutant library fragments to generate pbl116 derivatives encoding gfp-ftsn alleles with single amino acid substitutions within the FtsN(80-93) interval. For pbl137 [P BAD ::rfp-malf dedd ], a portion of pbl33 [P lac ::gfpmalf dedd ] was amplified with primers 5'- GAGCTGTACAAAGCTAGCATGACTGGTG-3' and 5'- CTGGTCGACAAAACGACGGCCAGTGCCAAGC-3', and the 773 bp BsrGI-SalI fragment was used to replace the 410 bp BsrGI-SalI fragment of pbl131. For pbl138 [P BAD ::rfp-malf ftsn ], a portion of pch310 [P lac ::gfpmalf ftsn ] was amplified with primers 5'- GAGCTGTACAAAGCTAGCATGACTGGTG-3' and 5'- CTGGTCGACAAAACGACGGCCAGTGCCAAGC-3', and the 377 bp BsrGI-SalI fragment was used to replace the 410 bp BsrGI-SalI fragment of pbl131. For pbl141 [P BAD ::ecrfp-ftsn le], a portion of pmg12 [P lac ::gfp-ftsn le] was amplified with primers 5'- GAGCTGTACAAAGCTAGCATGACTGGTG-3' and 5'- CTGGTCGACAAAACGACGGCCAGTGCCAAGC-3', and the 371 bp BsrGI-XhoI fragment was used to replace the 401 bp BsrGI-XhoI fragment of pbl131. For pbl142 [P BAD ::ecrfp-ftsn 1-90 ], a portion of pmg47 [P lac ::gfp-ftsn 1-90 ] was amplified with primers 5'- GAGCTGTACAAAGCTAGCATGACTGGTG-3' and 5'- CTGGTCGACAAAACGACGGCCAGTGCCAAGC-3', and the 320 bp BsrGI-XhoI fragment was used to replace the 401 bp BsrGI-XhoI fragment of pbl

94 For pbl143 [P lac ::gfp-ftsn (1-63)-glqg-(SphI_XhoI stuffer)-a-( ) ], a portion of pch201 [P lac ::gfp-ftsn] was amplified with primers 5'- CGAGGGCCTGCAAGGCCAGGCATGCAAAGTGACCGGAAACGGACTAC-3' and 5'- CACCGGCCGAAGGGGCCTCGAGTGTGGGCGCACGCACTCCCGGC- 3', and the 122 bp SfiI fragment of the 143 bp product was used to replace the 113 bp SfiI fragment of pbl116. In pbl143, ftsn codons are replaced by a non-coding SfiI fragment containing unique SphI and XhoI sites. The plasmid was used to facilitate directional cloning of 113 bp SfiI mutant library fragments to generate pbl116 derivatives encoding gfp-ftsn alleles with single amino acid substitutions within the FtsN(80-93) interval. For pbl145 [ci857 PλR::ftsN 1-90 ], a portion of pdb357 [P T7 ::ftsn] was amplified with primers 5'- GATCCCGCGAAATTAATACGACTCACTATAGGGG-3' and 5'- ATTTCTCGAGCTCTTTAATGTAGCGCCAGCGTTCTTCTGG-3', and the 306 bp XbaI- XhoI fragment was used to replace the 326 bp XbaI-XhoI fragment of pch287. For pbl153 [P syn135 ::ftsn], the 1127 bp XbaI-HindIII fragment of pch362 [P syn135 ::gfp-zapa] was replaced with the 1000 bp XbaI-HindIII fragment of pdb357. To construct pbl154 [repa ts P syn135 ::ftsn], the 2128 bp NarI-SphI fragment of pbl153 was replaced with the 2128 bp NarI-SphI fragment of pdb326 [repa ts ]. pbl160 and pbl161 [P lac ::gfp-ftsn Y85S ] were obtained by site directed mutagenesis of pmg59, using mutagenic primer 5'- CCAGAAGAACGCTGGCGCAGCATTAAAGAGCTGGAAAG-3' and its reverse complement. The 581 BamHI-AgeI fragment of pmg59(y85s) was next 94

95 exchanged with that of pch201, yielding pbl160, or with that of pmg59, yielding pbl161. pbl180 and pbl181 [P lac ::gfp-ftsn W83T ] were obtained by site directed mutagenesis of pmg59, using mutagenic primer 5'- CCACCAAAACCAGAAGAACGCACCCGCTACATTAAAGAGCTGGAA-3' and its reverse complement. The 581 BamHI-AgeI fragment of pmg59(w83t) was next exchanged with that of pch201, yielding pbl180, or with that of pmg59, yielding pbl181. For pbl190 [I-SceI ci857 PλR::ftsN 1-90 ], oligos 5'- AATTAGTTACGCTAGGGATAACAGGGTAATATA-3' and 5'- GATCTATATTACCCTGTTATCCCTAGCGTAACT-3' were annealed and the resulting fragment, with a unique I-SceI site and with EcoRI and BamHI compatible overhangs, was used to replace the 21 bp EcoRI-BamHI fragment of pbl145. For pbl191 [I-SceI ci857 P λr::i-scei], primers 5'- GCTCTAGACAGGAGGGTACCTATATGCATATG-3' and 5'- CAGGTCGACGCATGCGAATTCGACGTCGGGCCCTTATTTCAGGAAAGTTTCGGAGG-3' were used to amplify a portion of pacbsce [P BAD ::I-sceI] (121), and the 759 bp XbaI-SalI fragment was used to replace the 315 bp XbaI-SalI fragment of pbl190. For pbl193 [P lac ::gfp-ftsb], the 1780 bp SfiI fragment of pbl120 was replaced with the 325 bp SfiI fragment of pch

96 For pbl198 [aph P lac ::gfp-dedd], the 1806 bp ApaI-HindIII fragment of pbl81 [aph P lac :: TT gfp-ftsn ] was replaced with the 2176 bp ApaI-HindIII fragment of pfb236. pbl198(g11a) [aph P lac ::gfp-dedd G11A ], pbl198(p24a) [aph P lac ::gfpdedd P24A ], and pbl198(p42g) [aph P lac ::gfp-dedd P42G ] were obtained by Quickchange site-directed mutagenesis of pbl198 using, respectively, primers 5'- GTTTCAGAATCGGTTAGTGGCCACGATCGTGCTGGTGGCG-3', 5'- CTGGGGGTGATTGTACTTGCAGGGCTGCTGGACGGGCAG-3' and 5'- GATGAGTTCGCGGCTATCGGTCTGGTGCCGAAAGCGGGC-3' and their reverse complements. The 374 bp MfeI-BglII fragment of each mutated version of pbl198 was then used to replace the corresponding fragment of non-mutated pbl198. For pbl200 [repa ts P syn135 ::ftsn I-SceI ci857 PλR::I-sceI], the 685 bp SpeI- EcoRI fragment of pbl154 [repa ts P syn135 ::ftsn] was replaced with the 2880 bp SpeI-EcoRI fragment of pbl191 [I-SceI ci857 P λr::i-scei]. For pbl203 [P T7 ::gfp-ftsn(δ59-73)], one portion of pch201 was amplified with primers 5'- CAGCGAATTCCATATGGCACAACGAGATTATG-3' and 5'- GCTAGGCCGGCCTCTTCCTGCGGCCGCTTGTGATGCGTAATGAAGTACAG-3' and digested with EcoRI and FseI, yielding a 202 bp fragment. A second portion was amplified with 5'- GCGGCCGGCCTACCACCAAAACCAGAAGAACGC-3' and 5'- TGAGAAGCTTAACCCCCGGCGGCGAG-3' and digested with FseI and HindIII, yielding a 738 bp fragment. The two fragments were then used in a three-way ligation with the 6119 bp EcoRI-HindIII fragment of pch198 [P T7 ::gfp-ftsn ]. 96

97 pbl203 encodes a version of GFP-FtsN in which FtsN residues (~periplasmic helix 1) are replaced with the peptide RPQEEA. For pbl205 [P lac ::gfp-ftsn(δ59-73)], the 946 bp BamHI-HindIII fragment of pbl203 was used to replace the 973 bp BamHI-HindIII fragment of pch201. For pbl206 [teta repa ts ftsa(e124a)], the 133 bp EagI-ClaI fragment of pbl12 was replaced with the 1325 bp EagI-ClaI fragment of pah162 (140), yielding pbl204 [teta cat repa ts ftsa(e124a)]. Deletion of the 952 bp BstBI fragment of pbl204 then resulted in pbl206. For pbl209 [P lac ::gfp-ftsn::lacz], the 2480 bp ApaI-HindIII fragment of pch201 was used to replace the 2503 bp ApaI-HindIII fragment of ptb8 [P lac ::mincde]. For pbl210 [P lac ::gfp-ftsn (1-58)-P/Q95-(74-319) ], primers 5'- GTCAGCGGCCGCAACACCAGTACCAACCGCC-3' and 5'- GTCAGGCCGGCCTCCGCTACAGGCTCAGGCTGTGG-3' were used to amplify a portion of pch38 [P T7 ::zipa-h], and the 286 bp NotI-FseI fragment was used to replace the 19 bp NotI-FseI fragment of pbl205. pbl210 encodes a version of GFP-FtsN in which FtsN residues (~periplasmic helix 1) are replaced with a 95 residue P/Q-rich linker derived from ZipA For pbl211 [P lac ::gfp-ftsn (1-58)-P/Q15-(74-319) ], oligos 5'- GGCCGTCGCCGCAACACCAGTACCAACCGCCTTATGCGTCTGCCGG-3' and 5'- CAGACGCATAAGGCGGTTGGTACTGGTGTTGCGGCGAC-3' were annealed and the fragment with compatible overhangs was used to replace the 19 bp NotI-FseI fragment of pbl205. pbl211 encodes a version of GFP-FtsN in which FtsN 97

98 residues (~periplasmic helix 1) are replaced with a 15 residue P/Q-rich linker derived from ZipA Plasmids pbl214(y85c), pbl215(y85w), pbl216(w83l), pbl217(l89s), pbl218(w83m), and pbl219(l89h) [P lac ::gfp-ftsn (1-63)-glqg-(68-101)*-a-( ) ] encode mutant versions of GFP-FtsN in which a single FtsN residue has been replaced. They were obtained by replacing the 2503 bp ApaI-HindIII fragment of ptb8 [P lac ::mincde] with the 2483 bp ApaI-HindIII fragment of mutant pbl116 derivatives carrying the corresponding allele. For pbl221 [P BAD ::ftsq], the 24 bp XbaI-HindIII fragment of pbad33 was replaced with the 871 bp XbaI-HindIII fragment of pch252 [P lac ::ftsq]. For pbl222 [P BAD ::ftsl], the 24 bp XbaI-HindIII fragment of pbad33 was replaced with the 414 bp XbaI-HindIII fragment of pab12 [P T7 ::ftsl]. For pbl225 [P lac ::gfp-ftsn Y85S ::lacz], the 2480 bp ApaI-HindIII fragment of pbl161 was used to replace the 2503 bp ApaI-HindIII fragment of ptb8 [P lac ::mincde]. For pbl226 [P lac ::gfp-ftsn W83T ::lacz], the 2480 bp ApaI-HindIII fragment of pbl181 was used to replace the 2503 bp ApaI-HindIII fragment of ptb8 [P lac ::mincde]. For pbl236 [ftsa I143L ], primers 5'- GTAGTACGAATTCTGGAACTGGCGGAC-3' and 5'- GAGGCCGTAATCATCGTCGGCCTC-3' were used to amplify a chromosomal ftsqa fragment of BL86supKK-1/pBL215 [ftsa I143L / P lac ::gfp-ftsn Y85W ]. The 852 bp BglII-AscI fragment of the product was then used to replace that of pbl12 [ftsa E124A ]. 98

