Regulated Proteolysis of a Cross-Link–Specific Peptidoglycan Hydrolase Contributes to Bacterial Morphogenesis

Regulated proteolysis of a cross-link–specific peptidoglycan hydrolase contributes to bacterial morphogenesis

Santosh Kumar Singh1, Sadiya Parveen, L SaiSree, and Manjula Reddy2

Centre for Cellular and Molecular Biology, Hyderabad, India 500007

Edited by Joe Lutkenhaus, University of Kansas Medical Center, Kansas City, KS, and approved July 27, 2015 (received for review April 21, 2015) Bacterial growth and morphogenesis are intimately coupled to ex- (4, 5). The TP activity of PBP1a and PBP1b is activated, respec- pansion of peptidoglycan (PG), an extensively cross-linked macro- tively, by their cognate lipoprotein cofactors LpoA and LpoB, molecule that forms a protective mesh-like sacculus around the located in the outer membrane (6, 7). cytoplasmic membrane. Growth of the PG sacculus is a dynamic Given that the interconnecting peptide bridges in the PG sac- event requiring the concerted action of hydrolases that cleave the culus need to be cleaved for the insertion of new murein material, cross-links for insertion of new material and synthases that catalyze hydrolytic enzymes with such activity are expected to be critical for cross-link formation; however, the factors that regulate PG expan- PG expansion and thus for bacterial viability (2, 3, 8). Essential sion during bacterial growth are poorly understood. Here, we show cross-link–specific hydrolases have recently been identified in both that the PG hydrolase MepS (formerly Spr), which is specific to cleav- Gram-positive and -negative bacteria (9–13). E. coli possesses age of cross-links during PG expansion in Escherichia coli, is modulated several hydrolytic enzymes specific for D-ala−mDAP cross-links by proteolysis. Using combined genetic, molecular, and biochemical (termed D,D-endopeptidases based on their specificity) (14); of approaches, we demonstrate that MepS is rapidly degraded by a these peptidases, Spr, YebA, and YdhO (renamed MepS, MepM, proteolytic system comprising an outer membrane lipoprotein of and MepH, respectively, in which mep stands for murein endo- unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In peptidase) are critical for growth under normal physiological con- summary, our results indicate that the NlpI–Prc system contributes ditions because a mutant lacking all these three endopeptidases is to growth and enlargement of the PG sacculus by modulating the unable to incorporate new murein and exhibits rapid lysis (9). cellular levels of the cross-link–cleaving hydrolase MepS. Overall, Although cross-link cleavage is fundamental for PG growth, it this study signifies the importance of PG cross-link cleavage and its is obvious that such cleavage needs to be stringently regulated at regulation in bacterial cell wall biogenesis. the spatiotemporal level to avoid lethal breakage and rupture of the PG sacculus. In addition, to maintain the continuum and bacterial morphogenesis | peptidoglycan | regulated proteolysis | MepS | integrity of PG, cleavage must be tightly coupled to cross-link NlpI-Prc formation, but the underlying mechanisms are not known. To understand how this potentially lethal hydrolytic activity is eptidoglycan (PG or murein) is a unique and essential con- controlled in the cell, here, we examined the regulation of the Pstituent of eubacterial cell walls, thus making it an excellent D,D-endopeptidase MepS. We found that MepS is highly abundant target for several antimicrobial agents. It is a single, large, ex- in the exponential phase of growth, with levels falling sharply at tensively cross-linked macromolecule that forms a mesh-like the onset of the stationary phase. Using combined genetic, mo- sacculus protecting cells against intracellular turgor pressure in lecular, and biochemical approaches, we demonstrate that MepS addition to conferring cell shape. Structurally, the PG sacculus is made up of linear glycan strands cross-linked to each other by Significance short peptide chains forming a continuous layer around the cytoplasmic membrane. The glycan strands are made up of alter- Peptidoglycan (PG) is a unique and essential cross-linked, bag- nating N-acetyl muramic acid (NAM) and N-acetyl glucosamine like macromolecule that completely encases the cytoplasmic (NAG) disaccharide units in which NAM is covalently attached membrane and confers shape and rigidity to a bacterial cell. to a peptide chain containing 2- to 5-amino acid residues, with Therefore, bacterial cell growth is tightly coupled to PG ex- the pentapeptide consisting of L-alanine (ala)−D-glutamic acid pansion, requiring the coordinate activity of hydrolases that (glu)−meso-diaminopimelic acid (mDAP)−D-ala−D-ala. Normally, cleave the cross-links and synthases that catalyze the cross-link D-ala of one peptide chain is cross-linked to mDAP of another formation. This study highlights the importance of cross-link peptide chain of an adjacent glycan strand, resulting in an ex- cleavage and its regulation in PG biogenesis by demonstrating – tensively cross-linked single- or multilayered sacculus (1). the modulation of a cross-link specific PG hydrolytic enzyme, Because the murein sacculus totally encircles the cytoplasmic MepS, by a previously unidentified degradation system consist- membrane, growth of a cell is tightly coupled to expansion of PG. ing of an outer membrane lipoprotein, NlpI and a periplasmic Growth of the PG sacculus is a dynamic and coordinated event protease, Prc. These studies facilitate better understanding of bacterial cell wall synthesis, which is a target of several antimi- requiring concerted action both of murein hydrolases that fa- crobial therapeutic agents. cilitate cleavage of cross-links for the insertion of nascent murein

