Peptidoglycan Hydrolase of an Unusual Cross-Link Cleavage Specificity Contributes to Bacterial Cell Wall Synthesis

Peptidoglycan hydrolase of an unusual cross-link cleavage specificity contributes to bacterial cell wall synthesis

Pavan Kumar Chodisettia and Manjula Reddya,1

aCentre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India 500007

Edited by Carol A. Gross, University of California, San Francisco, CA, and approved March 13, 2019 (received for review October 2, 2018) Bacteria are surrounded by a protective exoskeleton, peptidoglycan Synthesis of PG is a complex process that occurs in two distinct (PG), a cross-linked mesh-like macromolecule consisting of glycan cellular compartments: the cytosol and the periplasm (8). The strands interlinked by short peptides. Because PG completely precursor UDP-MurNAc-pentapeptide is synthesized in the cytosol encases the cytoplasmic membrane, cleavage of peptide cross- by sequential enzymatic reactions catalyzed by MurA, -B, -C, -D, -E, links is a prerequisite to make space for incorporation of nascent and -F before being transferred to a membrane-bound lipid carrier glycan strands for its successful expansion during cell growth. In (C55-bactoprenol phosphate) to generate lipid I. Subsequently, a most bacteria, the peptides consist of L-alanine, D-glutamate, meso- molecule of UDP-GlcNAc is added to lipid I to yield lipid II, which diaminopimelic acid (mDAP) and D-alanine (D-Ala) with cross-links is then flipped across the inner membrane into the periplasm. Here, occurring either between D-Ala and mDAP or two mDAP residues. the membrane-bound D,D-transpeptidases catalyze the formation of Escherichia coli − 4 5 In ,theD-Ala mDAP cross-links whose cleavage by 4−3 cross-links by cleaving the D-Ala −D-Ala peptide bond of the 4 specialized endopeptidases is crucial for expansion of PG predomi- incoming disaccharide pentapeptide (donor) to link the D-Ala to − 3 nate. However, a small proportion of mDAP mDAP cross-links also the D-center of the mDAP residue of an adjacent peptide chain exist, yet their role in the context of PG expansion or the hydrolase(s) (acceptor) (6). On the contrary, L,D-transpeptidases (LdtD and capable of catalyzing their cleavage is not known. Here, we identi- LdtE) catalyze the formation of 3−3 cross-links by cleaving the

fied an ORF of unknown function, YcbK (renamed MepK), as an 3 4 BIOCHEMISTRY mDAP −D-Ala peptide bond of an existing tetrapeptide of the PG mDAP−mDAP cross-link cleaving endopeptidase working in conjunc- 3 3 sacculus (donor) to link the mDAP to the D-center of the mDAP tion with other elongation-specific endopeptidases to make space residue of an adjacent peptide (acceptor) (9). for efficient incorporation of nascent PG strands into the sacculus. Because the PG sacculus is a continuous molecular network E. coli mutants lacking mepK and another D-Ala−mDAP–specific en- that completely encircles the cytoplasmic membrane, the growth dopeptidase (mepS) were synthetic sick, and the defects were abro- of a cell is tightly coupled to expansion of PG, requiring the co- gatedbylackofL,D-transpeptidases, enzymes catalyzing the ordinated activity of hydrolases that cleave the cross-links and formation of mDAP cross-links. Purified MepK was able to cleave synthases that form the cross-links (1). Given that the PG is the mDAP cross-links of soluble muropeptides and of intact PG sac- – – culi. Overall, this study describes a PG hydrolytic enzyme with a interconnected by two types of cross-bridges (4 3 and 3 3), it is hitherto unknown substrate specificity that contributes to expansion expected that the cleavage of both of these cross-links is a pre- of the PG sacculus, emphasizing the fundamental importance of requisite to make space for the incorporation of incoming murein cross-link cleavage in bacterial peptidoglycan synthesis. strands for its successful expansion. We previously showed cleavage of 4−3 cross-links is crucial for PG enlargement as an E. coli bacteria | peptidoglycan | mDAP cross-link | MepK | YcbK mutant lacking three D,D-endopeptidases, MepS, MepM, and MepH

