Supplemental Data. Bacterial Birth Scar Proteins Mark Future Flagellum Assembly Site

Transcription

1 Supplemental Data Bacterial Birth Scar Proteins Mark Future Flagellum Assembly Site Edgar Huitema, Sean Pritchard, David Matteson, Sunish Kumar Radhakrishnan, and Patrick H. Viollier - 1 -

2 Figure S1. Z-Ring Constriction Is Required for Localization of TipN-GFP and TipF-GFP to the Division Site Early log phase cultures of tipf-gfp and tipn-gfp, treated with or without the FtsI inhibitor cephalexin, were grown for 150 (left panels) and 480 min (right) before being analyzed by DIC (left) and GFP (right) microscopy

3 Table S1. Analysis of Flagella in Wild Type, tipf and tipn Mutants by Transmission Electron Microscopy (TEM) Strain (Genotype) SW Pole ST Pole ST Tip ST Body Cell Body Total Cells w/ Flagella Total Cells NA1000 (wild type) 52 (100) (31) 166 NR1751 ( tipn) 205 (63) 29 (8) 29 (8) 61 (18) 6 (2) 326 (40) 824 NS142 (tipn::eztn5) 58 (80) 2 (3) 3 (4) 9 (12) 1 (1) 73 (47) 155 NS250 (tipn::hypermu) 54 (82) 4 (6) 3 (5) 5 (8) 0 66 (39) 170 NS267 (tipn::hypermu) 42 (69) 2 (3) 3 (5) 6 (10) 8 (13) 61 (44) 137 NR1584 ( tipf) 4 (100) (1) 344 NS46 (tipf::himar1) 2 (100) (1) 167 NS187 (tipf::himar1) (0) 176 The number of cells is displayed for each mutant, and the percentages are shown in brackets

4 Table S2. Stalk Analysis in Wild Type, tipf, tipn and tipn tipf Mutants by TEM Strain (Genotype) Total Cells Cells w/ Stalks Bistalked (Pole/Pole) Bistalked (Pole/Cell) NA1000 (wild type) (50) 0 0 NR1751* ( tipn) (76) 54 (15) 0 NR1584 ( tipf) (81) 1 0 NR1705 ( tipn; tipf) (77) 88 (15) 9 (2) *30% of the bistalked cells were unpinched and short (in the stalked cell stage or very early predivisional cell stage) Cell counts scored for the presence of two stalks. In parentheses are percentages. Note that there is a high incidence of stalks in the mutant cells possibly due to the filamentation defect associated with the loss of TipF and/or TipN. Stalked cells were subdivided into different classes with the percentages reflecting the fraction of the total cells with a stalk

5 Table S3. Localization of TipF-GFP in Wild Type and tipn Cells Strain (Genotype) NR1216 (NA1000; tipf-gfp) NR1760 ( tipn; tipf-gfp) Flagellar Pole Multiple & Mislocalized Foci Stalk Total Cells

6 Table S4. Localization of FliG-GFP in Wild Type (NA1000), tipf and tipn Cells Strain (Genotype) NR1744 (NA1000; xylx:: P xyl -flig-gfp) NR1740 ( tipf; xylx:: P xyl - flig-gfp) NR1770 ( tipn; xylx:: P xyl - flig-gfp) Flagellar Pole Bipolar Foci at Cell Body Stalk Total Cells DIC images as well as fluorescence images were used to score cells for localization of FliG-GFP to either the flagellated pole, both poles, the cell body, or the stalk

7 Table S5. CheA-GFP and PleC-GFP Localization in Wild Type (NA1000), tipn and tipf Mutant Strains Strain (Genotype) NR1212 (NA1000; chea-gfp NR1805 ( tipf chea-gfp NR1806 ( tipn; chea-gfp) PV760 (NA1000; plec-gfp) NR1885 ( tipf; plec-gfp) NR1792 ( tipn; plec-gfp) Flagellar Pole Bipolar Plane of Division Stalk Total Cells DIC images as well as fluorescence images were used to score cells for localization of CheA-GFP and PleC-GFP to either the flagellated pole, both poles, clustered near, or at the plane of cell division and accumulation in the stalk

8 Table S6. Localization of DivJ-GFP in Wild Type, tipn and tipf Mutant Strains Strain (Genotype) LS3200 (NA1000; divj-gfp) NR1890 ( tipf; divj-gfp) NR1891 ( tipn; divj-gfp) Flagellar Pole Bipolar Double Focus Stalked Pole Total Cells Experimental Procedures Strain Constructions The tipn, tipf, tipf , tipf , and tipf strains were constructed by creating an in-frame deletion of the sequence encoding residues of TipN, , , and of TipF with plasmids php001, pcwr207, php002, php003 and php004, respectively, using the standard two-step recombination sucrose-counterselection procedure. To construct the tipn tipf double mutant, the tipn deletion was created in the tipf mutant background. To construct the tipn-gfp and tipf-gfp strains in which 3` end of the endogenous tipn or tipf gene was replaced with one to which the gfp gene had been appended. Plasmids - 8 -