99 pbl247 [aph P lac ::gfp-dedd G11C ] and pbl248 [aph P lac ::gfp-dedd T12C ] were obtained by Quickchange site-directed mutagenesis of pbl198 using, respectively, primers 5'- GTTTCAGAATCGGTTAGTGTGCACGATCGTGCTGGTGGC-3' and 5'- CAGAATCGGTTAGTGGGCTGCATCGTGCTGGTGGCGCTG-3' and their reverse complements. The 374 bp MfeI-BglII fragment of each mutated version of pbl198 was then used to replace the corresponding fragment of non-mutated pbl198. For pbl270 [P lac ::t18-dedd 1-118; G11C ] and pbl271 [P lac ::t18-dedd 1-118; T12C ], primers 5'- GTACGGCCATTACGGCCGTGGCAAGTAAGTTTCAGAATCG-3' and 5'- GATCGGCCGAGGCGGCCTTACTTTGGTTTAGGCGGCTCC-3' were used to amplify fragments of pbl247 and pbl248, respectively. For each, the product was digested with SfiI, and the 370 bp fragment was used to replace the 1027 bp SfiI fragment of pch371. For pbl289 [P T7 ::ftsb], primers 5'- CGCATATGGGTAAACTAACGCTGCTGTTGC-3' and 5'- AAAGGGGGATGTGCTGCAAG-3' were used to amplify a portion of pbl193 [P lac ::gfp-ftsb], and the 334 bp NdeI-HindIII fragment of the product was used to replace the 63 bp NdeI-HindIII fragment of pet21a. For pbl294 [P lac ::ftsb], the 1000 bp XbaI-HindIII fragment of pmg21 [P lac ::ftsn] was replaced with the 374 bp XbaI-HindIII fragment of pbl289. For pbl295 [P lac ::ftsl], the 1000 bp XbaI-HindIII fragment of pmg21 [P lac ::ftsn] was replaced with the 414 bp XbaI-HindIII fragment of pbl222 [P BAD ::ftsl]. 99

100 For pbl301 [P lac ::ftsl D93G ], primers 5'- CCGAATTCCATATGATCAGCAGAGTGACAG-3' and 5'- CGTGTCGACTTATTTTTGCACTACGAT-3' were used to amplify chromosomal ftsl D93G from BL86-AK1/pBL215 [ftsl D93G / P lac ::gfp-ftsn Y85W ], and the 298 bp NsiI- SalI fragment was used to replace that of pbl295. For pbl304 [repa ts P syn135 ::ftsb D59H ], the 1000 bp XbaI-HindIII fragment of pbl154 was replaced with the 358 bp XbaI-HindIII fragment of pbl300. For pbl305 [repa ts P syn135 ::ftsl D93G ], the 1000 bp XbaI-HindIII fragment of pbl154 was replaced with the 414 bp XbaI-HindIII fragment of pbl301. For pbl306 [repa ts P syn135 ::ftsb D59H ispd 1-48 ], primers 5'- CGCATATGGGTAAACTAACGCTGCTGTTGC-3' and 5'- GCGTCGACTTAATGCGCCAGCAGCGCATGCACCG-3' were used to amplify chromosomal ftsb D59H and a portion of ispd from BL86-AK11/pBL216 [ftsb D59H / P lac ::gfp-ftsn W83L ], and the 209 bp MluI-SalI fragment was used to replace the 58 bp MluI-SalI fragment of pbl304. For pbl309 [P syn135 ::ftsl], the 1000 bp XbaI-HindIII fragment of pbl153 was replaced with the 414 bp XbaI-HindIII fragment of pbl295. For pbl312 [P lac ::gfp-malf ftsn le], primers 5'- GTCGGCCGAAATGTGCCTGCGGTTTCTCCC-3' and 5'- AGCGCTCGAGTTCTTCTGGTTTTGGTGGTAGTCCGTTTCC-3' were used to amplify a portion of pmg13 [P lac ::gfp-ftsn 1-81 ], and the 169 bp EagI-XhoI fragment was used to replace the 691 bp EagI-XhoI fragment of pfb261 [P lac ::gfp-malf rodz ]. 100

101 For pbl315 [P lac ::gfp-malf ftsn le], primers 5'- CTGTACGGATCCACGCATCACAAGAAAGAAGAGTCC-3' and 5'- AGCGCTCGAGTTCTTCTGGTTTTGGTGGTAGTCCGTTTCC-3' were used to amplify a portion of pch201 [P lac ::gfp-ftsn], and the 87 bp BamHI-XhoI fragment was used to replace the 207 bp BamHI-XhoI fragment of pch310 [P lac ::gfp-malf ftsn ]. For pbl330 [P lac ::gfp-ftsn D5S,Y6A ], a mutagenized 732 bp fragment of pmg59 was obtained by asymmetric amplification and overlap extension using primer pairs 5'- GCTGCTGGGATTACACATGGC-3' with 5'- CCGAATTCCATATGGCACAACGATCTGCTGTACGCCGCAGCCAACCGGC-3', and 5'- GCCGGTTGGCTGCGGCGTACAGCAGATCGTTGTGCCATATGGAATTCGG-3' with 5'- CGCAGGAGTTTGCAGCAGATC-3'. The 581 bp BamHI-AgeI fragment encoding the D5S and Y6A substitutions was next ligated to the 8804 bp BamHI-AgeI fragment of pbl210 [P lac ::gfp-ftsn (1-58)-P/Q95-(74-319) ]. For pbl331 [P syn135 ::ftsl H94Y ], the 138 bp BpmI-HindIII fragment of pjh2 [P syn135 ::ftsl] was replaced with that of a mutant derivative [pjh2(l9v, V27A, H94Y)-pBL215-L6], selected from a library of mutant ftsl alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. For pbl332 [P syn135 ::ftsl E88V ], the 138 bp BpmI-HindIII fragment of pjh2 [P syn135 ::ftsl] was replaced with that of a mutant derivative [pjh2(l77i, E88V)- pbl215-l7], selected from a library of mutant ftsl alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. 101

102 Plasmid pbl333 [P syn135 ::ftsl E88k ], was selected from a library of mutant ftsl alleles that allow growth of strain JH1/pBL200/pBL215 at 42 o C. For pbl334 [P syn135 ::ftsl N89S ], the 138 bp BpmI-HindIII fragment of pjh2 [P syn135 ::ftsl] was replaced with that of a mutant derivative [pjh2(k37r, N89S)- pbl215-l7], selected from a library of mutant ftsl alleles that allow growth of strain JH1/pBL200/pBL215 at 42 o C. For pbl335 [P lac ::gfp-ftsn 1-81;D5S,Y6A -le], primers 5'- GCTGCTGGGATTACACATGGC-3' and 5'- AGCGCTCGAGTTCTTCTGGTTTTGGTGGTAGTCCGTTTCC-3' were used to amplify a portion of pbl330, and the 291 bp NheI-XhoI fragment was used to replace the 777 bp NheI-XhoI fragment of pch354 [P lac ::gfp-ftsn le]. For pbl336 [P syn135 ::ftsb], the 414 bp XbaI-HindIII fragment of pjh2 [P syn135 ::ftsl] was replaced with the 374 bp XbaI-HindIII fragment of pbl294. Plasmid pbl338 [P syn135 ::ftsb E56V ], was selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pBL215 at 42 o C. For pbl339 [P syn135 ::ftsb E56A ], the 121 bp BstZ17I-SapI fragment of pbl336 [P syn135 ::ftsb] was replaced with that of a mutant derivative [pbl336(k3r, E56A, S92L)-pBL215-B2], selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. For pbl340 [P syn135 ::ftsb A55T ], the 238 bp XbaI-SapI fragment of pbl336 [P syn135 ::ftsb] was replaced with that of a mutant derivative [pbl336(a55t, T83S)-pBL215-B3], selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. 102

103 Plasmid pbl341 [P syn135 ::ftsb E56K ], was selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. Plasmid pbl342 [P syn135 ::ftsb E56G ], was selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pBL215 at 37 o C. Plasmid pbl343 [P syn135 ::ftsb D59V ], was selected from a library of mutant ftsb alleles that allow growth of strain JH1/pBL200/pJH10 at 37 o C. For pbl345 [P lac ::gfp-dedd ], the 1780 bp SfiI fragment of pbl147 [P lac ::gfp-ftsi] was replaced with the 370 bp SfiI fragment of pbl51 [P lac ::t18- dedd ]. Mutant versions of pbl345 (G11A, G11C, P24A, P42G) were obtained similarly, using the 370 bp SfiI fragments of the corresponding versions of pbl51 (G11A, P24A, P42G), or of pbl270 (G11C). For pbl353(e56a) [repa ts P syn135 ::ftsb E56A ispd 1-48 ], the 331 bp XbaI-ClaI fragment of pbl306 [repa ts P syn135 ::ftsb D59H ispd 1-48 ] was replaced with the 347 bp XbaI-ClaI fragment of pbl339 [P syn135 ::ftsb E56A ]. For pbl353(e56k) [repa ts P syn135 ::ftsb E56K ispd 1-48 ], the 331 bp XbaI-ClaI fragment of pbl306 [repa ts P syn135 ::ftsb D59H ispd 1-48 ] was replaced with the 347 bp XbaI-ClaI fragment of pbl341 [P syn135 ::ftsb E56K ]. For pbl353(e56v) [repa ts P syn135 ::ftsb E56V ispd 1-48 ], the 331 bp XbaI-ClaI fragment of pbl306 [repa ts P syn135 ::ftsb D59H ispd 1-48 ] was replaced with the 347 bp XbaI-ClaI fragment of pbl338 [P syn135 ::ftsb E56V ]. For pbl355 [SceI P syn135 ::ftsb E56V ispd 1-48 sacb], the 58 bp EcoRI-SalI fragment of pdoc-c [SceI sacb] (121) was replaced with the 573 bp EcoRI-SalI fragment of pbl353(e56v) [P syn135 ::ftsb E56V ]. 103

104 For pbl356 [SceI P syn135 ::ftsb E56K ispd 1-48 sacb], the 58 bp EcoRI-SalI fragment of pdoc-c [SceI sacb] was replaced with the 573 bp EcoRI-SalI fragment of pbl353(e56k) [P syn135 ::ftsb E56K ]. For pch252, the 20 bp BamHI-HindIII fragment of pmlb1113 was replaced with the 837 bp BglII-HindIII fragment of pgp2. For pch309 [P lac ::gfp-malf mrec le], primers 5'- GCAGAGATCTGATGTCATTAAAAAGAAACATTGGTGGC-3' and 5'- CGTTCTCGAGTTGCCCTCCCGGCGCACGCGCAGGC-3' were used to amplify a portion of pfb243 [P lac ::malf mrec ], and the 1116 bp BglII-XhoI fragment was used to replace the 330 bp BamHI-XhoI fragment of pmg12 [P lac ::gfp-ftsn le]. For pch310 [P lac ::gfp-malf ftsn ], the 1562 bp ApaI-BamHI fragment of pch288 [P lac :: TT gfp-ftsn ] was replaced with the 1627 bp ApaI-BamHI fragment of pch309 [P lac ::gfp-malf mrec le]. Plasmid pch312 [P T7 ::rfp-] encodes mcherry RFP codon-optimized for E.coli. To create it, the rfp ORF was amplified from pj1:g01080 (kind gift from Ken Marians) using primers 5 - AGGGCGCATATGGTTTCCAAGGGCGAGGAGGATAACATGGC-3 and 5 - ATGTTTGCTAGCTTTGTACAGCTCATCCATGCCACC-3. The product was treated with NdeI and NheI, and the 710 bp fragment was ligated to the 5422 bp NdeI- NheI fragment of pdr107a [P T7 ::gfpmut2-]. For pbl313 [P BAD ::rfp-malf ftsn le], the 972 bp XbaI-XcmI fragment of pbl138 [P BAD ::rfp-malf ftsn ] was used to replace the 1023 bp XbaI- XcmI fragment of pbl141 [P BAD ::ecrfp-ftsn le]. 104