ell envelopes of bacteria have a mesh-like exoskeleton called Significance Cpeptidoglycan (PG; also called murein) to protect them against turgor and environmental stress conditions. It also con- Bacteria contain peptidoglycan (PG) in their cell envelope to pro- fers mechanical strength and shape to bacterial cells. PG is a tect them against intracellular osmotic pressure and environmental single, large, covalently cross-linked macromolecule made up of stress. PG is a large elastic polymermadeupofglycanstrands interlinked by short peptide chains that form a mesh-like sacculus. multiple linear glycan strands that are interconnected by short In many bacteria, the peptide cross-links are of two types: the peptide chains (Fig. 1) (1–4). The glycan strands are polymers of predominant D-alanine−meso-diaminopimelic acid (mDAP) and the β alternating -1,4-linked N-acetylglucosamine (GlcNAc) and N- rare mDAP−mDAP cross-links. Here, we report the importance of acetylmuramic acid (MurNAc) disaccharide units in which the D- mDAP cross-links in PG synthesis during cell growth by identifying lactoyl moiety of each MurNAc residue is covalently attached to a previously unknown hydrolytic enzyme that cleaves such cross- the first amino acid of the peptide chain. Typically, the peptide links in the PG sacculi of Escherichia coli.Insummary,thisstudy chains consist of two to five amino acids, and in Escherichia coli, clarifies the role of PG hydrolysis in bacterial cell wall synthesis, 1 a pentapeptide consists of L-alanine (L-Ala )−D-glutamic acid (D- thereby rendering it an alternative drug target for development of 2 3 4 5 Glu )−meso-diaminopimelic acid (mDAP )−D-Ala −D-Ala . Nearly new antimicrobial agents. 40% of the neighboring peptide chains are cross bridged to each 4 3 − Author contributions: P.K.C. and M.R. designed research; P.K.C. performed research; other, either between the D-Ala and mDAP (D-Ala mDAP, or P.K.C. and M.R. analyzed data; and P.K.C. and M.R. wrote the paper. − 3 3 − − 4 3) or between mDAP and mDAP (mDAP mDAP, or 3 3) The authors declare no conflict of interest. − residues (2, 5). Of these, the 4 3 cross-links are more predominant This article is a PNAS Direct Submission. ∼ ( 93%) and are catalyzed by D,D-transpeptidase activity of high- Published under the PNAS license. molecular-weight penicillin-binding proteins (PBPs) that include 1To whom correspondence should be addressed. Email: [email protected]. − PBP1A, PBP1B, PBP2, and PBP3 (6). On the contrary, 3 3 cross- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. links are much less abundant (about 7%) and result from the ac- 1073/pnas.1816893116/-/DCSupplemental. tivity of L,D-transpeptidases LdtD and LdtE (7).

