Fig. S1. CrgA intracellular levels in M. tuberculosis. Ten and twenty micrograms of

Transcription

1 Supplementary data Fig. S1. CrgA intracellular levels in M. tuberculosis. Ten and twenty micrograms of cell free protein lysates from WT M. tuberculosis (Rv) together with various known concentrations of purified CrgA were resolved on a 15% NuPAGE gel, transferred to PVDF membrane and probed with α-crga antibodies. CrgA bands were quantitated by QuantityOne software. A standard curve was prepared using pure protein standards and used to determine the CrgA levels in the lysates. Arrowhead CrgA protein.

2 Fig. S2. (i) FtsZ levels in M. tuberculosis crga strains. WT, crga overexpression and crga antisense strains were grown for 48 hrs with 100 ng/ml anhydrotetracycline and cells processed for immunoblotting using indicated antibodies. (ii) CrgA levels do not change upon FtsZ depletion. M. smegmatis ftsz, Pami::ftsZ, Ptet::ecfp-crgA was grown with100 ng/ml anhydrotetracycline and without and with 0.2% acetamide for 6 hrs. Bacteria were pelleted by centrifugation and analyzed by immunoblotting using antibodies to indicated proteins.

3 Fig. S3. (A) FtsI and FtsZ levels in M. smegmatis WT and crga strains expressing Pami::gfp-ftsI. M. smegmatis strains were grown as described in the text and processed for immunoblotting. Blots were probed with antibodies to the indicated proteins as described in the text. FtsI and FtsI-GFP were probed with α-ftsi antibodies. - CrgA overproduction strain; Δ crga crga mutant strain. (B) Complementation of crga mutant. M. smegmatis WT (i), Δ crga (ii) and Δ crga PcrgA::crgA (iii) and Δ crga Pet::ecfp-crgA (iv and v) were grown in 7H9-AD and examined by brightfield (i - iv) and fluorescence (v) microscopy. Δ crga Pet::ecfp-crgA was grown with 100 ng/ml anhydrotetracycline. Black arrowheads swollen cells. White arrowheads ECFP-CrgA localization at midcell or quarter cell positions. White arrows polar localization of ECFP-CrgA. Bar 5 µms.

4 Fig. S4: (A) CrgA interactions with PBPB, PBPA and FtsQ are specific: crga, pbpa, ftsq, clpx and roda fusions to the T25 or T18 fragments of adenylate cyclase were cloned in various BACTH vectors (Table 1) and interactions examined as described in Fig. 4A legend. E. coli BTH101 recombinants bearing indicated combinations of plasmids were plated on LB agar containing IPTG and X-gal. Blue colonies indicate strong positive interactions

5 (Inset). RodA-PbpA is a positive interaction. Mean ± SD from 3 independent experiments are shown. (B) (i). Tag-free FtsZ and CrgA do not bind non-specifically to cobalt affinity resin. Tag-free purified FtsZ and CrgA were mixed together and allowed to bind cobalt affinity resin and bound proteins eluted as described for Fig. 4B (i). Load (L), wash (W) and elution (E) fractions were resolved on SDS-PA followed by immunoblotting with indicated antibodies. (ii). N-terminal 1-51 aa of CrgA are sufficient for interaction with FtsZ. Purified His-FtsZ was mixed with purified 1-51 aa CrgA protein and pull-down assay performed using cobalt affinity resin as described for Fig. 4A. Load (L), wash (W) and elution (E) fractions were resolved on SDS-PA followed by immunoblotting with indicated antibodies.

6 Fig. S5. (A) N-terminal 1-51 aa are sufficient for localization of CrgA to the septal sites. ECFP-CrgA or ECFP- C-term-CrgA fusion was localized in wild type M. smegmatis as described under Fig. 3A. CrgA domains cartoon: I and O are cytoplasmic and extracytoplasmic regions; T1 and T2 are 1 st and 2 nd transmembrane domain. Arrowhead midcell localization. Bar 5 µm. (B) Bacterial 2-hybrid assays with truncated FtsI and CrgA proteins. Shown BACTH assays were carried out as described for Fig. 4A. See table 1 for plasmid descriptions. FtsI679 is full-length FtsI. FtsI124 and FtsI298 are FtsI N-terminal and aa proteins, respectively. CrgA1-51aa is CrgA N-terminal 1-51 aa protein. GCN4-GCN4 is positive control.