material and of synthases that catalyze cross-link formation be- Author contributions: S.K.S., S.P., L.S., and M.R. designed research; S.K.S., S.P., and L.S. tween adjacent glycan strands (Fig. 1) (2, 3). performed research; M.R. analyzed data; and M.R. wrote the paper. Escherichia coli encodes multiple PG synthases that catalyze The authors declare no conflict of interest. − the formation of D-ala mDAP cross-links in the PG sacculus. This article is a PNAS Direct Submission. mrcA The class I enzymes (PBP1a and PBP1b, encoded by and 1Present address: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu mrcB, respectively) are bifunctional and possess both glycosyl University, Varanasi, India 221005. transferase (GT) and transpeptidase (TP) activities whereas class 2To whom correspondence should be addressed. Email: [email protected]. mrdA ftsI II enzymes (PBP2 and PBP3, encoded by and ,re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. spectively) are monofunctional and possess only the TP activity 1073/pnas.1507760112/-/DCSupplemental.

10956–10961 | PNAS | September 1, 2015 | vol. 112 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1507760112 Downloaded by guest on September 24, 2021 absence of NlpI or Prc. The data in Fig. 3A show that, in nlpI or prc mutants, the level of MepS is constitutively high throughout the growth cycle, indicating loss of regulation. To understand the basis of MepS regulation, a pulse–chase experiment was performed after inhibition of protein synthesis by treatment with spectinomycin. Fig. 3B shows that increased MepS in nlpI or prc mutants is a consequence of enhanced posttranslational stability. The half-life of MepS was substantially increased from ∼1–2 min in exponentially growing WT cells to more than 45 min in mutants lacking NlpI or Prc. Consistent with a posttranslational model of regulation, the expression of a chromosomal mepS-lacZ transcriptional fusion was altered neither during the growth cycle nor in nlpI or prc mutants (Table S3). To examine whether NlpI and Prc act in concert to regulate MepS, the level of MepS was measured in single and double mutants. As shown in Fig. 4A, MepS levels remain equally high in all of these mutants, suggesting that NlpI and Prc share a Fig. 1. Schematic of PG sacculus expansion. (A) Depiction of PG sacculus common pathway in regulation of MepS. Furthermore, a modest enlargement during growth of a bacterial cell. New murein strands (colored increase in NlpI led to a significant decrease of MepS, which in red) are incorporated into the preexisting murein strands (colored blue) to turn was dependent on the presence of functional Prc, demon- expand the PG sacculus (2, 3). (B) A model showing cleavage and resynthesis strating the combined requirement of both these factors in MepS of cross-links for successful insertion of new glycan strands for expansion of A the PG sacculus (8). The green bars indicate peptide stems whereas the small regulation (Fig. 4 ). dark red bars represent the cross-bridges between the peptide stems of adjacent glycan strands. Prc Does Not Process NlpI. In light of an earlier report that Prc protease processes NlpI into a mature functional derivative by cleaving the latter’s C-terminal 10- to 11-amino acid residues is degraded rapidly by a previously unknown proteolytic system (17), we measured the size of NlpI in WT and prc mutant strains comprising a tetratricopeptide repeat (TPR)-containing outer by Western analysis using anti-NlpI antisera. Fig. 4B shows that the membrane (OM) lipoprotein, NlpI, and a periplasmic protease, size and level of NlpI remain unaltered in the presence or