105 For pch425 [P lac ::t18-ftsb], ftsb was amplified with primers 5'- GATCGGCCATTACGGCCATGGGTAAACTAACGCTGCTG-3' and 5'- GTCAGGCCGAGGCGGCCTTATCGATTGTTTTGCCCCGC-3', and the 325 bp SfiI fragment was used to replace the 1027 bp SfiI fragment of pch371 [P lac ::t18- rodz]. For pch433 [P lac ::t25-ftsi], ftsi was amplified with primers 5'- GATCGGCCATTACGGCCATGAAAGCAGCGGCGAAAAC-3' and 5'- GATCGGCCGAGGCGGCCTTACGATCTGCCACCTGTC-3'. The product was digested with SfiI, and the 1780 bp fragment was used to replace the 1027 bp SfiI fragment of pch358 (148). For pch455 [P lac ::pal ecrfp-], the 7809 bp XhoI-HindIII fragment of pmg41 [P lac ::pal ecrfp] was ligated to the 750 bp XhoI-HindIII fragment of ptu136 [P lac ::dsba ecrfp-]. For pch535 [P lac ::gfp-t-malf tola ], a portion of pch520 [P lac :: ss toragfp-tola( )] was amplified with primers 5 - ACTGGATCCCTCGAGGCCATTACGGCCGATGATATTTTCGG -3 and 5 - AAAGGGGGATGTGCTGCAAG -3, and the 428 bp BamHI-HinDIII fragment of the product was used to replace the 230 bp BamHI-HinDIII fragment of pch310. For pch537 [P lac ::gfp-t-malf(2-39)-zipa(86-145)-pal(63-173)], the 563 bp XhoI-HinDIII fragment of plp112 was used to replace the 422 bp XhoI-HinDIII fragment of pch535. For pdb357 [P T7 ::ftsn], ftsn was amplified with primers 5'- CAGCGAATTCCATATGGCACAACGAGATTATG-3' and 5'- 105

106 TGAGAAGCTTAACCCCCGGCGGCGAG-3', and the 960 bp NdeI-HindIII fragment was used to replace the 58 bp NdeI-HindIII fragment of pet21a. For pez3, the -10 promoter element of the P syn1 promotor in pez1 was modified by the QuickChange procedure (Stratagene), using oligo 5 - TGCTTCCGGCTCGTATATTGTGTGGAGGTACC-3 and its reverse complement, resulting in a C to T mutation (underlined) and yielding pez3 [orir6k attλ P syn110 ::gfp-zapa]. For pjh1 [teta repa ts P syn135 ::ftsl], the 468 bp EcoRI-HindIII fragment of pbl309 [P syn135 ::ftsl] was ligated to the 5779 bp EcoRI-HindIII fragment of pbl206 [teta repa ts ftsa E124A ]. For pjh2 [teta P syn135 ::ftsl], the 2674 bp AflII-ApaLI fragment of pjh1 was replaced with the 2127 bp AflII-ApaLI fragment of pbl153 [aada P syn135 ::ftsn]. For plp8 [P lac ::t18-ponb], MG1655 ponb was amplified with primers 5'- GATCGGCCATTACGGCCATGGCCGGGAATGACCGCGAGCC-3' and 5'- GATCGGCCGAGGCGGCCTTAATTACTACCAAACATATCCTTGATCCAAC-3', and the 2548 bp SfiI fragment of the product was used to replace the 1027 bp SfiI fragment of pch371. For plp24 [atthk022 frt-cat-frt laci q P lac ::gfp-t-pona], the 4098 bp ApaI-HindIII fragment of plp14 (149) was used to replace the 1544 bp ApaI-HindIII fragment of pbl49 (28). For plp32 [atthk022 frt-cat-frt laci q P lac ::gfp-t-ponb], the 2548 bp SfiI fragment of plp8 was used to replace the 2566 bp SfiI fragment of plp24. An 106

107 integrated version of plp32 lacking cat [ilp32-cat] was obtained by eviction of cat after chromosomal integration of the plasmid in the desired strain. For plp95 [P lac ::dsba ecrfp-pal ], a portion of pmg36 was amplified with primers 5 - GGCTGGATCCTCGGCCATTACGGCCCAACAGCTGCAGCAGAACAACATCG-3 and 5 - GCTGAAGCTTGTCGACTGGCCGAGGCGGCCTTAGTAAACCAGTACCGCACGACGG-3, and the 377 bp BamHI-HinDIII fragment was used to replace the 229 bp BamHI- HinDIII fragment of pbl75. For plp102 [P lac ::pal ecrfp-pal ], the 978 bp EcoRI-BamHI fragment of pch455 was used to replace the 922 bp EcoRI-BamHI fragment of plp95. To create plp112 [P lac ::pal (1-35)-p/q(69)-(63-173) ], a portion of pch151 was amplified with primers 5 - CCGCTCGAGCCGTCGCCGCAACACCAG -3 and 5 - CGGGATCCCTGTGGCGAAACTGGCTGCTGC -3, and the 186 bp XhoI-BamHI fragment was used to replace the 732 bp XhoI-BamHI fragment of plp102. For plp160 [P BAD ::ecrfp-malf ftsn ], the 2232 bp XcmI-NcoI fragment of pbl313 [P BAD ::ecrfp-malf ftsn le] was replaced with the 2181 bp XcmI-NcoI fragment of pbl142 [P BAD ::ecrfp-ftsn 1-90 ]. For plp163 [P lac ::gfp-ftsn], a portion of pch201 [P lac ::gfp-ftsn] was amplified with primers 5 - GGTGAGATCTGTGGCACAACGAGATTATGTACGC-3 and 5 - TGAGAAGCTTAACCCCCGGCGGCGAG-3, and the 964 bp BglII-HindIII fragment was used to replace the 973 bp BamHI-HindIII fragment of pch201. plp163 is similar to pch201, but 3 codons in the linker between gfp and ftsn, as well as three restriction sites (BamHI, EcoRI, and NdeI), have been eliminated. 107

108 For plp164 [P lac ::gfp-ftsn le], a portion of pmg14 [P lac :: ss tora-gfp-ftsn le] was amplified with primers 5 - CATCGGATCCCTACCACCAAAACCAGAAGAACGC-3 and 5 - GCGATCGGCATAACCACCACGCTC-3, and the 955 bp BamHI-ClaI fragment of the product was used to replace the 1129 bp BamHI-ClaI fragment of pmg47 [P lac ::gfp-ftsn 1-90 ]. For plp165 [P lac ::gfp-ftsn le], a portion of pmg14 [P lac :: ss tora-gfp-ftsn le] was amplified with primers 5 - GAAGGGATCCGAAGAACGCTGGCGCTACATTAAAGAG-3 and 5 - GCGATCGGCATAACCACCACGCTC-3, and the 940 bp BamHI-ClaI fragment of the product was used to replace the 1129 bp BamHI-ClaI fragment of pmg47 [P lac ::gfp-ftsn 1-90 ]. For plp168 [P lac :: ss tora-gfp-ftsn le], the 1629 bp BamHI-ClaI fragment of pch282 [P lac :: ss tora-gfp-ftsn ] was replaced with the 955 bp BamHI-ClaI fragment of plp164 [P lac ::gfp-ftsn le]. For plp169 [P lac :: ss tora-gfp-ftsn le], the 1629 bp BamHI-ClaI fragment of pch282 [P lac :: ss tora-gfp-ftsn ] was replaced with the 940 bp BamHI-ClaI fragment of plp165 [P lac ::gfp-ftsn le]. For plp170 [P lac ::gfp-ftsn malf ftsn le], a portion of plp163 [P lac ::gfp-ftsn] was amplified with primers 5 - ATGACCATGATTACGAATTCCCG-3 and 5 - CCATCGGCCGGCAGGCAGATTTCGTTGCTTTTTCCG-3, and the 871 bp XbaI- EagI fragment of the product was used to replace the 817 bp XbaI-EagI fragment of pbl315 [P lac ::gfp-malf ftsn le]. 108

109 For plp171 [P lac ::gfp-ftsn le], a portion of pch201 [P lac ::gfp-ftsn] was amplified with primers 5 - GCTGCTGGGATTACACATGGC-3 and 5 - TAGGCTCGAGGGTCACTTTCTGGCTTTGCAG-3, and the 261 bp NheI-XhoI fragment was used to replace the 777 bp NheI-XhoI fragment of pch354 [P lac ::gfp-ftsn le]. For plp209 [P lac ::gfp-malf zipa ftsn ], oligos 5'- CCGGTCCCAAAACCAGAAGAACGCTGGCGCTACATTAAAGAGCTGGAATAAG-3' and 5'- TCGACTTATTCCAGCTCTTTAATGTAGCGCCAGCGTTCTTCTGGTTTTGGGA-3' were annealed, and the resulting 52 bp AgeI-SalI fragment was used to replace the 498 bp AgeI-SalI fragment of pch537 [P lac ::gfp-t-malf zipa pal ]. For plp213 [P BAD ::rfp-malf zipa ftsn ], the 688 bp EagI-SalI fragment of pbl137 [P BAD ::rfp-malf dedd ] was replaced with the 193 bp EagI-SalI fragment of plp209. For plp218 [P lac :: ss tora-gfp-ftsn le], a portion of plp168 [P lac :: ss tora-gfpftsn le] was amplified with primers 5 - GCTGCTGGGATTACACATGGC-3 and 5 - GGTCCTCGAGCTGGCGACTTTCCAGCTCTTTAATG-3, and the 96 bp NheI-XhoI fragment of the product was used to replace the 132 bp NheI-XhoI fragment of plp168. For plp219 [P lac :: ss tora-gfp-ftsn le], a portion of plp168 [P lac :: ss tora-gfpftsn le] was amplified with primers 5 - GCTGCTGGGATTACACATGGC-3 and 5 - GTGTCTCGAGGGGCGCACGCACTCCCGGCTG-3, and the 114 bp NheI-XhoI fragment of the product was used to replace the 132 bp NheI-XhoI fragment of plp

110 For plp221 [P lac :: ss tora-gfp-ftsn ], the 72 bp BamHI-SalI fragment of plp218 [P lac :: ss tora-gfp-ftsn le] was replaced with the 117 bp BamHI-SalI fragment of plp160 [P BAD ::ecrfp-malf ftsn ]. For pmg21 [P lac ::ftsn], the 1746 bp XbaI-HindIII fragment of pch201 [P lac ::gfp-ftsn] was replaced with the 1000 bp XbaI-HindIII fragment of pdb357 [P T7 ::ftsn]. For pmg39 [P BAD ::dedd], the 706 bp XbaI-HindIII fragment of pfb239 [P lac ::dedd] (28) was used to replace the 2743 bp XbaI-HindIII fragment of pfb174 [P BAD ::mreb mrec mred-le] (151). For pmg41 [P lac ::pal ecrfp], a portion of pmg36 [P lac ::pal-mcherry] was amplified with primers 5'- TCCCTCTAGACCCTGCCTGGTCGCCGTATCTGTG-3' and 5'- TCAGCTCGAGGCCTTCGCTGCCGTCATTGCTGGC-3', and the 170 bp XbaI-XhoI fragment was used to replace the 1026 bp XbaI-XhoI fragment of pch311 [P lac ::zipa-ecrfp]. For pmg62 [P lac ::gfp-ftsn ], the 1779 bp XbaI-HindIII fragment of ptb222 [P lac ::zipa-gfp] (148) was replaced with the 1174 bp XbaI-HindIII fragment of pch276 [P lac ::gfp-ftsn ] (28). For pmg63 [P lac ::gfp-ftsn le], the 1779 bp XbaI-HindIII fragment of ptb222 [P lac ::zipa-gfp] was replaced with the 1126 bp XbaI-HindIII fragment of pmg12 [P lac ::gfp-ftsn le] (28). For ppc1 [P lac ::gfp-dedd le], a portion of dedd was amplified with primers 5'- CGCGGATCCGTGGCAAGTAAGTTTCAGAATCGGTTAGTGGGC-3' and 5'- CCGCGCTCGAGCTTTGGTTTAGGCGGCTCCACC-3'. The product was digested with 110