9 pspx47 and pcwr169 that harbor the respective fragment and confer resistance to kanamycin were integrated by a one-step homologous recombination into NA1000. Plasmids pp xyl ::ParB N40 (Figge et al., 2003) or pp xyl ::ftsz (also known as puj142ftsz) (Din et al., 1998) were mobilized into Caulobacter by intergeneric conjugation from E. coli S17-1 (Ely, 1991). Exconjugants were isolated on PYE plates containing nalidixic acid (20 µg/ml) and glucose (0.2%). tipn-gfp and tipf-gfp cells containing pp xyl ::ParB N40 or pp xyl ::ftsz were grown to log phase, washed and re-suspended in PYEX (PYE supplemented with 0.3% xylose) or PYEG (PYE supplemented with 0.2% glucose). YB1585 (NA1000 ftsz::p xyl -ftsz) (Wang et al., 2001) was transduced with lysates from a strain bearing tipf-gfp or tipn-gfp allele marked with the apramycin resistance gene aac(3)iv (Blondelet-Rouault et al., 1997). These strains were created by ligating aac(3)iv on a SmaI fragment into the HpaI site of pspx47 and pcwr169 and transforming the resulting plasmids into NA1000. To make the CheA-GFP reporter strain, plasmid pcwr178 was introduced into NA1000, selecting for kanamycin resistant transformants. The reporter was subsequently transduced into NA1000, the tipf and the tipn mutant. Chromosomal PleC-GFP and DivJ-GFP reporter constructs were transduced from strain PV760 (Viollier et al., 2002) and LS3200 (Wheeler and Shapiro, 1999), respectively, into NA1000, the tipf and the tipn mutant

10 NA1000 harboring a translational fljk-lacz translational reporter, pfljk::lacz (Mangan et al., 1999), integrated at the fljk locus served as host for a phage lysate made from the generalized transducing phage ΦCr30. This lysate was transduced into NA1000 and the tipf mutant. The transcriptional fljk-lacz reporter plasmid, pfljklacz/290 (Wingrove et al., 1993), was transformed into NA1000 and the tipf mutant. The tipf mutation was introduced into the flbt650 (Johnson and Ely, 1979) mutant using plasmid pcwr207. Plasmids pcwr234 (tipf-h6) and pcwr235 (tipn-h6) were transformed into NA1000, yielding strains NR1904 and NR1905, respectively. The resulting strains were then transduced with a lysate from YB1585 and used in coimmunoprecipitation studies described below. The TipF-GFP and TipN-GFP reporter were introduced into strain CS606 for the cephalexin experiments, yielding NR1382 and NR1371, respectively. Plasmid Construction KOD thermostable DNA polymerase (EMD Biosciences, San Diego, CA) or PFU Turbo (Stratagene, La Jolla, CA) was used for amplifications using the polymerase chain reaction. The tipf and tipn deletion construct was made in

11 pnpts138 (M.R.K. Alley, unpublished) through amplification of a bp fragment upstream from the tipf start codon and extending 120 bp into the predicted open reading frame, and a 3 region containing the last 36 bp codons of tipf and extending 660 bp downstream of the gene. Amplification resulted in introduction of BamHI and HindIII for the 5 and BamHI and EcoRI sites for the 3 fragment, respectively. Similarly, a 599 bp 5 fragment and an 810 bp 3 fragment were amplified from the tipn locus and used to delete residues of TipN. The appropriate pairs of fragments were restricted and ligated into EcoRI and HindIII digested pnpts138 vector to generate pcwru207 ( tipf) and php001 ( tipn) respectively. A pnpts138 derivative harboring the tipf EAL allele to delete the sequences encoding residues that comprise the predicted EAL domain was also constructed in an identical approach. pspx47 (tipn-gfp) and pcwr169 (tipf-gfp) were made by cloning the 3 end of tipn and tipf (nucleotides of AE and of AE005746, respectively) as XbaI/BamHI fragments into SpeI/BamHI-restricted pxgfp5 (M.R.K. Alley, unpublished) and NheI/BamHI-restricted pxgfp4 (M.R.K. Alley, unpublished) respectively. The resulting plasmids, pspx47 and pcwr169, were cleaved with HpaI and ligated to a SmaI fragment harboring aac(3)iv (Blondelet- Rouault et al., 1997). To construct the P xyl -flig-gfp plasmid pcwr71, the flig coding sequence lacking the stop codon (nucleotides of AE005767) was cloned into pxgfp4 using NdeI/BamHI