absence – Prc. In summary, we show that the NlpI Prc system regulates PG of Prc. Importantly, a plasmid-borne copy of nlpI encoding a synthesis by altering the levels of MepS, a hydrolase that breaks polypeptide lacking the extreme C-terminal 10 amino acids (NlpI- the cross-links for insertion of new murein material during growth D284) was able to functionally complement an nlpI mutant but not of the PG sacculus. a prc or prc nlpI mutant, indicating that both NlpI and Prc con- Results tribute independently and directly to the stability of MepS (Fig. S2). Growth Phase Specificity of PG Hydrolase MepS. MepS is an OM NlpI Overexpression Phenocopies MepS Deletion. Because multiple lipoprotein, belonging to the NlpC/P60 superfamily of pepti- copies of NlpI led to lowered MepS levels, (Fig. 4A), we exam- dases, that contributes to the essential murein hydrolytic activity ined the effect of increased NlpI on the growth and viability of a in E. coli (9). Because this observation reflected a vital role for mutant deleted for the alternative endopeptidase MepM because MepS in growth and enlargement of the PG sacculus, we exam- a strain doubly deficient in MepS and MepM is not viable on rich ined the level of cellular MepS during various phases of growth, by media (9). A modest increase of NlpI resulted in severe cell lysis Western analysis using a functional C-terminal 3XFlag-tagged mepM CELL BIOLOGY derivative at the native chromosomal locus (Table S1). Fig. 2 in the mutant that was suppressed by introduction of A E. coli shows that the level of MepS is high throughout the exponential additional copies of MepS (Fig. 5 and Fig. S3). In WT , mepS phase of growth, with levels falling steeply at the onset of the as well, increased NlpI conferred the phenotypes of a stationary phase, with further gradual decrease as cells progress deletion mutant: i.e., inability to grow on media of low osmo- B into the late stationary phase. larity and altered cell shape from rods to fat ellipsoids (Fig. 5 ). Taken together, these data indicate that increased NlpI de- − Regulation of MepS Is Dependent on NlpI and Prc. To understand creases MepS levels very effectively in vivo and mimics the MepS the basis of the growth-phase specificity of MepS, we identified phenotype. mutations that altered the activity of alkaline phosphatase (PhoA) fusedtotheCterminusofmepS (Para::mepS-phoA)(Table S2). Two such mutations that increased the activity of MepS-PhoA were in loci encoding NlpI, an OM lipoprotein of unknown function, and Prc, a periplasmic protease (Fig. S1A). nlpI or prc deletion mutants do not grow on media of low osmotic strength in addition to forming long filaments (15, 16), and absence of MepS was able to suppress the growth and morphological defects of both these mutants (Fig. S1B). Suppression of nlpI or prc mutant phenotypes by mepS deletion was abolished by a plasmid-borne copy of WT mepS, but not by a derivative carrying a mutation, C68A, in the active site of MepS (Fig. S1C). These results sug- Fig. 2. Growth phase specificity of MepS. Strain MR802 (MG1655 mepS- Flag) was grown in LB and fractions were collected at various time intervals gest that the unfettered D,D-endopeptidase activity of MepS is nlpI prc (with indicated A600 values) during the growth cycle. Normalized cell extracts responsible for the phenotypes of or mutants. (corresponding to 0.25 OD cells) were separated by SDS/PAGE, and MepS was Because the experiments above were indicative of regulation detected by Western analysis using anti-Flag antisera. FtsZ was used as a of MepS by NlpI and/or Prc, we measured MepS levels in the loading control.