111 BamHI and XhoI, and the 360 bp fragment was used to replace the 345 bp BamHI-XhoI fragment of pfb252 [P lac ::gfp-rodz r]. For ptb12 [P lac ::daca], a daca fragment was amplified with primers 5'- GCCTGAATTCAACTCAGGAGATATACCATGAATACCATTTTTTCCGCTCGTATC-3' and 5'- CGCGCGAAGCTTTTAACCAAACCAGTGATGGAACATTAA-3' and digested with EcoRI and HindIII. The 1235 bp product was used to replace the 30 bp EcoRI- HindIII fragment of pmlb1113. Strains Relevant strains are listed in Table 10. BL17 was obtained by transduction of leu::tn10 from GC13109 (152) in TB28. BL19 was obtained by transduction of leu::tn10 from GC13109 in BL18. For BL23 [ponb<>cat], the cat cassette of pkd3 was amplified with primers 5'- AAATCGGGCTTTTGCGCCTGAATATTGCGGAGAAAAAGCCCATATGAATATC CTCCTTAG -3' and 5'- ATGGCAACTCGCCATCCGGTATTTCACGCTTAGATGTTAGTGTAGGCTGGA GCTGCTTCG -3', yielding a 1093 bp fragment with end sequences homologous to the chromosomal ponb locus (underlined). Recombination with the chromosome of TB10 yielded BL23, in which 2538 bp of the ponb gene (from bp +1 to bp +2538) is replaced with cat and transcription of the latter is in the same 111

112 direction as the replaced gene. Transduction of ponb<>cat from BL23 to TB28 resulted in BL24. BL73 variants were obtained by transduction of ftsn<>aph from CH34/pCH201 to MG14 carrying various plasmids. BL83 was obtained by transduction of ponb<>cat from BL23 to MG14, or to MG14 lysogenic for either λfb236 or λfb239. BL84(iMG62) was obtained by transduction of ponb<>cat from BL23 to TB77(iMG62). BL85 variants were obtained by transduction of ftsn<>aph from CH34/pCH201 into BL17 carrying various plasmids. BL86 variants were obtained by transduction of reca<>cat from BW10724 into BL85 variants. BL102 was obtained by transduction of dedd<>cat from FB71 (28) to LP11 carrying integrated CRIM construct ibl198 or ilp32 frt in the chromosome. For BL114, TB28 chromosomal ftsa was exchanged with the ftsa(i143l) allele on pbl236 by the method of Hamilton et al (120). BL120/pX were obtained by transduction of ftsn<>aph from CH34/pCH201 to BL120/pX. BL130 was obtained by eviction of aph from CH125. BL132 were then obtained by transduction of dedd<>cat from FB71 to BL130. For BL140, TB28 chromosomal ftsb was exchanged with the ftsb(d59h) allele on pbl306 by the method of Hamilton et al (120). 112

113 BL141/pX was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL140/pX. BL148 was obtained by transduction of leu::tn10 from GC13109 to BL114. BL149 was obtained by co-transduction of leu::tn10 and ftsa(i143l) from BL148 to BL140. BL150 was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL149. BL151 was obtained by co-transduction of leu::tn10 and ftsa(e124a) from BL19 to BL140. BL152 was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL151. For BL153/pBL215, the ftsl(d93g) allele of pbl305 was amplified with primers 5'- CCGAATTCCATATGATCAGCAGAGTGACAG -3' and 5'- CGTGTCGACTTATTTTTGCACTACGAT -3', yielding a 386 bp ftsl(d93g) fragment largely homologous to native chromosomal ftsl (underlined). Recombination with the chromosome of BL85/pBL200/pBL215/pTB51 and growth at 37 o C in the presence of ampicillin and 100 µm IPTG yielded BL153/pBL215. BL154 was obtained by co-transduction of leu::tn10 and ftsl(d93g) from BL153/pBL215 to TB28. BL155/pX was obtained by transduction of ftsb<>aph from NB946 to TB28/pX. 113

114 BL156/pX was obtained by transduction of ftsl<>aph from MDG277 to TB28/pX. BL157/pX was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL154/pX. BL159 was obtained by co-transduction of leu::tn10 and ftsl(d93g) from BL154 to BL140. BL163 was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL159. BL164 was obtained by co-transduction of leu::tn10 and ftsl(d93g) from BL154 to BL114. BL165 was obtained by transduction of ftsn<>aph from CH34/pCH201 to BL164. For BL167, TB28 chromosomal ftsb was exchanged with the ftsb(e56a) allele on pbl353(e56a) by the method of Hamilton et al (120). For BL171, the ftsb(e56v) allele of pbl355(e56v) was amplified with primers 5'- GTCGTCTTCGGATGCATGGGATGATGATGCCGTTTTTCAGGGGGCAGGATGGGTAAACT AACGCTGCTGTTGC -3' and 5'- GCGTCGACTTAATGCGCCAGCAGCGCATGCACCG-3', yielding a 533 bp ftsb(e56v) fragment largely homologous to native chromosomal ftsl (underlined). This fragment was recombined with the chromosomal ftsb<>aph allele of BL155/pBL355/pACBSCE with selection for growth on LB containing 5% sucrose, and for sensitivity to kanamycin, chloramphenicol and ampicillin. 114

115 For BL172, the ftsb(e56k) allele of pbl356(e56k) was amplified with primers 5'- GTCGTCTTCGGATGCATGGGATGATGATGCCGTTTTTCAGGGGGCAGGATGGGTAAACT AACGCTGCTGTTGC -3' and 5'- GCGTCGACTTAATGCGCCAGCAGCGCATGCACCG-3', yielding a 533 bp ftsb(e56k) fragment largely homologous to native chromosomal ftsl (underlined). This fragment was recombined with the chromosomal ftsb<>aph allele of BL155/pBL355/pACBSCE with selection for growth on LB containing 5% sucrose, and for sensitivity to kanamycin, chloramphenicol and ampicillin. BL173, BL174, and BL175 were obtained by transduction of ftsn<>aph from CH34/pCH201 to BL167, BL171, and BL172, respectively. BL177 was obtained by transduction of dedd<>cat from FB71 to BL167. LP11 was obtained by eviction of cat from BL24. LP31 was obtained by transduction of reca<>aph from JW2669 (153) to TB28. Growth conditions. Cells were grown at 30 C in LB medium with 0.5% NaCl or in M9 minimal medium containing 0.2% Casamino Acids, 50µg/ml L-tryptophan, 50µM thiamine, and 0.2% of maltose or glucose, unless stated otherwise. The media were supplied with antibiotics at the following concentrations: ampicillin (50 µg ml -1 for multicopy plasmids or 15 µg ml -1 for bla integrated in the chromosome), kanamycin (25 µg ml -1 ), spectinomycin (50 µg ml -1 ), chloramphenicol (25 µg ml

116 for multicopy plasmids or 12.5 µg ml -1 for cat integrated in the chromosome), tetracycline (12.5 µg ml -1 ), and cephalexin (15 µg ml -1 ). Microscopy and image analyses. Cell imaging was performed on a Zeiss Axioplan-2 microscope as previously described (154). Live cells were imaged on agar pads of M9 salt solution containing 1.2% agarose. For cell dimension measurements, cells were chemically fixed (151) and imaged on poly-l-lysine-coated coverslips using phase contrast microscopy. Cell length, cell diameter, and constriction site were measured using ImageJ 1.48 and ObjectJ 1.03 (155). The volume of rod cells were calculated using formula V = 4/3πr 3 + πr 2 h, where r and h represent radius and cylinder length, respectively. The average age of cells showing septal localization of a fluorescent marker or cell constriction was calculated according to the methods in ref (38). The average time (t x ) after cell birth when a fluorescent ring appears was calculated by using tt x =!! ln [1 0.5FF xx ]!"! where Td is the doubling time and F(x) is the fraction of cells with neither a constriction site nor a fluorescent ring. The average time (t x ) before the end of cell cycle when a fluorescent ring disassembles was calculated by using tt x =!! ln [1 + FF xx ]!"! where F(x) is the fraction of cells with a constriction site but without a fluorescent ring. 116

117 Data analysis was performed in Microsoft Excel and Origin 9 (OriginLab Corporation). Sequence alignments and secondary structure predictions For the FtsN sequence alignment in FIG. 11A, FtsN sequences from the following species (UniPortKB accession number) in the OMA (156) group were aligned using the program ClustalW2 (157): Photorhabdus luminescens (Q7MYC3), Proteus mirabilis (BF4173), Escherichia coli (P29131), Shigella flexneri (F5R4C6), Citrobacter rodentium (D2TU51), Salmonella typhimurium (Q8ZKN9), Enterobacter asburiae (G2S6I3), Klebsiella pneumonia (B5XZ35), Pantoea sp. (E6WAF5), Erwinia tasmaniensis (B2VI86), Serratia plymuthica (G0BC89), Yersinia pestis (D0A5K0), Dickeya dadantii (E0SFN8), Sodalis glossinidius (Q2NQY3), Edwardsiella ictaluri (C5BB83), Mannheimia succiniciproducens (Q65VF3), Actinobacillus pleuropneumoniae (B0BTF0), Aggregatibacter aphrophilus (C6ALF4), and Haemophilus influenza (A5UDH7). Sequence logo was created using WebLogo 3 (158, 159). For the DedD sequence alignment in FIG. 20A, DedD sequences from the following species (UniPortKB accession number) in the OMA group and were aligned using the program ClustalW2: Pectobacterium carotovorum (C6DA70), Dickeya dadantii (E0SL87), Erwinia tasmaniensis (B2VIW1), Pantoea sp. (E6W8J4), Serratia plymuthica (G0BB41), Yersinia enterocolitica (A1JL80), Edwardsiella ictaluri (C5B8M4), Sodalis glossinidius (Q2NSI5), Salmonella typhimurium (Q8ZNC1), Citrobacter rodentium (D2THN8), Enterobacter asburiae 117

118 (G2S279), Klebsiella pneumoniae (B5XNS3), Shigella flexneri (Q0T2H1), Escherichia coli (P09549), Cronobacter turicensis (C9XWU5), Shimwellia blattae (I2B732), Photorhabdus luminescens (Q7N2B7), Proteus mirabilis (B4EZF3), Oceanimonas sp. (H2FWY9), Aeromonas hydrophila (A0KLN4), Shewanella baltica (A3D664), Acidovorax sp. (A1W9F4), Methylococcus capsulatus (Q604P7). Relative conservation of residues in FtsB, FtsL and FtsN in FIG. 13A is based on the sequence alignments of Hogenom gene families HOG (FtsB), HOG (FtsL), and HOG (FtsN), repectively (160). Secondary structures were predicted using GOR secondary structure prediction method version IV (161). Identification of critical residues in E FtsN. To identify individual residues critical to FtsN function, each residues in the FtsN interval was subjected to site-scanning and/or site-directed mutagenesis, and resulting mutants were tested for their ability to rescue growth of the FtsN-depletion strain CH31 [P BAD ::ftsn] on LB plates without arabinose. Specific site-directed mutants were generated in the context of pch201 [P lac ::gfp-ftsn], using Quickchange site-directed mutagenesis. Random site- T64G, S67G, scanning mutants were obtained in the context of pbl116 [P lac ::gfp-ftsn +A102 ]. This plasmid is similar to pch201, except that ftsn codons are flanked by two unique SfiI sites that were introduced to facilitate the generation of site-scanning libraries. It encodes a fully functional variant of GFP-FtsN in which 118

