SummaryBacterial peptidoglycan (PG or murein) is a single, large, covalently cross-linked macromolecule and forms a mesh-like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre-existing crosslinks for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross-bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross-link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.

Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion in Escherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI-Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link-cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.bacterial morphogenesis | peptidoglycan | regulated proteolysis | MepS | NlpI-Prc P eptidoglycan (PG or murein) is a unique and essential constituent of eubacterial cell walls, thus making it an excellent target for several antimicrobial agents. It is a single, large, extensively cross-linked macromolecule that forms a mesh-like sacculus protecting cells against intracellular turgor pressure in addition to conferring cell shape. Structurally, the PG sacculus is made up of linear glycan strands cross-linked to each other by short peptide chains forming a continuous layer around the cytoplasmic membrane. The glycan strands are made up of alternating N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) disaccharide units in which NAM is covalently attached to a peptide chain containing 2-to 5-amino acid residues, with the pentapeptide consisting of L-alanine (ala)−D-glutamic acid (glu)−meso-diaminopimelic acid (mDAP)−D-ala−D-ala. Normally, D-ala of one peptide chain is cross-linked to mDAP of another peptide chain of an adjacent glycan strand, resulting in an extensively cross-linked single-or multilayered sacculus (1).Because the murein sacculus totally encircles the cytoplasmic membrane, growth of a cell is tightly coupled to expansion of PG. Growth of the PG sacculus is a dynamic and coordinated event requiring concerted action both of murein hydrolases that facilitate cleavage of cross-links for the insertion of nascent murein material and of synthases that catalyze cross-link formation between adjacent glycan strands (Fig. 1) (2, 3).Escherichia coli encodes multiple PG synthases that catalyze the formation of D-ala−mDAP cross-links in the PG sacculus. The class I enzymes (PBP1a and PBP1b, encoded by mrcA and mrcB, respectively) are bifunctional and possess both glycosyl transferase (GT) and tr...

The outer membrane of Gram-negative bacteria is an asymmetric lipid bilayer consisting of an essential glycolipid lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the inner leaflet. Here, we show that yciM, a gene encoding a tetratricopeptide repeat protein of unknown function, modulates LPS levels by negatively regulating the biosynthesis of lipid A, an essential constituent of LPS. Inactivation of yciM resulted in high LPS levels and cell death in Escherichia coli; recessive mutations in lpxA, lpxC or lpxD that lower the synthesis of lipid A, or a gain of function mutation in fabZ that increases the formation of membrane phospholipids, alleviated the yciM mutant phenotypes. A modest increase in YciM led to significant reduction of LPS and increased sensitivity to hydrophobic antibiotics. YciM was shown to regulate LPS by altering LpxC, an enzyme that catalyses the first committed step of lipid A biosynthesis. Regulation of LpxC by YciM was contingent on the presence of FtsH, an essential membrane-anchored protease known to degrade LpxC, suggesting that FtsH and YciM act in concert to regulate synthesis of lipid A. In summary, this study demonstrates an essential role for YciM in regulation of LPS biosynthesis in E. coli.

The function of SufI, a well-studied substrate of the TatABC translocase in Escherichia coli, is not known. It was earlier implicated in cell division, based on the finding that multiple copies of sufI suppressed the phenotypes of cells with mutations in ftsI (ftsI23), which encodes a divisomal transpeptidase. Recently, sufI was identified as both a multicopy suppressor gene and a synthetic lethal mutant of ftsEX, which codes for a division-specific putative ABC transporter. In this study, we show that sufI is essential for the viability of E. coli cells subjected to various forms of stress, including oxidative stress and DNA damage. The sufI mutant also exhibits sulA-independent filamentation, indicating a role in cell division. The phenotypes of the sufI mutant are suppressed by factors that stabilize FtsZ ring assembly, such as increased expression of cell division proteins FtsQAZ or FtsN or the presence of the gain-of-function ftsA* (FtsA R286W) mutation, suggesting that SufI is a divisomal protein required during stress conditions. In support of this, multicopy sufI suppressed the divisional defects of mutants carrying the ftsA12, ftsQ1, or ftsK44 allele but not those of mutants carrying ftsZ84. Most of the division-defective mutants, in particular those carrying a ⌬ftsEX or ftsI23 allele, exhibited sensitivity to oxidative stress or DNA damage, and this sensitivity was also abolished by multiple copies of SufI. All of these data suggest that SufI is a division component involved in protecting or stabilizing the divisomal assembly under conditions of stress. Since sufI fulfils the requirements to be designated an fts gene, we propose that it be renamed ftsP.

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