Singh et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 10957 Downloaded by guest on September 24, 2021 both MepS and Prc were copurified with NlpI (Fig. 7B), and these interactions were independent of Prc and MepS, respectively (Fig. 7B). Taken together, these results indicate that NlpI is able to formbinarycomplexeseachwithMepSandPrcinvivo,butMepS is itself unable to form a binary complex with Prc, suggesting that NlpI is required as an adapter protein to bring together the Prc protease with its target protein MepS, an interpretation that is consistent with the genetic and biochemical data. Discussion In this study, we demonstrate that a previously unknown proteolytic system comprising NlpI and Prc contributes to growth of the PG sacculus by rapidly degrading the cross-link–specific key murein hydrolytic enzyme MepS. This stringent regulation sug- gests that the cross-link cleavage activity of MepS is a crucial determinant of PG synthesis and may indeed be the rate-limiting step in PG expansion. To our knowledge, MepS is the first ex- Fig. 3. Regulation of MepS is dependent on NlpI and Prc. (A) MepS-Flag ample of an enzyme involved in PG metabolism to be regulated levels in nlpI and prc mutants. Strains MR802 (MG1655 mepS-Flag), MR803 by proteolysis. (MR802 ΔnlpI), or MR804 (MR802 Δprc) were grown in LB, and fractions The mechanism of degradation of MepS by the NlpI–Prc were collected at different time points during growth and analyzed by system seems to be analogous to that described earlier on the Western blotting as described in the legend to Fig. 2. (B) Determination of stability of MepS by pulse–chase experiment (in vivo degradation assay). To proteolysis of the rate-limiting enzyme of the lipopolysaccharide

rapidly growing cultures of the above strains in LB (at an OD600 of ∼0.4), biosynthesis, LpxC, by an essential membrane-anchored protease spectinomycin (Spec) was added at a concentration of 300 μg/mL to block FtsH and the TPR-containing adapter protein YciM (19). translation, and fractions were collected at indicated time points and analyzed as described in the legend to Fig. 2. Role of NlpI–Prc in E. coli. Contrary to an earlier report that had suggested a proteolytic processing role for Prc to activate NlpI into a mature functional derivative (17), our results show that NlpI Enhances the Proteolysis of MepS by Prc. Our results thus far NlpI is not processed by Prc either in vivo (Fig. 4B) or in vitro suggested that NlpI and Prc together modulate the level of MepS (Fig. 6A). Rather, we find that NlpI binds both Prc and MepS, in vivo. To test this observation directly in vitro, we purified MepS, NlpI, and Prc, as C-terminal hexahistidine fusion proteins (Fig. 6A, lanes 2, 3, and 4) lacking their signal sequences, and performed degradation assays (Fig. 6A). NlpI and MepS were stable in the presence of one another (Fig. 6A, lane 5) as was Prc in the presence of NlpI (Fig. 6A, lane 6). Coincubation of Prc and MepS led to degradation of the latter to a minimal extent (Fig. 6A, compare lanes 7 and 8), and this process was substantially accelerated in the presence of NlpI (Fig. 6A, compare lanes 9 and 10). A time course experiment also indicated that MepS degradation by Prc is enhanced to a large extent by addition of NlpI (Fig. S4 A and B). Degradation of MepS by Prc was abolished by introduction of mutations at the active site residues of Prc (S452A or K477A) (18), even in the presence of NlpI (Fig. 6B, compare lanes 5, 6, and 7). The proteolytic activity of Prc toward MepS was specific because two other putative periplasmic peptidases, NlpC (a paralog of MepS belonging to the NlpC/P60 family) and YfiH, were not considerably degraded by Prc (Fig. S4C). Overall, these results demonstrate that MepS is a substrate of Prc protease and that NlpI facilitates this degradation.