119 FtsN residues T64 and S67 are replaced with glycine, and an alanine residue is inserted between FtsN residues 101 and 102. To make the site-scanning mutant library, a library of oligos in the context of CGTGGCCTGCAAGGCCAGAAAGTGACCGGAAACGGACTACCACCAAAACCAGAAGAACG CTGGCGCTACATTAAAGAGCTGGAAAGTCGCCAGCCGGGAGTGCGTGCGCCCACAGAGG CCCCTTCGGCCTGC and their reverse complements were order from DNA2.0, Inc., which cover the FtsN interval flanked by two SfiI sites but carry a mutation in each of the ftsn codons substituted by a random codon. The oligos were either directly digested with SfiI, or amplified with 5'- GTGGCCTGCAAGGCCAGAAAGTGACCGGAAACGG-3' and 5'- CAGGCCGAAGGGGCCTCTGTGGGCGCACGCACTCCCGG-3' before SfiI digestion, yielding a 113 bp fragment that was used to replace the 122bp SfiI fragment of pbl143. The ligation mixture was transformed to DH5α cells and amplified to make the plasmid library with site-scanning ftsn mutants. To screen for mutations disrupting the essential function of FtsN, plasmids with site-directed mutations or a fraction of the site-scanning plasmid library was transformed to strain CH31 [P BAD ::ftsn] on LB plates supplemented with 0.5% arabinose. The transformants were then streaked on LB plates with or without arabinose, and the ones that only grow in the presence of arabinose were qualified as carrying a nonfunctional ftsn mutant. The mutations were identified by sequencing the ftsn region on the plasmid. Screens for extragenic suppressors of nonfunctional ftsn alleles. 119

120 Strain BL86 [ΔftsN ΔrecA ΔlacIZYA leu::tn10] carrying plasmid pbl200 [aada repa ts P syn135 ::ftsn I-SceI ci857 P λr::i-scei] and one of the mini-f plasmids [bla laci P lac ::gfp-ftsn*::lacz] encoding mutant GFP-FtsN* variants as well as LacZ under control of the lac promotor was constructed to serve as host in these screens. For screens on LB-IX plates (LB plates containing 100 µm IPTG and 60 µg ml -1 Xgal), host strains carrying one of the mini-f plasmids were grown overnight in LB medium supplemented with 50 µg ml -1 ampicillin and 100 µm IPTG at 30 C. 100 µl of 10- or 100-fold dilution of the overnight culture (about 3 X 10 6 or 3 X 10 7 cells per plate for 10- or 100-fold dilution respectively) was plated on LB-IX plates. The plates were incubated at 42 C overnight and solid blue colonies were picked and then subjected to several tests as described later to ensure that they indeed propagated without native FtsN but still required FtsN*. For screens in liquid medium, 100 µl of the overnight culture (about 3 X 10 8 cells) was diluted in 50 ml LB medium containing 100 µm IPTG and 50 µg ml -1 ampicillin or 25 µg ml -1 kanamycin. After growth at 42 C overnight, 100 µl of the new culture was diluted in another 50 ml LB medium containing 100 µm IPTG and 25 µg ml -1 kanamycin and grown at 42 C overnight. Several µl of the resulting culture was then streaked on LB-IX plates. The resulting suppressors were named as BL86-AK or BL86-KK strains according to whether they were selected in LB medium containing ampicillin (AK) or kanamycin (KK) in the first round of selection at 42 C. The suppressors were subjected to several tests and the ones qualify for the following conditions were then picked to map the suppressing mutations: 120

121 IPTG-dependent cell division phenotype, sensitive to 50 µg ml -1 spectinomycin, and retention of gfp-ftsn* allele on mini-f plasmid. To determine whether the suppressor mutations were linked with the leu::tn10 marker, P1-phage lysates obtained from the suppressor strains were used to introduce the leu::tn10 marker to CH34 [ΔftsN] cells carrying pbl200 and the corresponding mini-f plasmids. 48 transductants were streaked on LB plates supplemented with 100 µm IPTG and tested for their ability to grow at both 30 C and 42 C. The co-transduction frequency was determined as the percentage of transductants growing at 42 C. For mutations linked to leu::tn10, PCR products amplifying the ftsl-ftsi, mray, ftsw-murg, and ftsq-ftsa regions were sequenced to map the mutations. For the ones that was not co-transducible with leu::tn10, the entire chromosome of the suppressor strain was subjected to deep sequencing, and compared to that of unsuppressed BL86 to identify the suppressor mutation. Deep sequencing was performed at the CWRU Genomics Sequencing Core. Total DNA was extracted using the Epicenter MasterPure DNA Purification kit and the libraries were then prepared using the Epicentre Nextera DNA Sample Prep Kit before sequencing using the Illumina HiScan system. Illumina reads were mapped to the reference genome (MG1655) using the Bowtie program (162) and SNPs were called using the SAMtools package (163). Identify ftsb and ftsl suppressing mutations using randomly mutagenized libraries. 121

122 Randomly mutagenized ftsb and ftsl libraries were in the context of plasmids pbl336 [P syn135 ::ftsb] and pjh2 [P syn135 ::ftsl], respectively, following an Errorprone PCR method with modifications (164). For the ftsb library, ftsb sequence on plasmid pbl294 was amplified with primers 5 - CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAC-3 and 5 -GCCAAGCTTGTCGACTGGCCGAGGCGGCC-3 using error-prone PCR with Taq polymerase. The products were then digested with XbaI and HindIII and used to replace the 414bp XbaI-HindIII fragment on plasmid pjh2. The ligation mixture was transformed to DH5α cells to make the plasmid library carrying randomly mutagenezied ftsb mutants. The plasmid library of randomly mutagenezied ftsl mutants was made similarly, except that primers 5 - CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAC-3 and 5 -CATGATTACGCCAAGCTTGTCGAC-3 were used to amplify the ftsl sequence on plasmid pjh1 in error-prone PCR. For the screens, libraries were introduced into strain JH1 [ΔftsN ΔrecA ΔlacIZYA] harboring pbl200 [aada repa ts P syn135 ::ftsn I-SceI ci857 PλR::i-sceI], and one of the mini-f derivatives encoding mutant GFP-FtsN* variants. Transformants were then plated on LB plates supplemented with 200 µm IPTG at 37 o C (or 42 o C) to induce loss of pbl200 and, hence, select for plasmid-borne ftsb or ftsl alleles that allow survival of E FtsN cells. Suppressors were tested for their sensitivity to spectinomycin before the plasmids carrying ftsb or ftsl mutants were sequenced to identify the mutations. Subsequent subcloning was performed to resolve clones with multiple silent and/or missense mutations in 122

123 ftsb (9/16) or ftsl (9/13), then allowed us to identify a total of 6 and 5 relevant single residue substitutions in FtsB and FtsL, respectively. Other methods. Bacterial two-hybrid (BACTH) assays (148) and western analyses with anti-green fluorescent protein (GFP) antibodies (15) were performed as previously described. 123

124 FIGURES FIG. 1 Schematic overview of SR assembly in E. coli. Shown are an enlarged representation of the SR during cell constriction, the SR assembly pathway, and the approximate step at which the SR components join the SR. Essential cell division proteins are underlined. The Min and NO systems that regulate Z-ring position are in red. Proteins residing at the cytoplasmic face of the inner membrane are in blue, trans-membrane inner membrane proteins are in black, periplasmic proteins in orange, and outermembrane (lipo-)proteins are in purple. Some SR components were listed in a box because pertinent information on their assembly is missing. 124

125 FIG. 2 Further definition of the essential domain of FtsN. (A) E. coli ftsn locus, FtsN domains, and properties of genetic constructs. The deletion-replacement in ΔftsN<>kan (ΔftsN) strains and EZTnKan-2 insertion site in ftsn slm117 strains are shown above the ftsn gene. The domain structure of FtsN is illustrated below the ftsn gene, and an enlarged view of the N-terminal part of FtsN is shown below that. Indicated are the transmembrane domain ( TM FtsN), helices H1, H2, and H3, and the C-terminal SPOR domain ( S FtsN). Also shown are inserts on plasmids that produce fusions of various portions of FtsN to GFP, RFP, or TT GFP (Tat-targeted periplasmic GFP) under the control of the P lac or P BAD regulatory regions. Columns indicate the FtsN residues present in each fusion, and whether the fusion could (+) or could not (-) compensate for the absence of FtsN. (B) Spot titer analyses of strain CH34 (ΔftsN) harboring plasmids pmg14 (P lac :: TT gfp-ftsn ), plp168 (P lac :: TT gfp-ftsn ), plp

126 (P lac :: TT gfp-ftsn ), or plp218 (P lac :: TT gfp-ftsn ). Cells were grown overnight in M9-maltose medium with 250 µm IPTG, and resuspended in M9-maltose to an optical density at 600 nm of , , , , , and Five microliters of each dilution was then spotted on LB or M9-Maltose agar with or without 250 µm IPTG, as indicated, and plates were imaged after incubatation at 30 C for 18 (LB plates) or 24 hours (M9-maltose plates). (C) Spot titer analyses of strain CH34 (ΔftsN) harboring plasmids pch201 (P lac ::gfp-ftsn), pbl205 (P lac ::gfp-ftsn Δ(59-73)<>5 ), pbl211 (P lac ::gfp-ftsn Δ(59-73)<>15 ), pbl210 (P lac ::gfp-ftsn Δ(59-73)<>95 ). Cells were grown overnight in LB medium with 25 µm IPTG, and resuspended in LB medium to an optical density at 600 nm of , , , , and Five microliters of each dilution was then spotted on LB agar with or without 500 µm IPTG, as indicated, and plates were incubated at 30 C overnight. 126

127 FIG. 3 Localization of TT GFP- S FtsN at constriction sites depends on E FtsN and PBP3 activities. 127

128 (A-C) E FtsN-dependent localization of S FtsN. Cells of E FtsN-depletion strain CH34/pMG20 [ΔftsN/ P BAD :: TT bfp-ftsn ] harboring ptb6 [P lac :: TT gfp] (A) or pmg4 [P lac :: TT gfp-ftsn ] (B, C) were grown to OD 600 = in M9- maltose supplemented with 5 µm IPTG and 0.1% of either arabinose (A and B) or glucose (C). Note the absence of fluorescent TT GFP- S FtsN rings in the E FtsNdepleted filament in panel C. (D and E) In contrast to unfused TT GFP (E), TT GFP- S FtsN (D) accumulates at constriction sites of ΔftsN cells that manage to divide in the complete absence of E FtsN, due to production of the FtsA E124A protein. Strains BL20(iBL6)/pBL12 [ΔftsN ftsa E124A (P lac :: TT gfp-ftsn )/ ftsa E124A ] (D) and BL20(iBL5)/pBL12 [ΔftsN ftsa E124A (P lac :: TT gfp)/ ftsa E124A ] (E) were grown to OD 600 = 0.3 in M9-maltose supplemented with 25 µm IPTG. (F and G) Unlike ZipA (G), S FtsN (F) fails to accumulate in rings in cephalexintreated cells. Overnight cultures of TB28(iBL48) [WT(P lac ::zipa-gfp)] and TB28(iBL50) [WT(P lac :: TT gfp-ftsn )] were diluted 100-fold into M9-maltose with 10 µm IPTG and incubated for 2 hr. Cephalexin was added to 15 µg/ml, and growth was continued to OD 600 =0.5. Fluorescence and corresponding DIC images of live cells are shown in each case. Bar equals 2 (A, B) or 4 (C-G) µm. 128

