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.

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.

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.

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

(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.

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 1st and 2nd 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 1-

124 and 1-298 aa proteins, respectively. CrgA1-51aa is CrgA N-terminal 1-51 aa protein.

GCN4-GCN4 is positive control.

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 H2O+TFA with a Acetonitrile Gradient (0-20%)

(Table S1; see also Lavollay et al. 2008 for more details).

Table S1: Muropeptide composition

A Tripeptide

B Tetrapeptide -GlcNac

C Tetrapeptide

D 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)

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

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 crgAMtb antisense

aCrgA-SwaI ATCGGATTTAAATATGCCCAAGTCCAAGGTCCG reverse for crgAMtb antisense

CrgA-BamHI TCTAGAGGATCCCATGCCCAAGTCCAAGGTCCGC forward for crgA BACTH

CrgA-KpnI TTACTTAGGTACCCGATGCCAGCGCATCGTGAGCA reverse for crgA BACTH

Yfp XbaI TATAGTCTAGATTATTACTTGTACAGCTCGTCCA reverse for ftsZ-yfp

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

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

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 OD600 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.