NlpI Facilitates Interaction of MepS and Prc in Vivo. To examine the in vivo interactions of MepS with Prc and/or NlpI, pull-down assays were performed in strains carrying a functional chromosomal Prc-HA fusion allele, along with a plasmid-encoded Fig. 4. Requirement of both NlpI and Prc for regulation of MepS. (A) Levels functional MepS bearing a hexahistidine tag at its C terminus of MepS in single and double mutants of nlpI and prc. Strains MR802 (MG1655 mepS-Flag), MR803 (MR802 ΔnlpI), MR804 (MR802 Δprc), and (Para::mepS-His). Immunoblot analysis of the MepS-His pull- MR806 (MR802 ΔnlpI Δprc) were grown in LB to an A600 of 1.0, and MepS down fractions showed that NlpI, but not Prc, is copurified with was detected by Western analysis as described in the legend to Fig. 2. Plas- MepS (Fig. 7A and Fig. S5A). The MepS–NlpI interaction oc- mid-carrying strains were grown with appropriate antibiotic (Spec) and curred even in absence of Prc (Fig. 7A). A mutant derivative of 1 mM IPTG. FtsZ was used as a loading control. (B) NlpI is not processed by Prc-HA carrying an active site mutation, S452A, that is expected Prc. The indicated strains MG1655 (WT), MR810 (MG1655 ΔmepS), MR812 to bind but not cleave MepS (Fig. S5B) also could not be cop- (MG1655 Δprc), MR814 (MG1655 ΔmepS Δprc) were grown in LB and har- ∼ urified with MepS, suggesting that Prc does not directly interact vested at an A600 of 1.0. Normalized cell extracts were analyzed by Western A blotting using anti-NlpI antisera. Plasmid-carrying strains were grown with with MepS in vivo (Fig. 7 ). Spec and 1 mM IPTG. The C-terminal truncations of NlpI (bands in lanes 8 and Pull-down assays were also done using a plasmid-borne NlpI- 9) served as controls to indicate the size of processed NlpI. Processed NlpI is His (Para::nlpI-His) in strains carrying functional MepS-Flag and expected to be of 284 amino acids in length (17) whereas full-length is 294 Prc-HA fusion alleles at their native chromosomal loci. Here, amino acids.

10958 | www.pnas.org/cgi/doi/10.1073/pnas.1507760112 Singh et al. Downloaded by guest on September 24, 2021 interactions to facilitate assembly of multiprotein complexes (21). NlpI binding to Prc has been shown earlier (17). FtsI, the essential division-specific transpeptidase, is also shown to be processed by Prc although the physiological significance of such processing is not clear (22). A previous report had indicated that Prc is a periplasmic protein associated with the cytoplasmic membrane (16); however, as predicted by several algorithms, we find that Prc is a soluble periplasmic protein (Fig. S6). In this context, it is to be noted that Prc is specific to proteins with nonpolar C termini and thus is also called a Tail-specific protease (Tsp) (23). However, in many of our in vitro and in vivo experiments, we have used MepS derivatives with various C-terminal tags; therefore, it is possible that the extent of MepS degradation observed here is an un- derestimate and that the native cellular MepS in vivo may perhaps be more susceptible to regulation by the NlpI–Prc system. Regulation of MepS seems to be the primary role of NlpI and − − Prc because most NlpI or Prc phenotypes are abrogated by the absence of MepS (this study and refs. 24–27). It is to be noted