129 FIG. 4 Localization of TT GFP- S FtsN and TT GFP in murein amidase mutants. Shown are fluorescence and corresponding DIC images of live cells of strains BL11 [amia amic] (A and B) and BL12 [amia amib amic] (C and D) harboring pmg4 [P lac :: TT gfp-ftsn ] (A, C), or ptb6 [P lac :: TT gfp] (B, D). Cells were grown to OD 600 = in M9-glucose medium supplemented with 2.5 µm IPTG. Note the sharp accumulation of TT GFP- S FtsN at constriction sites in panel A. Bar equals 2 µm. 129

130 FIG. 5 Western analyses of TT GFP- S FtsN. Whole cell extracts were fractionated by SDS-PAGE, and the blot was probed with anti-gfp antibodies. Each lane received equivalent amounts of cell material (corresponding to 200 ml of a culture at OD 600 =1). The positions of 66, 45, 36, 29, 24, 20, and 14 kd (top to bottom) migration standards are indicated to the left of the panel. The blot contained extracts of TB28/pMG4 [wt/ P lac :: TT gfp-ftsn ] (lane 1), BL12/pMG4 [ΔamiABC/ P lac :: TT gfp-ftsn ] (lane 2), CH34/pMG20/pMG4 [ΔftsN/ P BAD :: TT bfp-ftsn / P lac :: TT gfp-ftsn ] (lanes 3 and 4), TB28/pMG5 [wt/ P lac :: TT gfp-ftsn ] (lane 5), and TB28/pMLB1113 [wt/vector] (lane 6). Cells were grown to OD 600 = at 30 o C in M9 with 0.2% glucose and 5 µm IPTG except for strain CH34/pMG20/pMG4, which was grown in M9 containing 130

131 0.2 % maltose, 5 µm IPTG, and 0.1 % of either arabinose (lane 3) or glucose to deplete TT BFP- E FtsN (lane 4). Arrowheads and arrows point to positions of immature and periplasmic (exported and processed) forms, respectively, of TT GFP-FtsN (black, see lanes 1-4) and TT BFP-FtsN (grey, see lane 3). The position of the immature form of TT GFP-FtsN in lane 5 is marked (<), as is the expected position of the exported form of this fusion (*), which is likely too unstable to be detected. 131

132 FIG. 6 Model for self-enhanced FtsN activity during the initiation of cell constriction. Shown are schematic representations of the constriction site during the initiation of cell constriction. The cell envelope layers are indicated as the Innermembrane (IM) and Outer-membrane (OM) in dark grey and the peptidoglycan layer (PG) in red. The Z-ring is shown as assembled at the inner side of the 132

133 cytoplasmic membrane, with the FtsZ (Z) polymers attatched to the IM by the two essential proteins FtsA (A) and ZipA. FtsN (N) molecues are indicated in brown with the C-terminal SPOR domain only binding the PG layer at the constriction site. The transpeptidase FtsI (PBP3, I) is indicted in light blue, which is essential for setpal peptidoglycan (spg) synthesis perpendibular to the long axis of the cell. The murein amidases (Ami) required for splitting spg are indicated in cyan. 133

134 FIG. 7 Western analyses of periplasmic FtsN fusions. Whole cell extracts of TB28 (wt) cells harboring plasmid pmlb1113δh3 (vector control, lane 1), pmg14 (P lac :: TT gfp-ftsn , lane 2), plp168 (P lac :: TT gfpftsn , lane 3), plp169 (P lac :: TT gfp-ftsn , lane 4), plp219 (P lac :: TT gfpftsn 75-99, lane 5), plp218 (P lac :: TT gfp-ftsn 75-93, lane 6), pmg50 (P lac :: TT gfp-ftsn 71-90, lane 7), or plp221 (P lac :: TT gfp-ftsn 55-90, lane 8) were fractionated by SDS- PAGE, blotted to nitrocellulose, and probed with anti-gfp antibodies. Cells were grown at 30 C in M9-maltose medium supplemented with 50 µm IPTG to OD 600 = before extraction preparation. Each lane received equivalent amounts of cell material. The positions of 66, 45, 36, 29 and 24 kd (top to bottom) migration standards are indicated to the left of the panel. 134

135 FIG. 8 Western analyses of FtsN fusions with internal deletions. Whole cell extracts of TB28 (wt) cells harboring plasmid pmlb1113δh3 (vector control, lane 1), pch201 (P lac ::gfp-ftsn, lane 2), pbl136 (P lac ::gfp-ftsn Δ(64-101)<>22, lane 3), pbl205 (P lac ::gfp-ftsn Δ(59-73)<>5, lane 4), pbl211 (P lac ::gfp-ftsn Δ(59-73)<>15, lane 5), or pbl210 (P lac ::gfp-ftsn Δ(59-73)<>95, lane 6) were fractionated by SDS-PAGE, blotted to nitrocellulose, and probed with anti-gfp antibodies. Cells were grown at 30 C in LB medium supplemented with 5 µm IPTG to OD 600 = before extraction preparation. Each lane received equivalent amounts of cell material. The positions of 66, 45, and 36 kd (top to bottom) migration standards are indicated to the left of the panel. 135

136 FIG. 9 Genetic screens for extragenic suppressors of nonfunction ftsn alleles. (A) Summary of the screen for critical residues in E FtsN. Residues of FtsN are shown with their relative conservation in the OMA Database represented by different colors (red for 100%, blue for 95%, green for 90%, magenta for 85%, black for 60% or less). Permissible or non-permissible substitutions of each residue are listed above or below the FtsN sequence, respectively. (B) Scheme of the extragenic suppressor screens. The host strain 136

137 BL86 [ΔftsN ΔrecA ΔlacIZYA leu::tn10] carrying plasmid pbl200 [aada repa ts P syn135 ::ftsn I-SceI ci857 P lr ::i-scei] and a mini-f plasmid [bla laci P lac ::gfpftsn*::lacz] grown at 30 C or restrictive temperature like 42 C is shown. Plasmid pbl200 was lost at 42 C and a suppressor mutation (red star) appeared on the chromosome to ensure growth of the strain. (C) A table summarizing the results of the extragenic suppressor screens. 137

138 FIG. 10 Suppressing mutations rescue division of cells producing otherwise non-functional FtsN variants. (A) Shown are live cells of strain BL120 (ΔftsN ftsa I143L ) harboring plasmid pbl215 (P lac ::gfp-ftsn Y85W ). Cells were grown for about four doublings to OD 600 = at 30 C in LB medium supplemented with 0.2% glucose (-) or 100 µm IPTG (+), as indicated. (B) Shown are live cells of strain BL120/pBL236 (ΔftsN ftsa I143L /ftsa I143L ). Cells were grown in LB medium to OD 600 = at 30 C before imaging. (C-D) Spot titer analyses of strain BL120 (ΔftsN ftsa I143L ) 138

139 (C), BL20 (ΔftsN ftsa E124A ) (D), BL157 (ΔftsN ftsl D93G ) (E), or BL141 (ΔftsN ftsb D59H ) (F) harboring plasmids pbl209 (P lac ::gfp-ftsn), pbl215 (P lac ::gfpftsn Y85W ), pbl216 (P lac ::gfp-ftsn W83L ), pbl217 (P lac ::gfp-ftsn L89S ), or pbl225 (P lac ::gfp-ftsn Y85S ). Cells were grown overnight in LB medium with 100 µm IPTG, and resuspended in LB medium to an optical density at 600 nm of , , , and Five microliters of each dilution was then spotted on M9-maltose agar containing 0.2% glucose or 100 µm IPTG, as indicated, and plates were incubated at 30 C overnight. Bar equals 4 µm (A and B). 139

140 FIG. 11 Conserved residues in N FtsN are important for its function and localization. (A) A sequence logo representing sequence alignments of N-terminal FtsN. The first 93 residues of E. coli K12 FtsN sequence are given below the sequence logo. The transmembrane domain ( TM FtsN) is underlined. Conserved residues D5 and Y6 are highlighted in orange. FtsN sequences representing every genus of Gammaproteobacteria listed in the OMA group were used for sequence alignment. (B) Bacterial two-hybrid analyses of FtsN and a mutant variant (D5S, Y6A). Strain BTH101 (cya-99) was cotransformed with plasmid pairs encoding the indicated T18- and T25-fusion proteins, and individual colonies were streaked on M9 plates containing 0.2% glucose, 40 µg/ml X-Gal and 250 µm IPTG. Plates were imaged after 24 hour incubation at 30 C and 30 hour at room temperature. (C) Shown are GFP images of wildtype strain TB28 140

141 carrying plasmid pch201 (P lac ::gfp-ftsn), pmg13 (P lac ::gfp-ftsn 1-81 ), plp170 (P lac ::gfp-ftsn malf ftsn ), pbl312 (P lac ::gfp-malf ftsn ), or pbl335 (P lac ::gfp-ftsn 1-81, DY>SA ). Note that FtsN 1-81 fusions ( S FtsN - ) with native N FtsN (GFP-FtsN 1-81 and GFP-FtsN MalF FtsN ) accumulate at the constriction sites; while ring-like accumulation is absent when N FtsN is deleted (GFP-MalF FtsN ) or when the two conserved residues in N FtsN (D5 and S6) are mutated (GFP-FtsN 1-81,DY>SA ). Cells were grown to OD 600 = at 30 C in M9-glucose medium supplemented with 10 µm IPTG. Bar equals 4 µm. 141

142 FIG. 12 Western analyses of FtsN 1-81 fusions. Whole cell extracts of strain LP31/pMLB1113ΔH3 (ΔrecA/vector control, lane 1), LP31/pMG13 (ΔrecA/P lac ::gfp-ftsn 1-81, lane 2), LP31/pBL335 (ΔrecA/P lac ::gfp-ftsn 1-81,DY>SA, lane 3), BL140/pMG13 (ftsb D59H /P lac ::gfp-ftsn 1-81, lane 4), BL140/pBL335 (ftsb D59H /P lac ::gfp-ftsn 1-81,DY>SA, lane 5), BL114/pMG13 (ftsa I143L /P lac ::gfp-ftsn 1-81, lane 6), and BL114/pBL335 (ftsa I143L /P lac ::gfp-ftsn 1-81,DY>SA, lane 7) were fractionated by SDS-PAGE, blotted to nitrocellulose, and probed with anti-gfp antibodies. Cells were grown at 30 C in M9-maltose medium supplemented with 50 µm IPTG to OD600= before extraction preparation. Each lane received equivalent amounts of cell material. The positions of 66, 45, 36, 29 and 24 kd (top to bottom) migration standards are indicated to the left of the panel. 142

143 FIG. 13 Viability of ΔftsN cells due to (combinations of) compensating mutations in ftsa, ftsb, and/or ftsl. (A) Suppressing mutations in ftsb and ftsl. Sequences of E. coli K12 FtsB, FtsL and the first 100 residues of FtsN are shown. Relative conservation of residues is indicated by colors (RED>Blue>Green>Purple), based on sequence alignments of Hogenom gene families HOG (FtsB), HOG (FtsL), and HOG (FtsN), repectively (160). Substitutions are listed above the protein sequence in orange color. The three essential residues in E FtsN are indicated by carets. Secondary structures and the domains involved in protein interaction (63, 64, 130, 161, 165) were also indicated (transmembrane domains in gray color, alpha helices in underline, random coils in wave underline, 143