www.pnas.org/cgi/doi/10.1073/pnas.1816893116 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 and SI Appendix,Fig.S1A). However, the ΔmepK single mutant did not exhibit any discernible phenotype except a moderate increase in sensitivity to certain β-lactam antibiotics such as ampicillin and cephalexin (ref. 13, SI Appendix,Fig.S1B). MepK belongs to the M15 family of peptidases that also comprises DdpX, a peptidase that cleaves D-Ala−D-Ala di- peptide in the cytosol of E. coli (14, 15). In addition, a mepK homolog of Klebsiella pneumoniae is predicted to be a PG hydrolase (16). Hence, we tested whether increased expression of mepK is able to compensate the loss of MepS and/or MepM, the elongation-specific PG hydrolases (10). The absence of MepS results in sensitivity on NA at 42 °C (17), and this growth defect was significantly suppressed when mepK (Ptrc::mepK) was introduced in multiple copies (Fig. 2C). In addition, mepK overexpression suppressed the vancomycin sensitivity of a ΔmepM deletion mutant, indicating that MepK compensates for the loss of either mepS or mepM (Fig. 2D). A double Fig. 1. Schematic representation of the PG sacculus of E. coli. The structure mutant lacking mepS and mepM does not grow on rich media and composition of glycan strands and peptide chains are shown. Listed are the known PG hydrolases of E. coli (3, 4, 10). Cleavage sites of the hydrolases such as LB but grows well on minimal media (10). Additional are indicated by the scissors symbol. MepK (indicated in red) is identified in copies of mepK could also support, albeit partially, the growth this study. of a ΔmepS ΔmepM double mutant on LB (SI Appendix,Fig. S2A). Moreover, deletion of mepK conferred significant additive sickness to the ΔmepS ΔmepM strain growing on min- (specific to D-Ala−mDAP cross bridges), is unable to incorporate imal media, with the triple-deletion strain growing very poorly new murein and undergoes rapid lysis (10). However, it is not clear with extensive lysis and cell death (SI Appendix,Fig.S2B and how the 3−3 cross-linkages affect PG enlargement because they are C). These observations collectively indicated contribution of also expected to hinder opening of the mesh for the incorporation of MepK to the functions of elongation-specific D,D-endopepti- new PG material. dases, MepS and MepM. In this study, we show that 3−3 cross-link cleavage contributes to PG enlargement by identifying an enzyme of previously unknown MepK Modulates mDAP−mDAP Cross-Links of Peptidoglycan. As the specificity, YcbK (renamed murein endopeptidase K, MepK), as a above observations suggest that MepK is a PG peptidase, we murein hydrolase that cleaves 3−3 cross-links in E. coli. Extensive examined the composition of PG of a mutant lacking MepK. The genetic and molecular analyses indicate that mepK functions in PG sacculi from WT and the ΔmepK mutant were prepared and conjunction with other elongation-specific D,D-endopeptidases to digested with a muramidase (mutanolysin), and the resulting contribute to synthesis of PG. Biochemical studies demonstrate muropeptides were separated by reverse-phase high pressure MepK is a PG hydrolase with the ability to cleave the 3−3 cross- links of soluble muropeptides and of intact PG sacculi. MepK orthologs from other gram-negative bacteria also exhibit similar substrate specificity. To the best of our knowledge, MepK is the only enzyme reported so far in any bacterial genera to exhibit 3−3 cross-link–specific catalytic activity. Results MepK Functions in Conjunction with Other Elongation-Specific Endopeptidases. To understand the mechanism of cross-link cleavage during PG enlargement, we made an attempt to identify mutations that confer additive sickness to an E. coli strain lacking a major elongation-specific endopeptidase, MepS (10, 11). We observed that deletion of tatB, a gene encoding a component of the twin-arginine translocase (TatABC) secretion pathway, resulted in synthetic sickness in a strain deleted for MepS on media of low osmolarity such as LB without NaCl and nutrient agar (NA) (Fig. 2A). Deletions in tatA or tatC also behaved similarly to that of tatB, indicating the importance of a functional Tat system in the growth of the mepS deletion mutant. TatABC is a secretion system that facilitates transport of folded proteins from the cytosol into the ∼ Fig. 2. Genetic interactions between mepS, mepK,andmepM.(A)WT periplasm across the inner membrane. In E. coli, 27 proteins are (MG1655) and its mutant derivatives were grown in LB and serially diluted, and known to be transported by the TatABC pathway (12), and we 5 μL of each dilution were spotted on indicated plates and incubated overnight tested the genes which are crucial for growth of the mepS mutant by at 30 °C. (B) Cultures of WT and its mutant derivatives grown overnight were examining the phenotypes of several double-deletion mutants diluted 1:100 into fresh LB and grown until A600 of 0.3 at 30 °C. Cultures were lacking each of the TAT substrates. Interestingly, a double mutant pelleted, washed, and resuspended in nutrient broth (NB) and grown at 30 °C. lacking MepS and YcbK, an ORF of unknown function (hereafter Growth was monitored every 1 h. The arrow indicates the time of the shift to Δ designated MepK), behaved exactly like that of the ΔmepS ΔtatB NB. (C)WTand mepS strains carrying either vector (pTrc99a; Ptrc::) or pPK2 (Ptrc::mepK) were grown, and viability was tested on NA plates at 37 °C with or strain, suggesting the deficiency of MepK in the periplasm as the without Isopropyl thiogalactopyranoside (IPTG) (0.5 mM) as described above. (D) basis of synthetic sickness of the ΔmepS ΔtatB double mutant (Fig. WT and ΔmepM strains carrying vector or pPK2 (Ptrc::mepK)weregrowninLB 2A). Consistent with the viability assay,thecellsofthedoublemutant and tested for viability at 37 °C on LB plates supplemented with vancomycin (ΔmepS ΔmepK) bulge and lyse in low-osmolarity conditions (Fig. 2B (250 μg/mL) and IPTG (0.5 mM).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1816893116 Chodisetti and Reddy Downloaded by guest on September 24, 2021 liquid chromatography (RP-HPLC). Analysis of the chromato- absence of mepK), indicating that L,D-transpeptidase activity leads grams revealed a single significant alteration in the PG of the to the accumulation of 3−3 cross-links in the mepK mutant (Fig. Δ mepK strain compared with the WT. A muropeptide peak eluted 4A). Consistent with this, overexpression of LdtD (PT5-lac::ldtD) at 47 min (denoted as 4 in Fig. 3A) was found to be increased 1.75- was extremely toxic in a ΔmepK mutant, with its PG sacculi ac- fold in the ΔmepK mutant (with peak 4 constituting about 4% of cumulating significantly higher levels of 3−3 cross-links compared the total soluble muropeptides in the WT and 7% in the ΔmepK with the WT (Fig. 4 B and C). mutant) (Fig. 3A and SI Appendix,TableS3). Mass spectrometry Similarly, introduction of LdtDE deletion abrogated the (MS) data indicated the molecular mass of peak 4 is 1,794 Da, growth defects of the mepS mepK double mutant (Fig. 4D). suggesting it is a cross-linked dimer of a disaccharide tetrapeptide LdtDE deletion was also able to confer partial growth advantage and a disaccharide tripeptide (Fig. 3B). Further analysis by tandem to a ΔmepS single mutant, indicating that the 3−3 cross-links are mass spectrometry revealed these dimers are cross-linked by the deleterious in the absence of MepS (Fig. 4 D and E). The above − − rare mDAP mDAP linkages (3 3; tri-tetra; Fig. 3B). On the other results clearly indicate that ldtDE is epistatic to mepK; however, hand, peak 3, which also had an identical molecular mass of 1,794 − − we observed that overexpression of mepK was able to confer Da, was a dimer cross-linked by D-Ala mDAP linkages (4 3; tetra- further growth to a mepS ldtDE double mutant, suggesting MepK C mepK tri; Fig. 3 ). In addition, multiple copies of plasmid-borne may have an additional function which is independent of LdtDE (P ::mepK) were able to decrease the tri-tetra peak to 2% of total trc (Fig. 4E). These observations are discussed below. soluble muropeptides in both the WT and ΔmepK strain (Fig. 3D and SI Appendix,TableS3), suggesting that MepK directly or in- − MepK Contributes to PG Synthesis Along with MepS and MepM. Our directly modulates the level of 3 3 cross-links in PG sacculi. results thus far indicated MepK is a putative 3−3 cross-link– In light of an earlier report that MepA, a member of the LAS specific PG hydrolase functioning in conjunction with MepS metallopeptidase family (18) and a known 4−3 endopeptidase, is and MepM, the elongation-specific 4−3 endopeptidases. Hence, also able to cleave 3−3 cross-links in vitro (5), we examined PG we reasoned that cleavage of 3−3 cross-links may also contribute composition of a double-deletion mutant lacking mepA and to PG expansion and therefore measured PG synthesis in various mepK and found it to be comparable to that of the single mepK 3 deletion mutant (SI Appendix, Fig. S3). Moreover, mepA de- mutants deleted for MepK/MepS/MepM/LdtDE using a H- letion did not confer any additive sickness to the mepS mepK mDAP incorporation assay (ref. 10, Fig. 5). Lack of MepK mutant, suggesting MepA may not have a role in the turnover of alone resulted in a somewhat lower rate of PG synthesis; how- Δ Δ 3−3 cross-linkages under normal laboratory growth conditions. ever, its absence in mepS or mepM strains led to a further BIOCHEMISTRY decrease in 3H-mDAP incorporation compared with the WT, ldtDE Is Epistatic to mepK. L,D-transpeptidases, LdtD and LdtE, clearly indicating that the combined activity of these peptidases catalyze the formation of 3−3 cross-links in the PG sacculi of E. contributes to optimal PG synthesis. Deletion of ldtDE in the coli (7); hence, we examined the effect of LdtDE activity on the ΔmepS ΔmepK mutant was able to restore PG synthesis to that phenotypes of mepK. The tri-tetra peak (peak 4) was completely of a single ΔmepS mutant, validating the earlier findings of L,D- absent in strains lacking ldtDE (irrespective of the presence or transpeptidase activity being epistatic to MepK (Fig. 4D).