12 To make pcwr178 (chea-gfp) the 3 end of the chea gene (nucleotides of AE005716) was amplified as XbaI/BamHI fragment and cloned into NheI/BamHIrestricted pxgfp4. The resulting plasmid was cleaved with HpaI and ligated to a SmaI fragment harboring aac(3)iv, yielding pcwr178. Plasmids pcwr234 (tipf-h6) and pcwr235 (tipn-h6) were made by cloning PCR fragments containing the 3 end of tipf or tipn, respectively, into XbaI/EcoRI.- restricted phpv465 (Viollier et al., 2004). These fragments were amplified using the same upstream primer as for pspx47 and pcwr169 and a different downstream primer fusing a sequence encoding HHHHHHGYKDDDDK-tag to the penultimate codon of either tipn or tipf. Construction of pcwr208 (P xyl -tipf) and pcwr239 (P xyl -tipfe211a) involved several steps. The tipf coding sequence (nt of AE5746) was PCR amplified with primers that introduced an NdeI restriction site overlapping the start codon (changed from TTG to ATG) and an EcoRI site after the stop codon. The fragment was cloned into pok12 (Vieira and Messing, 1991) using the restriction sites in the primers, yielding pcwr206. The tipf fragment was excised using NdeI/EcoRI and ligated along with the xylx promoter (P xyl ) fragment isolated from prw432 (R. Wright, unpublished) using SpeI/NdeI into the low-copy vector plac290 (Wingrove et al., 1993) that had been restricted with XbaI and EcoRI. The resulting plasmid, pcwr208, harboring P xyl tipf, was used in the complementation studies with the tipf mutant

13 The tipf(e211a) mutant allele was made by inverse PCR using pcwr206 as a template along with primers that changed the codon for glutamate (GAG) at position 211 to one encoding alanine (GCG). The resulting plasmid was named pcwr226. The mutant tipf allele was placed under P xyl control in plac290 using the same strategy as the one used for construction of pcwr208. The resulting P xyl - tipf(e211a) plasmid was named pcwr239. Analysis of the TipF and TipN Primary Amino Acid Sequence The DAS ( and the Coils ( server were used for prediction of transmembrane segments and coiled-coils in TipF and TipN. Expression of Wild Type TipF and TipF (E211A) To test if the tipf(e211a) allele could correct the phenotypes of the tipf mutant, pcwr239 and pcwr208 were introduced into strain NR1584 and cultivated in PYEG. Under these conditions, NR1584/pCWR208 swim, have polar flagella, and secrete FljK and FlgE. Under the same conditions, NR1584/pCWR239 cells are nonmotile, lack external flagellar structures, and do not secret FljK and FlgE. Transposon Mutagenesis and Isolation of tipn and tipf Mutants To maximize genome-wide coverage of transposon mutagenesis, we used four different mobile elements (1) HyperMu <R6Kγori/KAN-1> (Epicentre, Madison,

14 WI); (2) EZ-Tn5 <R6Kγori/KAN-2> (Epicentre); (3) the Himar1-derived mariner transposon (Viollier et al., 2004); and (4) the Tn5 IS50L-derived ISlacZ/hah transposon (Jacobs et al., 2003) containing an outwardly directed promoter to create nonpolar insertions collecting a library of ~17,000 mutants arrayed in 96-well plates. This library was replica stamped on swarm agar plates to screen for mutants with motility defects. A total of 194 mutants were identified, and the site of the insertion of each transposon was mapped by first recovering HinP1I partiallyrestricted neighboring chromosomal sequences as plasmids that transformed E. coli EC100D-pir116 (Epicentre) to kanamycin resistance and then by sequencing across the junction. Five mutants (NS46, NS142, NS187, NS250, NS267) were chosen for further study. References Blondelet-Rouault, M. H., Weiser, J., Lebrihi, A., Branny, P., and Pernodet, J. L. (1997). Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. Gene 190, Din, N., Quardokus, E. M., Sackett, M. J., and Brun, Y. V. (1998). Dominant C- terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA. Mol Microbiol 27, Ely, B. (1991). Genetics of Caulobacter crescentus. Methods Enzymol 204, Figge, R. M., Easter, J., and Gober, J. W. (2003). Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol Microbiol 47, Jacobs, M. A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., et al. (2003). Comprehensive transposon

15 mutant library of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 100, Johnson, R. C., and Ely, B. (1979). Analysis of nonmotile mutants of the dimorphic bacterium Caulobacter crescentus. J Bacteriol 137, Mangan, E. K., Malakooti, J., Caballero, A., Anderson, P., Ely, B., and Gober, J. W. (1999). FlbT couples flagellum assembly to gene expression in Caulobacter crescentus. J Bacteriol 181, Vieira, J., and Messing, J. (1991). New puc-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, Viollier, P. H., Sternheim, N., and Shapiro, L. (2002). A dynamically localized histidine kinase controls the asymmetric distribution of polar pili proteins. Embo J 21, Viollier, P. H., Thanbichler, M., McGrath, P. T., West, L., Meewan, M., McAdams, H. H., and Shapiro, L. (2004). From The Cover: Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. PNAS 101, Wang, Y., Jones, B. D., and Brun, Y. V. (2001). A set of ftsz mutants blocked at different stages of cell division in Caulobacter. Mol Microbiol 40, Wheeler, R. T., and Shapiro, L. (1999). Differential localization of two histidine kinases controlling bacterial cell differentiation. Mol Cell 4, Wingrove, J. A., Mangan, E. K., and Gober, J. W. (1993). Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter. Genes Dev 7,