144 and beta stands in dotted underline). The positions in the heptad repeat in the coiled coil regions of FtsB and FtsL were labeled abcdefg, with the hydrophobic a and d positions highlighted in green and yellow (166). (B-C) ΔftsN strains with suppressing mutations. Shown are DIC images of strain BL150 (ΔftsN ftsa I143L ftsb D59H ), BL165 (ΔftsN ftsa I143L ftsl D93G ), BL163 (ΔftsN ftsb D59H ftsl D93G ), BL173 (ΔftsN ftsb E56A ), BL175 (ΔftsN ftsb E56K ), and BL174 (ΔftsN ftsb E56V ). Cells were grown to OD 600 = at 30 C in LB medium before imaging. Bar equals 4 µm. 144

145 FIG. 14 FtsB E56A promotes septal murein synthesis in ftsn + cells. (A) Mutants with suppressing mutations. Shown are DIC images of strain TB28 (wt), BL114 (ftsa I143L ), BL167 (ftsb E56A ), BL140 (ftsb D59H ), BL154 (ftsl D93G ), and BL159 (ftsb D59H ftsl D93G ). The mutants grow normally at 30 C in LB medium 145

146 and they are slightly shorter than wildtype cells. Cells were grown to OD 600 = at 30 C in LB medium before imaging. (B) Localization of GFP-ZapA and RFP- S FtsN in wildtype or ftsb E56A cells. Shown are DIC and corresponding fluorescent images of strain TB28(iEZ3)(iBL92) [wt (P syn110 ::gfp-zapa) (P lac :: ss dsba-rfp-ftsn )] and BL167(iEZ3)(iBL92) [ftsb E56A (P syn110 ::gfp-zapa) (P lac :: ss dsba-rfp-ftsn )]. Arrowheads indicate RFP- S FtsN rings in cells without corresponding visible constrictions. Cells were grown to OD 600 = at 30 C in LB medium supplemented with 50 µm IPTG before imaged. Bar equals 4 µm (A and B). (C) Schematic overview of the cell division cycle in wildtype or ftsb E56A cells growing in LB medium at 30 C with a doubling time of 43 minutes. The average time periods for the appearance of visible cell constrictions, GFP- ZapA rings, and RFP- S FtsN rings are indicated in grey, green, and red respectively. 146

147 FIG. 15 Cell shape and lysis phenotypes of various strains in LB ΔNaCl medium at 42 C, and suppression by ΔftsN. Shown are DIC images of strain BL156/pJH2 (ΔftsL/P syn135 ::ftsl), and BL156/pBL333 (ΔftsL/P syn135 ::ftsl E88V ) (A), TB28 (wt), BL140 (ftsb D59H ), and 147

148 BL154 (ftsl D93G ) (B), BL159 (ftsb D59H ftsl D93G ), and BL163 (ΔftsN ftsb D59H ftsl D93G ) (C), BL167 (ftsb E56A ), and BL173 (ΔftsN ftsb E56A ) (D), BL114 (ftsa I143L ), and BL18 (ftsa E124A ) (E), or TB28 (wt) with plasmids pch201 (P lac ::gfp-ftsn), pbl136 (P lac ::gfp-ftsn Δ(64-101) ), or pbl330 (P lac ::gfp-ftsn DY>SA ) (F). For panels B-E, cells were grown in LB ΔNaCl medium at 42 C for 3.5 hours before imaging. In panel A, cells were grown in LB medium at 30 C or in LB ΔNaCl medium at 42 C to OD 600 of around 0.6 as indicated. Cells in panel F were grown at 42 C in LB ΔNaCl medium supplemented with 100 µm IPTG till OD 600 of around 0.6. Bar equals 4 µm. 148

149 FIG. 16 Lethality of Δ E FtsN-suppressing mutations in ftsb and/or ftsl on LB ΔNaCl medium at 42 C in the presence of E FtsN. (A) Spot titer analyses of cells carrying Δ E FtsN-suppressing mutations in ftsb and ftsl. The genotypes of the strains tested are indicated on the right. Cells were grown overnight in LB medium 30 C, and resuspended in LB medium to an 149

150 optical density at 600 nm of , , , , , , , and Five microliters of each dilution was spotted on LB or LB DNaCl agar, and plates were incubated at 30 C or 42 C overnight, as indicated. (B) Spot titer analyses of BL173 (ΔftsN ftsb E56A ) cells carrying plasmids pmlb1113δh3 (vector control), pch201 (P lac ::gfp-ftsn), pbl136 (P lac ::gfp-ftsn Δ(64-101) ), or pbl330 (P lac ::gfp-ftsn DY>SA ). Cells were grown overnight in LB medium with 0.2% glucose at 30 C, and resuspended in LB medium to an optical density at 600 nm of , , , , , and Five microliters of each dilution was spotted on LB ΔNaCl agar, and plate was incubated at 42 C overnight. 150

151 FIG. 17 The model for the SR to initiate septal murein synthesis upon initiation of cell constriction. Shown are schematic overviews of the model for FtsN, the FtsBLQ subcomplex, and FtsA to trigger septal murein synthesis and cell constriction. The essential SR components, FtsZ (Z), FtsA (A), ZipA (ZipA), FtsB (B), FtsL(L), FtsQ (Q), FtsW (W), Fts(K), FtsI (PBP3), and FtsN (N) are shown with their topology relatively to the inner membrane (grey). The FtsBLQ subcomplex and FtsA are in their off state before accumulation of FtsN at the SR (A). FtsN stimulates the on state of the FtsBLQ subcomplex and FtsA through its essential domain ( E FtsN) and cytoplasmic domain ( N FtsN), respectively, leading to initiation of spg synthesis and Z-ring contraction (B). An alternative model for FtsA is shown in panel C, where the activated FtsA stimulates PBP3 through the FtsBLQ subcomplex or another route from the Z-ring to PBP3. The essential domain and cytoplasmic domain of FtsN are highlighted in red and purple, respectively. Arrows indicated the possible pathways for the activation signal. 151

152 152

153 FIG. 18 Domain analysis of DedD. (A) E. coli dedd locus, DedD domains, and properties of genetic constructs. The deletion-replacement in ΔdedD<>cat (ΔdedD) strains is shown above the dedd gene. The domain structure of DedD is illustrated below the dedd gene. Indicated are the transmembrane domain (TM), and the C-terminal SPOR domain (SPOR). Also shown are inserts on plasmids that produce fusions of various portions of DedD to RFP, GFP, TT GFP (Tat-targeted periplasmic GFP, hatched box), or the first transmembrane segment of MalF (MalF 2-39, M) under the control of the P lac or P BAD regulatory regions. Columns indicate the DedD residues present in each fusion, whether the fusion could fully (+++) or partially (+--) or could not (---) compensate for the absence of native DedD, and whether it accumulated at constriction sites sharply (+++) or poorly (+--) or appeared evenly distributed in the cytosol (Cyt) in BL40 (ΔdedD ΔdamX) cells. (B-H ) Shown are live cells of strain BL40 harboring plasmid pmlb1113δh3 (vector control) (B), pfb236 (P lac ::gfp-dedd ) (C and C ), pbl95 (P lac ::gfp-dedd 1-54 ) (D and D ), pbl33 (P lac ::gfp-malf dedd ) (E and E ), ppc1 (P lac ::gfp-dedd ) (F and F ), pbl37 (P lac ::gfp-dedd 1-35 ) (G and G ), or pbl101 (P lac ::gfp-dedd rfp) (H and H ). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 50 µm IPTG. The squares in panel C-H correspond to the fluorescent images in panel C -H. Bar equals 4 µm (B, C, D, E, F, G, H), or 2 µm (C, D, E, F, G, H ). 153

154 FIG. 19 Septal localization of GFP-DedD depends on E FtsN and FtsI. (A to B ) E FtsN-dependent septal localization of GFP-DedD. Overnight culture of CH34/pMG20/pFB236 (ΔftsN/P BAD :: TT bfp-ftsn /P lac ::gfp-dedd) grown in M9-maltose medium containing 0.01% arabinose was washed twice 154

155 before it was diluted 100-fold to fresh M9-maltose medium containing 0.01% arabinose (A and A ) or glucose (B and B ). Cells were imaged at OD 600 of 0.5. (C to D ) FtsI-dependent septal localization of GFP-DedD. An overnight culture of JE7947/pBL87/pFB236 (ΔftsI/cI857 λp R ::ftsi/p lac ::gfp-dedd) grown at 37 C in LB was diluted 200-fold to fresh LB medium containing 10 µm IPTG. In panels C and C, cells were grown at 37 C to OD 600 of 0.5. For FtsI depletion, cells were grown at 37 C for 2.5 hrs and then shifted to 30 C for another 3 hrs when OD 600 reached 0.5 (D and D ). Bar equals 4 µm. 155

156 FIG. 20 Conserved residues in TM DedD are important for DedD s function in cell division. 156

157 (A) A sequence logo representing sequence alignments of DedD. DedD sequences representing every genus of Gammaproteobacteria listed in the OMA groups and were used for sequence alignment. The E. coli K12 DedD residue sequence is given underneath the sequence logo. The transmembrane domain is underlined. Conserved residues mutated in this study are in orange. (B-E ) Shown are live cells of strain BL40(iBL345) [ΔdedD ΔdamX (P lac ::gfp-dedd )] (B and B ), BL40(iBL345)(G11A) [ΔdedD ΔdamX (P lac ::gfpdedd 1-118, G11A )] (C and C ), BL40(iBL345)(P24A) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, P24A )] (D and D ), and BL40(iBL345)(P42G) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, P42G )] (E and E ). Cells were grown to OD 600 = at 30 C in M9-maltose medium supplemented with 250 µm IPTG. The squares in panel B-E correspond to the fluorescent images in panel B -E. Bar equals 4 µm (B, C, D, E), or 2 µm (B, C, D, E ). 157

158 FIG. 21 Western analyses of GFP-DedD fusions in BL40 cells. Whole cell extracts of BL40 cells harboring plasmid pfb236 (lane 1), ppc1 (lane 2), pbl95 (lane 3), pbl33 (lane 4), pbl37 (lane 5), pbl101 (lane 6) or pmlb1113 ΔH3 (lane 7) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-gfp antibodies. Cells were grown at 30 C in LB medium supplemented with 50 µm IPTG to OD600= before extraction preparation. Each lane received equivalent amounts of cell material. The bands for intact DedD fusions are indicated by arrowheads and their relative intensities are indicated below each lane (GFP-DedD band as 100). The positions of 250, 150, 100, 75, 50, 37, 25, and 20 kd (top to bottom) migration standards are 158

159 indicated to the left of the panel. FIG. 22 Western analyses of GFP-DedD mutants in BL40 cells. Whole cell extracts of cells from strains BL40(iBL345) [ΔdedD ΔdamX (P lac ::gfp-dedd )] (lane 1), BL40(iBL345)(G11A) [ΔdedD ΔdamX (P lac ::gfpdedd 1-118, G11A )] (lane 2), BL40(iBL345)(G11C) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, G11C )] (lane 3), BL40(iBL345)(P24A) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, P24A )] (lane 4), or BL40(iBL345)(P42G) [ΔdedD ΔdamX (P lac ::gfp-dedd 1-118, P42G )] (lane 5) were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-gfp antibodies. Cells were grown at 30 C in M9 medium supplemented with 250 µm IPTG to OD600= before extraction preparation. Each lane 159

160 received equivalent amounts of cell material. The bands for intact GFP-DedD variants are indicated by the arrowhead. The positions of 250, 150, 100, 75, 50, 37, 25, and 20 kd (top to bottom) migration standards are indicated to the left of the panel. 160