Fig. 3. MepK modulates mDAP cross-links of PG sacculi. (A) HPLC chromatograms depicting the soluble muropeptides of WT and ΔmepK mutants. Muropeptide peaks (1–5) were collected and analyzed by MS, and structures are depicted. The red arrow represents peak 4; the blue arrow represents peak 3. (B and C) + Mass spectra of peaks 3 and 4 show identical molecular mass (M+H) of 1,795.78 Da (Inset). Tandem MS indicated peak 4 is a 3−3 cross-linked dimer (tri-tetra) and peak 3 is a 4−3 cross-linked dimer (tetra-tri). Fragments specific to the 3−3 dimer are shown in blue. (D) HPLC chromatograms of WT and ΔmepK mutant

strains carrying pPK2 (Ptrc::mepK). Cultures were grown to an A600 of ∼1 in LB containing 0.5 mM IPTG followed by isolation and analysis of PG sacculi. The red arrow represents peak 4.

Chodisetti and Reddy PNAS Latest Articles | 3of6 Downloaded by guest on September 24, 2021 Fig. 4. Genetic interactions between mepK, mepS, and ldtDE.(A) HPLC chromatograms of PG sacculi isolated from ldtDE and ldtDE mepK mutants. The absence of peak 4 is indicated by a red arrow. (B) HPLC chromatograms of WT and ΔmepK mutant carrying vector alone (pCA24N) or vector carrying ldtD

(PT5-lac::ldtD). Strains were grown to an A600 of ∼1 in LB containing IPTG (20 μM for WT and 6 μMforΔmepK mutant) followed by isolation and analysis of PG sacculi. MS analysis indicated peaks a, b, and c are tetra (Gly4), tri-tetra (Gly4), and tri-tri, respectively (SI Appendix, Fig. S6). (C) WT and ΔmepK mutant

carrying vector (pCA24N) or its derivative (PT5-lac::ldtD) were grown, and their viability was tested on indicated plates at 37 °C with or without IPTG (20 μM). (D) Growth of WT and its mutant derivatives was tested on indicated plates at 37 °C as described above. (E) ΔmepS and ldtDE ΔmepS strains carrying either vector

or pPK2 (Ptrc::mepK) were grown in LB, and viability was examined on NA plates with or without IPTG (0.5 mM) at 37 °C.