7 Fig. S6. Reverse-phase HPLC profile of peptidoglycan from WT or CrgA overproducing M. smegmatis strains. Cells from overnight cultures of both strains were mechanically disrupted, boiled in SDS and digested with proteases and muramidases as described (Lavollay et al. 2008). Muropeptides were reduced with Sodium borohydrate and injected onto C18 nucleosil column and separated in H 2 O+TFA with a Acetonitrile Gradient (0-20%) (Table S1; see also Lavollay et al for more details). Table S1: Muropeptide composition A B C D Tripeptide Tetrapeptide -GlcNac Tetrapeptide Peptide contaminant m=522 (always here) E Tri-Tetra -GlcNAc 3-3 crosslink (L,D-transpeptidation) F Tri-Tri 3-3 crosslink (L,D-transpeptidation) G Tri-Tetra 3-3 crosslink (L,D-transpeptidation) H Tetra-Tetra 4-3 crosslink (D,D-transpeptidation) I Peptide contaminant m=679 (always here) J Anhydro Tri-Tetra 3-3 crosslink (L,D-transpeptidation)

8 Table S2: Oligonucleotide used in the study Oligo Name Sequence (5 -> 3 ) Plasmid/ gene Reference CrgA-PacI AGAACCTTAATTAAGAGCCCCACCAGGGAGGAA GCCGAACGATGCCCAAGTCCAAGGTCCG forward for crga in pds5 and ppp91 CrgA-SwaI ATCGGATTTAAATATCAATGCCAGCGCATCGTGA reverse for crga pds5, ecfp fusion in ppp93 CFP-PacI F AACCTTAATTAAGAGCCCCCACCAGGGAGGAAG CCGAACGATGGTGAGCAAGGGCGAGGA forward for ecfp CFP-BamHI CGGGATCCCTGCAGGTTGTTGTTCTTGTACAGCT CGTCCATG reverse ecfp

9 CrgA-BamHI GCGGATCCATGCCCAAGTCCAAGGTCCG forward for crga in pmal and ecfp fusion CrgA-end- HindIII GAGCCCAAGCTTTCATCAATGCCAGCGCATCGTGA reverse for crga in pmal acrga-paci AGAACCTTAATTAAGAGCCCCACCAGGGAGGAA GCCGAACGTCAATGCCAGCGCATCGTGA forward for crga Mtb antisense acrga-swai ATCGGATTTAAATATGCCCAAGTCCAAGGTCCG reverse for crga Mtb antisense CrgA-BamHI TCTAGAGGATCCCATGCCCAAGTCCAAGGTCCGC forward for crga BACTH CrgA-KpnI TTACTTAGGTACCCGATGCCAGCGCATCGTGAGCA reverse for crga BACTH Yfp XbaI TATAGTCTAGATTATTACTTGTACAGCTCGTCCA reverse for ftsz-yfp

10 FtsZ-NdeI CAGCCATATGACCCCCCCGCACAACTACC forward for ftsz-yfp PBPA- BamHI TCTAGAGGATCCCATGAACGCCTCTCTGCGCCGA forward for pbpa BACTH PBPA-KpnI TTACTTAGGTACCCGTGGTTCCCCCTGCAGTGCGGC reverse for pbpa BACTH RodA-XbaI TTGCTCTAGAGATGACGACACGACTGCAAGC forward for roda BACTH RodA-KpnI TTACTTAGGTACCCGTACGCGCCTGATGACCTCGG reverse for roda BACTH FtsI-BamHI GCGGATCCGTGAGCCGCGCCGCCCCCAG forward for gfp-ftsi FtsI298- XbaI TTGCTCTAGATTACTATTGGCCGGCCGCCTGCGCACG reverse for ftsi BACTH FtsI 124 XbaI TTGCTCTAGATTACTAGTAGCTGCCGGGGATGACGAC reverse for ftsi BACTH FtsI XbaI TTGCTCTAGATTACTAGGTGGCCTGCAAGACCAA reverse for gfp ftsi

11 FtsI NdeI GGAATTCCATATGAGCCGCGCCGCCCCCAGGC forward for ftsi pet-19b FtsI BamHI CGCGGATCCCTAGGTGGCCTGCAAGACCAA reverse for ftsi pet-19b CrgA 51aa SwaI TCGGATTTAAATTCAAAACACCATTAACCAGATGAG reverse for crga ppp91 CrgA 27aa PacI CACTGGATCCATGGGACCGTCGAGCGTATGGTT forward for crga ppp93

12 Supplementary methods Intracellular levels of CrgA in M. tuberculosis. Intracellular lysates of M. tuberculosis strains were prepared and the CrgA levels were quantitated by immunoblotting and normalized to SigA as described (Dziedzic et al. 2010). Cell lysates were resolved on NuPAGE polyacrylamide gels, transferred to PVDF membrane and probed with anti-crga antibodies diluted to 1: 25,000. Immunoblots were processed with the ECF Western blotting kit (GE life sciences, Piscataway, NJ) and scanned on a Bio-Rad Molecular Imager. For quantitative immunoblotting, known amounts of purified CrgA protein were quantified by volume analysis function of the QuantityOne software and standard curves were plotted. Lysates loaded on the same gel as the standards were then quantitated using the standard curve as described previously (Dziedzic et al. 2010). Concentration of purified CrgA protein was determined using the BCA protein assay kit (Pierce). The number of colony forming units per ml culture at appropriate OD 600 was used to calculate the number of CrgA molecules per cell. The calculated intracellular CrgA levels are average from lysates derived from 3 independent cultures of M. tuberculosis.