− that the levels of both NlpI and Prc are constitutive and not Fig. 5. NlpI overexpression phenocopies MepS phenotype. (A) Lethal ef- dependent on growth cycle (Fig. S7). Interestingly, mepS (for- fect of overexpressed NlpI in ΔmepM mutants. Strains MR816 (BW27783 spr s pr Δ merly, ) was identified initially as a uppressor of c (24). Our mepM) carrying either pTRC99a (Ptrc) or pMN204 (Ptrc::nlpI) were grown in results provide a mechanistic basis for earlier observations from LB with Amp, and 5 μL of various dilutions were placed on indicated plates μ several groups that absence of MepS suppresses various pheno- and grown. IPTG was used at 100 M. (B) Effect of overexpressed NlpI in WT prc nlpI – E. coli. MG1655 carrying either pTRC99a (Ptrc) or pMN204 (Ptrc::nlpI) was types of and mutants (24 27). cultured and grown on nutrient agar (NA) at 42 °C. IPTG was used at 100 μM. ΔmepS strains are known not to grow on NA at 42 °C (24). For microscopy, Models for Regulation of MepS. The data from Fig. 2 show that the the cultures were grown, diluted 1:100 either in LBON (for WT and ΔmepS levels of MepS significantly decline at the onset of the stationary strains) or in LBON plus 100 μM IPTG (for plasmid-carrying strains) and grown phase. At the same time, the pulse–chase experiments (Fig. 3B) ∼ – at 42 °C until an OD600 of 0.4 0.6. Differential interference contrast (DIC) indicate that MepS has a short half-life of ∼1–2 min, even in images were taken after concentrating and spotting the culture on agarose exponentially growing cultures, suggesting a rapid and constitu- pads. Overexpression of NlpI in WT is known to result in loss of rod shape tive degradation of MepS throughout the growth cycle. Based on and formation of prolate ellipsoid cells (15). It is not clear why the mutants lacking MepS (or those overproducing NlpI) exhibit fat and wide cell mor- the above findings, the following two scenarios for degradation phology; these cells may possibly be partially deficient in cross-link cleavage, of MepS can be envisaged. In the first case, the synthesis of leading to defective growth and elongation of the PG sacculus. MepS could be reduced in the stationary phase, therefore leading to the observed decline in steady-state levels as it is rapidly degraded. However, then the question arises about why cells thereby acting like an adapter protein to facilitate degradation of should indulge in a futile cycle of simultaneous synthesis and MepS by Prc (Figs. 6 and 7). NlpI is an OM lipoprotein with degradation unless such a mechanism has evolved to permit in- multiple TPR motifs (which is conserved in γ-proteobacteria of stantaneous changes in the levels of MepS in individual cells the Enterobacteriaceae, Pasteurellaceae, and Vibrionaceae line- within a population. The second scenario would be that the ob- ages) (20). It is known that TPR proteins mediate protein–protein served half-life of MepS represents an average of different cell CELL BIOLOGY

Fig. 6. MepS is a substrate of Prc. (A) In vitro degradation assays. The C-terminal hexahistidine-tagged MepS, NlpI, and Prc proteins were mixed in all combinations (as indicated) and incubated for 3 h at 37 °C followed by SDS/PAGE and Coomassie blue staining. Each reaction contained the following (approximately): MepS, 15 μg; NlpI, 2 μg; and Prc, 4 μg. The mixtures in lanes 7 and 9 served as controls (0 h; no incubation). M is a protein marker with the following molecular size range (in kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10. Purified MepS was always seen on SDS/PAGE as a doublet, and N-terminal sequencing confirmed that both bands belong to MepS itself. (B) Specificity of Prc. Two mutant derivatives of Prc (carrying K477A or S452A alterations at the active site residues) did not show any proteolytic activity (even when higher concentrations were used) against MepS in the presence of NlpI.

Singh et al. PNAS | September 1, 2015 | vol. 112 | no. 35 | 10959 Downloaded by guest on September 24, 2021 at cell division (12–14). Even as murein hydrolytic activity is essential for bacterial growth and division, it needs also to be tightly regulated to prevent otherwise lethal degradation of PG. Acti- vation of division-specific amidases in E. coli has been shown to be coupled to formation of a cytokinetic ring at the midcell in which a divisomal component, FtsEX, activates EnvC, a catalyti- cally inactive LytM family peptidase that in turn activates the amidases to cleave septal PG for separation of daughter cells (28). In Bacillus subtilis, two L,D-endopeptidases, CwlO and LytE, form a minimal essential set of hydrolases for PG enlargement (10, 12, 13), of which CwlO is activated by the FtsEX complex (29) and LytE is regulated by MreBH (30). Likewise, FtsEX activates the hydrolytic activity of the peptidases PcsB and RipC in Streptococcus pneumoniae and Mycobacterium tuberculosis,respectively (31, 32). In contrast, here, we find that MepS is regulated by proteolysis, possibly providing an advantage of a rapid response to changing environmental conditions.