MepK Is an mDAP−mDAP Cross-Link–Specific L,D-Endopeptidase. To Based on homology modeling, an earlier study predicted an examine the in vitro activity of MepK, we overexpressed and purified MepK ortholog of K. pneumoniae to be a metal-binding PG hy- 31-182 a histidine-tagged signal-less MepK derivative (MepK -His6). drolase (ref. 16, SI Appendix,Fig.S8A). To validate the function of Purified MepK was able to show a zone of clearance in a zymo- gram assay, indicating either PG binding or hydrolysis (SI Appen- dix,Fig.S4). Incubation of total soluble muropeptides (obtained by mutanolysin digestion of PG sacculi from WT or the mepK deletion mutant) with MepK resulted in complete loss of tri-tetra (peak 4) with a concomitant increase in tripeptide disaccharide (tri, peak 1) and tetrapeptide disaccharide (tetra, peak 2) (Fig. 6 A and B). Cleavage of tri-tetra into tri and tetra was also confirmed using purified tri-tetra as a substrate (Fig. 6C). MepK was also able to weakly cleave the tetra-tetra dimer (peak 5) (Fig. 6 A, B,andD) but had no activity on the tetra monomer (Fig. 6E). MepM, a D,D- endopeptidase specific to 4−3 cross-links (10), was not able to cleave the 3−3 cross-links of peak 4 but was able to cleave the 4−3cross- linked muropeptides (peaks 3 and 5) (SI Appendix,Fig.S5). To further confirm the catalytic specificity of MepK, we used soluble muropeptides of a strain carrying more copies of LdtD (WT/PT5-lac::ldtD) as its substrates. As expected, LdtD overexpression resulted in accumulation of a large number of 3−3 cross-linked muropeptides and also tetrapeptides with glycine at position 4 (Fig. 4B and SI Appendix, Fig. S6). Consistent with earlier results, MepK completely hydrolyzed all of the 3−3 cross- linked muropeptides (peaks b, c, and 4) and also partially cleaved the tetra-tetra dimer (peak 5) (Fig. 6F), clearly indicating that MepK is predominantly a 3−3 cross-link–specific PG hydrolase with a minor activity on 4−3 cross-linked muropeptides. Further, we purified two types of 3−3 cross-linked muropeptides designated as b (tri-tetra with Gly4) and c (tri-tri). Incubation of MepK with b and c yielded tri and tetra (Gly4) and monomers of Fig. 5. MepK contributes to optimal PG synthesis. Incorporation of new murein 3 tri, respectively, confirming the ability of MepK to effectively using H-mDAP was measured in WT and its various mutant derivatives. Strains cleave 3−3 cross-linked muropeptides (Fig. 6 G and H). In ad- were grown in minimal A media supplemented with 0.5% casamino acids (CAA) dition, MepK was able to cleave the 3−3 cross-links of intact PG and 0.2% glucose at 37 °C. In this medium, all strains grew equally well, and their growth rates were comparable. At an A600 of ∼0.7, a 0.5-mL aliquot was pulsed sacculi as well as those of the soluble muropeptides (SI Appendix, with 3H-mDAP and processed as described in Materials and Methods. All strains Fig. S7). Taken together, these results demonstrate that MepK is carried lysA deletion to prevent conversion of mDAP into lysine. Values are primarily a 3−3 cross-link–specific L,D-endopeptidase and also a expressed in percentages, and WT is normalized to 100. Data shown represent weak D,D-endopeptidase. three independent experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1816893116 Chodisetti and Reddy Downloaded by guest on September 24, 2021 of cross-link cleavage being an important determinant of PG expansion during growth of a bacterium.