161 FIG. 23 More E FtsN activity is required for cell division in the absence of DedD. 161

162 (A) Spot titer analyses of strain MG19/pMG39 (ΔdedD ftsn slm117 /P BAD ::dedd) harboring plasmids pfb236 (P lac ::gfp-dedd ), pbl33 (P lac ::gfp-malf dedd ), ppc1 (P lac ::gfp-dedd ), pbl95 (P lac ::gfp-dedd 1-54 ), pbl37 (P lac ::gfp-dedd 1-35 ), or pmlb1113 ΔH3 (vector). Cells were grown overnight in LB medium with 0.5% arabinose, and resuspended in LB medium to an optical density at 600 nm of , , , and Five microliters of each dilution was then spotted on LB agar containing 0.5% Arabinose, 0.2% Glucose, or 50 µm IPTG, as indicated, and plates were incubated at 30 C overnight. (B) Spot titer analyses of strains CH34(iMG59) [ΔftsN(P lac ::gfp-ftsn)], CH34(iMG62) [ΔftsN(P lac ::gfp-ftsn )], CH34(iMG63) [ΔftsN(P lac ::gfp-ftsn )], BL73(iMG39) [ΔftsN ΔdedD(P lac ::gfp-ftsn)], BL73(iMG62) [ΔftsN ΔdedD(P lac ::gfp-ftsn )], and BL73(iMG63) [ΔftsN ΔdedD(P lac ::gfp-ftsn )]. Cells were grown overnight in M9-maltose with 5 (ΔftsN strains) or 250 (ΔftsN ΔdedD strains) µm IPTG, and resuspended in M9- maltose to an optical density at 600 nm of , , , , , and Five microliters of each dilution was then spotted on LB agar containing 0, 50, or 500 µm IPTG, as indicated, and plates were incubated at 30 C overnight. (C) Shown are live cells of strain TB77 (ftsn slm117 ) harboring pmlb1113δh3 (vector control, -) or pfb236 (P lac ::gfp-dedd, +). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 50 µm IPTG. (D) Shown are live cells of strain BL40 (ΔdedD ΔdamX) harboring pmlb1113δh3 (vector control, -) or pch201 (P lac ::gfp-ftsn, +). Cells were grown 162

163 to OD 600 = at 30 C in LB medium supplemented with 50 µm IPTG. Bar equals 4 µm (C and D). 163

164 FIG. 24 Western analyses of DedD and FtsN fusions in ΔdedD ftsn slm117 cells. (A) Western analyses of DedD fusions in MG19/pMG39 (ΔdedD ftsn slm117 /P BAD ::dedd) strains harboring pfb236 (P lac ::gfp-dedd ) (lanes 1 and 2), ppc1 (P lac ::gfp-dedd ) (lanes 3 and 4), pbl33 (P lac ::gfp-malf dedd ) (lanes 5 and 6), pbl95 (P lac ::gfp-dedd 1-54 ) (lanes 7 and 8), or pmlb1113 ΔH3 (vector) (lane 9). (B) Western analyses of FtsN fusions in MG19/pMG39 (ΔdedD ftsn slm117 /P BAD ::dedd) strains harboring pch201 (P lac ::gfp-ftsn ) (lanes 1 and 2), pmg12 (P lac ::gfp-ftsn ) (lanes 3 and 4), pch310 (P lac ::gfpmalf ftsn ) (lanes 5 and 6), pbl128 (P lac ::gfp-dedd ftsn ) (lanes 7 and 8), or pmlb1113δh3 (vector) (lane 9). Whole cell extracts were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-gfp antibodies. Prior to extraction preparation, cells were grown at 30 C in LB medium containing 50 µm IPTG and with (lanes 1, 3, 5, 7, and 9) or without (lanes 2, 4, 6, and 8) 0.5% arabinose to OD600= Each lane received 40 µg of total protein. The positions of 66, 45, 36, 29, and 24 kd (top to bottom) migration standards are indicated to the left of the panel. 164

165 FIG. 25 Mutations in TM DedD affect interactions between N DedD and FtsL. Strain BTH101 (cya-99) was cotransformed with plasmid pairs encoding the indicated T18- and T25-fusions, and individual colonies were streaked on M9 plates containing 0.2% glucose, 40 µg/ml X-Gal and 250 µm IPTG. Plates were imaged after 43 hour incubation at 30 C. 165

166 FIG. 26 BATCH analyses for DedD and FtsN. Strain BTH101 (cya-99) was cotransformed with plasmid pairs encoding the indicated T18- and T25-fusions, and individual colonies were streaked on M9 plates containing 0.2% glucose, 40 µg/ml X-Gal and 250 µm IPTG. Plates were imaged after 43 hour incubation at 30 C. 166

167 FIG. 27 Combining ΔdedD with ΔponB leads to massive cell lysis. (A) Shown are BL83 (ΔdedD ΔponB) cells from an overnight culture grown at 30 C in LB medium. Bar equals 2 µm. (B) Growth curves of strain TB28 (wt), LP11 (ΔponB), MG14 (ΔdedD), BL83 (ΔdedD ΔponB), and BL102(iBL198) [ΔdedD ΔponB(P lac ::gfp-dedd)]. Overnight cultures in LB were diluted to an optical density at 600 nm of 0.02 in LB, or LB supplemented with 100 µm IPTG [BL102(iBL198) 100µM IPTG]. Cells were grown in 96-well plates with 167

168 continuous shaking at 30 C. Optical density at 600 nm was measured every 20 minutes for 10 hours. Error bar denotes standard deviation (n =3). (C) Shown are DIC images of BL83 cells taken at various time points after dilution in fresh medium. An overnight culture of BL83 in LB was diluted 200-fold in fresh LB and live cells were imaged at one-hour intervals. Bar equals 4 µm. (D) Shown are DIC images of BL84(iMG62) [ftsn slm117 ΔponB(P lac ::gfp-ftsn )] cells grown in LB medium containing 0.1% glucose (-) or 500 µm IPTG (+). Bar equals 4 µm. 168

169 FIG. 28 Model for E FtsN and N DedD in the regulation of spg synthesis. We propose that E FtsN and N DedD activate two independent targets (T1 and T2, respectively) at the SR, ultimately leading to septal murein (spg) synthesis. spg is processed by murein hydorlases, most prominently the murein amidases, AmiA, AmiB, and AmiC (controlled by EnvC and NlpD), to form the polar PG for the daughter cells. Duing spg processing, a transient form of spg is generated, providing the substrate for the SPOR domains of FtsN and DedD. Accumulation of FtsN and DedD molecules at the SR driven by their SPOR domains leads to increased E FtsN and N DedD activities at the SR and sustained spg synthesis. 169

170 FIG. 29 Massive cell lysis in ΔdedD ΔponB cells is suppressed by PBP1B or DedD. (A) Shown are live cells of strain BL102(iLP32-cat) [ΔdedD ΔponB(P lac ::gfp-pbp1b)]. Cells were grown to OD 600 = at 30 C in LB medium supplemented with 100 µm IPTG. (B) Shown are live cells of strain BL102(iBL198) [ΔdedD ΔponB(P lac ::gfp-dedd)]. Cells were grown to OD 600 = at 30 C in LB medium supplemented with 25 µm IPTG. (C and D) Shown are live cells of strain BL24 (ΔponB, C) or TB28 (wt, D) treated with cephalexin. Cells were grown to OD 600 =0.3 at 30 C in LB medium. Cephalexin was added to 15 µg/ml and cells were imaged 1hr after cephalexin. Bar equals 4 µm. 170

171 FIG. 30 Toxicity of excess FtsQ, B, or L molecules in ΔdedD cells. (A) Shown are live cells of strain MG14 (ΔdedD) or TB28 (wt) harboring pch195 (P lac ::gfp-ftsl). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 0, 10, or 100 µm IPTG. Bar equals 4 µm. (B) Spot titer analyses of TB28 (wt), MG14 (ΔdedD), BL40 (ΔdedD ΔdamX), and TB77 (ftsn slm117 ) harboring pmlb1113 (vector control), pch196 (P lac ::gfp-ftsq), 171

172 pch195 (P lac ::gfp-ftsl), or pbl193 (P lac ::gfp-ftsb). Cells were grown overnight in LB with 0.1% glucose, and resuspended in LB to an optical density at 600 nm of , , , , , , and Five microliters of each dilution was then spotted on LB agar with or without 500 µm IPTG, as indicated, and plates were incubated at 30 C overnight. 172

173 FIG. 31 Suppressing the toxicity of excess FtsQ in ΔdedD cells. Spot titer analysis of MG14/pBL221 (ΔdedD/P BAD ::ftsq) harboring various pmlb plasmids expressing various GFP fusion proteins. Cells were grown overnight in LB with 0.1% glucose, and resuspended in LB to an optical density at 600 nm of , , , , , , , and Five microliters of each dilution was then spotted on LB agar supplemented with arabinose and/or IPTG, as indicated, and plates were incubated at 30 C overnight. 173

174 FIG. 32 Overexpression of FtsQ inhibits cell division in the absence of DedD. 174

175 (A) Shown are live cells of strain TB28 (wt) and MG14 (ΔdedD) haboring pch196 (P lac ::gfp-ftsq). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 100 µm IPTG. The average cell length per GFP-FtsQ ring is indicated on the right. (B) Overproduction of FtsQ induces mislocalization of GFP-FtsN in ΔdedD cells. Shown are live cells of strain MG14/pBL221/pCH201 (ΔdedD/P BAD ::ftsq /P lac ::gfp-ftsn). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 25 µm IPTG and with (+) or without (-) 0.2% arabinose. The squares in the DIC images correspond to the fluorescent images. Bar equals 4 µm. 175

176 FIG. 33 Z-ring formation is not affected by overexpression of GFP-FtsQ in the absence of DedD. Shown are live cells of strain BL132 (ΔyfeN zipa-rfp-zipa ΔdedD) harboring pch196 (P lac ::gfp-ftsq). Cells were grown to OD 600 = at 30 C in LB medium supplemented with 100 µm IPTG. Bar equals 4 µm. 176

177 FIG. 34 Overexpression of certain septal ring proteins is toxic to ΔdedD cells. Spot titer analysis of TB28 (wt) or MG14 (ΔdedD) harboring plasmids expressing various untagged septal ring proteins, as indicted. Cells were grown overnight in LB with 0.1% glucose, and resuspended in LB to an optical density at 600 nm of , , , , , , , and Five microliters of each dilution was then spotted on LB agar 177

178 supplemented with 100 or 500 µm IPTG, as indicated, and plates were incubated at 30 C overnight. 178

179 FIG. 35 FtsB E56A suppresses the division defects in ΔdedD cells. Shown are live cells of strain MG14 (ΔdedD) (left) and BL177 (ΔdedD ftsb E56A ) (right). Cells were grown to OD 600 = at 30 C in LB medium. Bar equals 4 µm. 179

180 FIG. 36 The model for E FtsN and N DedD to initiate septal murein synthesis upon initiation of cell constriction. Shown are schematic overviews of the model for E FtsN and N DedD to trigger septal murein synthesis. SR components, FtsZ (Z), FtsA (A), ZipA (ZipA), FtsB (B), FtsL(L), FtsQ (Q), FtsW (W), Fts(K), FtsI (PBP3), DedD and FtsN (N) are shown with their topology relatively to the inner membrane (grey). E FtsN and N DedD are highlighted in red and magenta, respectively. E FtsN and N DedD may activate PBP3 and/or FtsW indirectly via stimulating the on state of FtsBLQ subcomplex, which then triggers spg synthesis (A). Alternatively, E FtsN and N DedD may directly interact with PBP3 and/or FtsW to stimulate spg synthesis together with the FtsBLQ subcomplex (B). Arrows indicated the possible pathways for the activation signal. 180