Coupling of Cross-Link Cleavage and Cross-Link Formation. For an effective enlargement of the PG sacculus, hydrolysis is as im- portant as synthesis although the coupling between these pro- Fig. 7. Interactions of MepS, NlpI, and Prc. (A) Western blot showing in- cesses is not well understood. In Gram-negative organisms with teraction of MepS-His with NlpI and Prc. Strains MR818 (MG1655 ΔmepS prc- HA-Cam), MR819 (MG1655 ΔmepS prcS452A-HA-Cam), or MR814 (MG1655 monolayered PG sacculus, cleavage and resynthesis are believed Δ Δ to be either concomitant or tightly coupled to maintain PG in- mepS prc) carrying pMN217 (Para::mepS-His) were grown overnight in – LB with Amp and next morning diluted 1:100 into fresh LB and at A600 of tegrity (2, 3, 33 36). On the other hand, in Gram-positive organ- ∼0.2–0.3 expression of MepS was induced by addition of 0.05% L-arabinose. isms such as B. subtilis, in which the PG sacculus is multilayered,

Cultures were further grown until an A600 of ∼0.8–1.0, and MepS-His was hydrolysis is not coupled to synthesis even though PG hydrolytic + purified using Ni2 -NTA beads as described in SI Materials and Methods. The activity is essential for growth (10, 12, 13, 29). purified fractions of MepS-His (pull-down fractions, PL) along with the input It is not yet clear whether cross-link cleavage initiates or fol- samples (IN) were separated on SDS/PAGE, and immunoblot analysis was lows the cross-link formation in E. coli. However, because some performed to detect MepS, NlpI, and Prc with anti-His, anti-NlpI, and anti-HA of our preliminary results suggest additional roles for NlpI and/or antibodies, respectively. All strains had a deletion of mepS to reduce the – interference from the endogenous MepS protein. A strain carrying plasmid- Prc in PG cross-link formation, we believe that the NlpI Prc system may facilitate concomitant cleavage and resynthesis of cross- borne, untagged MepS (MG1655 ΔmepS/Para::mepS) was used as a negative control (Fig. S5A). (B) Western blot showing interaction of NlpI-His with links, resulting in successful expansion of the PG sacculus during MepS and Prc. Strains MR805 (MG1655 ΔnlpI mepS-Flag), MR806 (MG1655 growth of bacteria. ΔnlpI Δprc mepS-Flag), MR820 (MG1655 ΔnlpI mepS-Flag prc-HA-Cam), MR821 (MG1655 ΔnlpI ΔmepS prc-HA-Cam), and MR822 (MG1655 ΔnlpI prc- Materials and Methods HA-Cam) carrying pMN218 (Para::nlpI-His) were grown with 0.05% L-arabi- Detailed strain and plasmid constructions, additional materials, methods, nosetoanODof0.8–1.0. NlpI-His was purified from all these strains using an – – + Tables S1 S3,andFigs. S1 S7 are listed in SI Materials and Methods. Ni2 -NTA column, and the purified fractions (PL) along with the input fractions (IN) were separated on SDS/PAGE. Western analysis was done using Media and Growth Conditions. Strains were normally grown in LB (1% tryp- anti-His, anti-Flag, and anti-HA antibodies to detect NlpI, MepS, and Prc, tone, 0.5% yeast extract, 1% NaCl) unless otherwise indicated. LBON has no ∼ respectively. In both these experiments, input sample corresponds to 0.3 OD added NaCl. Nutrient broth has 0.5% peptone and 0.3% beef extract. Solid ∼ cells whereas the pull-down fraction is 20-fold concentrated (i.e., from media had agar to a concentration of 1.5% (wt/vol). Antibiotics were used at ∼ − − 6 OD cells). the following concentrations (μg·mL 1): ampicillin (Amp), 50 μg·mL 1;chlor- − − amphenicol (Cam), 25 μg·mL 1; kanamycin (Kan), 25 μg·mL 1; and spectino- −1 mycin (Spec), 50 μg·mL .ConcentrationsofL-arabinose and isopropyl β-D- cycle-specific values for the asynchronously growing culture. In thiogalactopyranoside (IPTG) were indicated. Growth temperature was 37 °C this model, MepS is relatively stable in cells that are in the unless otherwise specified. elongation mode of PG synthesis compared with the cells that are in the division mode of PG synthesis (that is, when the re- Western Blotting. Samples were separated using SDS/PAGE and transferred quirement for expansion of the PG sacculus is low). The above onto a nitrocellulose or PVDF membrane. Membrane was blocked by 5% two possibilities may not be mutually exclusive and need to be skimmed milk in 1× TBS-T (Tris-NaCl-Tween 20) for 2 h and then incubated overnight with primary antibodies (1:10,000 for α-NlpI, 1:3,000 for α-His, experimentally tested further. α α α However, additional questions that remain to be addressed -FLAG, and -HA, and 1:5,000 for -FtsZ) at 4 °C. Membrane was washed three times with 1× TBS-T and then probed with secondary antibodies relate to the nature of the signal generated during expansion of (1:10,000) tagged with horseradish peroxidase (HRP) and incubated for 1 h the PG sacculus and how it is transmitted to NlpI, an OM li- at room temperature. Membrane was overlaid with ECL Prime detection poprotein. The causes of signal generation may include inputs substrate (Amersham) for 5 min, and the blots were developed. from the PG biosynthetic machinery, alterations in membrane turgor, or the process of cross-link formation itself. Other instances Pull-Down Experiments. Pull downs were performed as described earlier, but of regulation of PG synthesis by OM lipoproteins are known: The with modifications (6). Strains were grown overnight in LB broth supple- TP activity of PG synthases PBP1a and PBP1b is activated by mented with Amp and next morning diluted 1:100 into fresh LB (200 mL) and grown at 37 °C until an OD600 of ∼0.2. At this point, 0.05% arabinose was binding of OM lipoproteins LpoA and LpoB, respectively (6, 7). – How these OM lipoproteins sense, bind, and regulate their cognate added, and growth was continued until OD600 was 0.8 1.0. Cells were re- covered by centrifugation, and the cell pellet was washed, resuspended in effectors is an interesting question to be addressed. 25 mL of the lysis buffer (50 mM Tris·Cl, 10 mM MgCl2, 100 mM NaCl, 20% glycerol, 2% Triton X-100, 1 mg/mL lysozyme, 1× mixture protease inhibitor, Regulation of Other PG Hydrolases. Most eubacterial genomes en- 20 units of DNase, 20 units of RNase, and 10 mM imidazole; pH 8.0), and code a multitude of PG hydrolases that function in murein growth, incubated on ice for 2 h. This mixture was subjected to brief sonication, and maturation, turnover, recycling, autolysis, and cleavage of septum the lysate was gently stirred overnight with glass beads at 4 °C to solubilize