Role of MepK in PG Enlargement. PG hydrolysis is an indispensable step of cell wall synthesis and cell growth in several gram-negative and gram-positive bacterial genera (10, 19–22). In E. coli and other gram-negative bacteria, cleavage of D-Ala−mDAP cross-links mediated by D,D-endopeptidases is crucial for PG enlargement (10, 20, 22). In this context, our results show that cleavage of mDAP cross- links also plays a role in PG enlargement. The synthetic and additive phenotypes of the mepS mepK double mutant (Figs. 4D and 5) indicate that in the absence of MepS, a major 4−3 endopeptidase, activity of MepK is required. Loss of these synthetic phenotypes in the absence of L,D-transpeptidases suggests that the 3−3 cross-links are deleterious and are capable of hindering the prog- ress of nascent PG incorporation during growth of the PG sacculus (Fig. 4D). Consistent with this, the absence of 3−3 cross-links partially relieves the growth defects of the mepS mutant (Fig. 4 D and E). As shown in Fig. 4E, overexpression of mepK was able to further confer growth to an ldtDE mepS double mutant. This LdtDE- independent activity of MepK is most likely due to its weak 4−3 cross-link cleaving ability (Fig. 6 D and F). However, this weak activity may not significantly contribute to PG enlargement under normal physiological conditions. In a recent study, a mutant of E. coli was constructed which contains exclusively 3−3 cross-links in its PG sacculus (9). As this strain is completely dependent on LdtD Fig. 6. HPLC chromatograms showing the biochemical activity of MepK. for the formation of cross-links, MepK may become indispensable BIOCHEMISTRY Soluble muropeptides of WT (A), soluble muropeptides of mepK mutant (B), in this strain to open up the 3−3 cross-links for PG expansion and purified tri-tetra (C), purified tetra-tetra (D), purified tetra (E), soluble mur- 4 hence for its growth and viability. opeptides of WT/PT5-lac::ldtD (F), purified tri-tetra (Gly )(G), and purified tri-tri (H) were incubated either with buffer or with MepK (5 μM) for 20 h and − separated by RP-HPLC. Note that MepK cleaved peaks 4, b, and c completely How Is 3 3 Cross-Linkage Regulated? Several studies reported al- (red arrows) and partially cleaved peak 5 (F). All peaks were identified by MS terations in the frequency of 3−3 linkages in E. coli growing under and tandem MS (SI Appendix,Fig.S6). different physiological conditions, the stationary phase, and stress (5, 23, 24). It is likely that MepK activity may also be regulated under these conditions. In a transcriptomic study, mepK levels were found MepK, we created site-directed mutations in the predicted metal- to be slightly up-regulated during σE stress response (25). However, binding site by modifying each of the amino acid residues H133, preliminary studies done using a mepK::lacZ transcriptional fusion D140, and H173 into an L-alanine in a Ptrc::mepK plasmid system. did not reveal any noticeable alterations during envelope stress or Complementation assays (Fig. 7A) and the analysis of PG com- the stationary phase. It would be interesting to examine how 3−3 position (SI Appendix,Fig.S8B) showed that these mutant plas- cross-link formation is modulated by the combined activities of mids are not functional, although they are able to express MepK LdtDE and MepK. In this context, it is noteworthy that the bicistronic to a similar extent (SI Appendix, Fig. S8C), indicating the impor- mepK-ycbL operon is located immediately downstream to ldtD in tance of these active-site amino acid residues. the E. coli genome (and also in many gram-negative bacteria), MepK is conserved in most classes of gram-negative bacteria raising an interesting possibility of their coevolution. but not in gram-positive organisms. To examine the function of mepK, we cloned its orthologs from two model organisms, Cau- Conservation of MepK Across Bacteria. MepK belongs to the M15 family of peptidases which includes the VanX type of peptidases lobacter crescentus and Klebsiella pneumoniae. Viability assays indicated that both of the homologs complement ΔmepK ΔmepS growth defects (Fig. 7B). However, we found weak complementation with mepK of C. crescentus, most likely due to its poor expression (SI Appendix,Fig.S8D). In addition, the recombinant MepKKp-His protein was able to cleave the 3−3cross-linksof soluble muropeptides of E. coli (SI Appendix,Fig.S8E), suggesting a conserved function of MepK in these bacteria. Discussion In this study, we report identification of a previously unknown peptidoglycan hydrolase, MepK, which cleaves the 3−3cross- linked muropeptides of PG sacculi in E. coli. To the best of our knowledge, MepK is the only enzyme known so far across the Fig. 7. Functionality of mepK variants and orthologs. (A) ΔmepS ΔmepK bacterial genera to exhibit such catalytic specificity. We also show strain carrying either vector (Ptrc), pPK17 (Ptrc::mepK), pPK19 (Ptrc::mep- that cross-link cleavage mediated by the combined activity of both KH133A), pPK20 (Ptrc::mepKD140A), or pPK21 (Ptrc::mepKH173A) was grown in LB, and viability was tested on NA plates with IPTG (0.5 mM) at 30 °C. (B) 3−3-specific (MepK) and 4−3-specific (MepS/MepM) endopep- ΔmepS ΔmepK strain carrying vector (Ptrc), pPK17 (Ptrc::mepK-HisE. coli;Ec), tidases is required to make optimal space for the incorporation of pPK23 (Ptrc::mepK-HisC. crescentus;Cc), or pPK25 (Ptrc::mepK-HisK. pneumoniae;Kp) new murein material, thereby confirming the earlier observations was grown in LB and processed as described above.

Chodisetti and Reddy PNAS Latest Articles | 5of6 Downloaded by guest on September 24, 2021 as well as DdpX (14, 15). MepK is conserved across most families Sacculi were again boiled with 8% SDS, collected by ultracentrifugation, and of gram-negative but not gram-positive bacteria. It has been ob- washed with water to remove SDS. Pellet was resuspended and stored served that the proportion of 3−3 and 4−3 cross-links is extremely at −30 °C. See SI Appendix. variable across bacteria (2, 26). For example, 3−3 cross-links ac- count for about 1.2% in Pseudomonas putida,7%inE. coli,and Analysis of PG Sacculi. Analysis of PG sacculi was done as previously described 40% in Aeromonas sp. and Agrobacterium tumefaciens (26). In (5). Intact PG sacculi were digested with mutanolysin and centrifuged to members of the Mycobacteriaceae family, PG has extremely high remove the insoluble material. Soluble fraction was reduced with sodium borohydride; samples were loaded onto a reverse-phase C18 column, and levels of 3−3 cross-links, accounting for almost 60% of the total fractions were collected. Absorbance was detected at 205 nm. See SI Appendix. cross-linkages (27). In these organisms, the 3−3 cross-link hydrolases may have a higher significance. Determination of Enzymatic Activity. Soluble muropeptides were incubated with either buffer or MepK (5 μM) at 30 °C for 20 h with shaking. Samples Materials and Methods were heat inactivated, reduced with sodium borohydride, and separated Detailed strain and plasmid constructions, additional materials, methods, by RP-HPLC. Tables S1–S3, and Figs. S1–S8 are listed in SI Appendix. Measurement of 3H-mDAP Incorporation into PG Sacculi. Incorporation of 3H- Media, Bacterial Strains, and Plasmids. Bacteria were grown either in LB media mDAP into murein sacculi was done as described (10, 31). Strains were grown (0.5% yeast extract, 1% tryptone, and 1% NaCl) or minimal A media sup- overnight in LB, washed, and diluted 1:100 into prewarmed minimal A me- plemented with 0.2% glucose (28). NA has 0.5% peptone and 0.3% beef dium supplemented with 0.2% glucose and 0.5% casamino acids. Cells were extract. Antibiotics were used at the following concentrations (μg/mL): am- grown to an A of 0.7, and 0.5-mL aliquots were incubated with 5 μCi/mL picillin, 50; chloramphenicol, 30; vancomycin, 250; and kanamycin (Kan), 50. 600 of 3H-mDAP (14.6 Ci/mmol; Moravek Biochemicals) for 10 min at 37 °C. Reac- Growth temperature was 37 °C unless otherwise indicated. The bacterial tion was stopped by boiling cells with SDS and then cooling and filtering them. strains and plasmids are listed in SI Appendix. Filters were washed and dried, and the radioactivity was measured using a liquid scintillation counter. Molecular and Genetic Techniques. Recombinant DNA techniques and P1 phage-mediated transductions and transformations were performed using standard methods (28). Deletion mutants are from the Keio mutant collec- ACKNOWLEDGMENTS. We thank Sujata Kumari, Vaishnavi Kunteepuram, Aisha tion (29), and wherever necessary, the KanR gene was flipped out using Hamid, Nilanjan Som, and Raj Bahadur for plasmids and strains; V. Krishna Kumari andC.SubbalakshmiforHPLC;B.Raman and Y. Kameshwari for mass pCP20 plasmid encoding Flp recombinase (30). spectrometry; N. Madhusudhana Rao for advice; and G. Shambhavi for help with the manuscript. We thank National BioResource Project:: E. coli for the Preparation of PG Sacculi. Isolation of PG was done as described earlier (5). Keio mutant collection and the ASKA plasmid library. This work is supported Briefly, cells are grown to an A600 of 1.0, harvested, resuspended in water, and by funds from the Department of Biotechnology (Centre of Excellence on boiled with 8% SDS to solubilize membranes. PG sacculi were collected by high- Microbial Biology) and the Council of Scientific and Industrial Research, gov- speed centrifugation and washed with water to remove SDS, followed by ernment of India (to M.R.). P.K.C. acknowledges financial support from the treatment with α-amylase and pronase to remove bound glycogen and proteins. University Grants Commission of India.