10960 | www.pnas.org/cgi/doi/10.1073/pnas.1507760112 Singh et al. Downloaded by guest on September 24, 2021 the membrane proteins. The next morning, insoluble material of the lysate The eluate was electrophoresed using 12% SDS/PAGE, and the proteins were was removed by centrifugation (14,000 × g, 15 min, 4 °C), at which point detected by Western blotting. 100 μL was set aside for input fractions, and then the supernatant was mixed + with 200 μLofNi2 -NTA agarose and mixed for 2 h at 4 °C. The agarose ACKNOWLEDGMENTS. We thank Sujata Kumari and M. R. Sunayana for beads were washed twice with 10 mL of wash buffer (50 mM Tris·Cl, 100 mM plasmid constructions; Nilanjan Som for Prc localization studies; Tom Bernhardt for FtsZ antisera; and J. Gowrishankar and N. Madhusudhana Rao for advice. NaCl, and 20 mM imidazole; pH 8.0) and twice with wash buffer containing This work is supported by the Department of Biotechnology and the Council of 50 mM imidazole, and the bound proteins were eluted with 250 μLofthe Scientific and Industrial Research, Government of India. S.P. acknowledges elution buffer (50 mM Tris·Cl, 100 mM NaCl, and 300 mM imidazole; pH 8.0). financial support from the University Grants Commission of India.

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