1. Höltje JV (1998) Growth of the stress-bearing and shape-maintaining murein sacculus 17. Hara H, Abe N, Nakakouji M, Nishimura Y, Horiuchi K (1996) Overproduction of of Escherichia coli. Microbiol Mol Biol Rev 62:181–203. penicillin-binding protein 7 suppresses thermosensitive growth defect at low osmo- 2. Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. larity due to an spr mutation of Escherichia coli. Microb Drug Resist 2:63–72. FEMS Microbiol Rev 32:149–167. 18. Marcyjaniak M, Odintsov SG, Sabala I, Bochtler M (2004) Peptidoglycan amidase 3. van Heijenoort J (2011) Peptidoglycan hydrolases of Escherichia coli. Microbiol Mol MepA is a LAS metallopeptidase. J Biol Chem 279:43982–43989. – Biol Rev 75:636 663. 19. Hashimoto M, Ooiwa S, Sekiguchi J (2012) Synthetic lethality of the lytE cwlO ge- 4. Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hy- notype in Bacillus subtilis is caused by lack of D,L-endopeptidase activity at the lateral drolases. FEMS Microbiol Rev 32:259–286. cell wall. J Bacteriol 194:796–803. 5. Glauner B, Höltje JV, Schwarz U (1988) The composition of the murein of Escherichia 20. Dörr T, Cava F, Lam H, Davis BM, Waldor MK (2013) Substrate specificity of an coli. J Biol Chem 263:10088–10095. elongation-specific peptidoglycan endopeptidase and its implications for cell wall 6. Sauvage E, Kerff F, Terrak M, Ayala JA, Charlier P (2008) The penicillin-binding pro- – teins: Structure and role in peptidoglycan biosynthesis. FEMS Microbiol Rev 32: architecture and growth of Vibrio cholerae. Mol Microbiol 89:949 962. 234–258. 21. Ng WL, Kazmierczak KM, Winkler ME (2004) Defective cell wall synthesis in Strep- 7. Magnet S, Dubost L, Marie A, Arthur M, Gutmann L (2008) Identification of the L,D- tococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or transpeptidases for peptidoglycan cross-linking in Escherichia coli. J Bacteriol 190: the VicR (YycF) response regulator. Mol Microbiol 53:1161–1175. 4782–4785. 22. Srivastava D, et al. (2018) A proteolytic complex targets multiple cell wall hydrolases 8. Bouhss A, Trunkfield AE, Bugg TD, Mengin-Lecreulx D (2008) The biosynthesis of in Pseudomonas aeruginosa. MBio 9:e00972-18. peptidoglycan lipid-linked intermediates. FEMS Microbiol Rev 32:208–233. 23. Bernal-Cabas M, Ayala JA, Raivio TL (2015) The Cpx envelope stress response modifies 9. Hugonnet JE, et al. (2016) Factors essential for L,D-transpeptidase-mediated pepti- peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein doglycan cross-linking and β-lactam resistance in Escherichia coli. eLife 5:e19469. YgaU. J Bacteriol 197:603–614. 10. Singh SK, SaiSree L, Amrutha RN, Reddy M (2012) Three redundant murein endo- 24. Delhaye A, Collet JF, Laloux G (2016) Fine-tuning of the Cpx envelope stress response peptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escher- is required for cell wall homeostasis in Escherichia coli. MBio 7:e00047-16. ichia coli K12. Mol Microbiol 86:1036–1051. 25. Rhodius VA, Suh WC, Nonaka G, West J, Gross CA (2006) Conserved and variable – 11. Singh SK, Parveen S, SaiSree L, Reddy M (2015) Regulated proteolysis of a cross-link functions of the σE stress response in related genomes. PLoS Biol 4:e2. specific peptidoglycan hydrolase contributes to bacterial morphogenesis. Proc Natl 26. Quintela JC, Caparrós M, de Pedro MA (1995) Variability of peptidoglycan structural – Acad Sci USA 112:10956 10961. parameters in gram-negative bacteria. FEMS Microbiol Lett 125:95–100. 12. Lee PA, Tullman-Ercek D, Georgiou G (2006) The bacterial twin-arginine translocation 27. Lavollay M, et al. (2011) The peptidoglycan of Mycobacterium abscessus is pre- pathway. Annu Rev Microbiol 60:373–395. dominantly cross-linked by L,D-transpeptidases. J Bacteriol 193:778–782. 13. Liu A, et al. (2010) Antibiotic sensitivity profiles determined with an Escherichia coli 28. Miller JH (1992) A Short Course in Bacterial Genetics: A Laboratory Manual and gene knockout collection: Generating an antibiotic bar code. Antimicrob Agents Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Lab Press, Chemother 54:1393–1403. 14. Rawlings ND, Barrett AJ (1999) MEROPS: The peptidase database. Nucleic Acids Res Cold Spring Harbor, NY). 27:325–331. 29. Baba T, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene 15. Lessard IA, Walsh CT (1999) Mutational analysis of active-site residues of the en- knockout mutants: The Keio collection. Mol Syst Biol 2:2006.0008. terococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala- 30. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Es- D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY. Chem Biol 6:177–187. cherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645. 16. Teh BA, et al. (2014) Structure to function prediction of hypothetical protein 31. Wientjes FB, Pas E, Taschner PEM, Woldringh CL (1985) Kinetics of uptake and in- KPN_00953 (Ycbk) from Klebsiella pneumoniae MGH 78578 highlights possible role in corporation of meso-diaminopimelic acid in different Escherichia coli strains. cell wall metabolism. BMC Struct Biol 14:7. J Bacteriol 164:331–337.

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