Manjula Reddy

SUMMARY: Bacterial cell walls are made up of peptidoglycan (PG), a primary load-bearing layer that forms a protective exoskeleton around the cytoplasmic membrane. PG is a heteropolymer composed of glycan chains attached to short peptides that are crosslinked to each other, forming a mesh-like macromolecule that prevents osmotic lysis of the cell. Far from being a static exoskeleton, PG is a dynamic living polymer that undergoes continuous synthesis, expansion, remodeling, and turnover throughout the bacterial cell cycle. Central to the dynamic nature of PG is a finely tuned balance between two seemingly opposite processes—synthesis and hydrolysis. The PG synthases, which are essential for bacterial viability, have long been recognized as excellent drug targets and have therefore been studied extensively for decades. On the other hand, the significance of PG hydrolysis in diverse fundamental PG processes has become increasingly evident in recent years. Bacteria encode several highly conserved PG hydrolases with distinct substrate specificities that contribute to critical cellular processes, including cell wall expansion during growth, cell division, remodeling, and recycling, as well as predation and pathogenesis. Consequently, PG hydrolases represent promising targets for the development of novel antibacterial therapeutics. This review provides a comprehensive overview of the classification, physiological functions, and regulatory mechanisms governing the PG hydrolases in the model organism Escherichia coli and highlights parallels among related taxa across the bacterial kingdom.

To establish infection, Salmonella confronts a dynamic barrage of host‐induced stresses. The peptidoglycan layer is essential for maintaining bacterial cell integrity and counteracting these environmental stress. Its synthesis relies on the lipid carrier undecaprenyl phosphate, which is generated by the enzyme undecaprenyl pyrophosphate phosphatase (UppP). While UppP is linked to virulence in other pathogens, its role in Salmonella remains unclear. We show that an uppP mutant in S. Typhimurium exhibits altered cell morphology, reduced stiffness, and impaired survival in RAW 264.7 macrophages. The mutant is also attenuated in systemic infection in C57BL/6 mice. These defects are associated with increased sensitivity to nitrosative stress. Notably, iNOS inhibition or deficiency restores intracellular survival of the uppP mutant in both RAW 264.7 macrophages and the mouse model, implicating UppP in resistance to nitrosative stress. Our findings reveal a critical role for UppP in promoting Salmonella survival within macrophages and contributing to systemic pathogenesis.

Peptidoglycan (PG), a defining feature of the bacterial cell envelope, is crucial for cell integrity and morphology. PG is a macromolecular mesh consisting of glycan polymers crosslinked by short peptides encasing the cytoplasmic membrane. PG peptides contain two to five amino acids of both L- and D-configuration, with a conserved L-alanine residue at the first position in most bacteria. We previously identified a β-lactam hypersensitive mutant of Escherichia coli lacking yfiH (renamed pgeF ) that shows incorporation of L-serine or glycine instead of L-alanine. Here, we demonstrate that PgeF is an editing enzyme that imparts fidelity to the biosynthesis of PG peptides across bacterial genera. Using extensive mass spectrometry, we find accumulation of misincorporated cytoplasmic PG precursors in the absence of pgeF . Detailed biochemical analysis of several bacterial PgeF homologs reveals that PgeF specifically cleaves serine or glycine but not alanine from the PG precursors. Additionally, expression of heterologous ligases that incorporate L-serine or glycine is lethal in the absence of pgeF , indicating that L-alanine at the first position is crucial for wall integrity. Interestingly, PgeF is selectively conserved in bacteria and vertebrates; however, we find that the PG editing activity is exclusive to bacterial homologs. Furthermore, homologs from both taxa were previously characterized as purine nucleoside phosphorylases (PNP). Here, we find that they indeed have a weak PNP activity, but with no physiological relevance in bacterial systems. Overall, our study demonstrates the existence of a conserved proofreading pathway that is fundamental to the integrity of the bacterial cell wall.

Gram-negative bacterial cell envelope consists of a surface-exposed lipid bilayer (outer membrane or OM) that serves as a permeability barrier to maintain the cellular integrity. Beneath the OM is the periplasmic space that harbours peptidoglycan (PG), a highly cross-linked mesh-like glycan polymer closely encasing the inner membrane (IM). During growth of a bacterium balanced synthesis of the envelope components is required to maintain the cellular integrity, of which little is known. In this study, we identify sanA, an ORF of unknown function encoding a predicted IM-anchored protein as a factor contributing to balanced synthesis of PG in E. coli. Absence of SanA increased the rate of nascent PG strand incorporation, and restored growth and viability to several mutants defective in either cell division or cell elongation. Detailed mutant analysis of sanA showed that it is defective in the envelope barrier properties. Interestingly, overexpression of the periplasmic endopeptidases that cleave the cross-links of the PG mesh was able to alleviate the phenotypes of sanA mutant implying the envelope defects are due to alterations in the PG sacculus. Additionally, a SanA variant (^SSDsbA-SanA) targeted to the periplasm, complemented the SanA⁻ phenotypes suggesting it functions in the periplasmic phase of the PG synthesis. Further, we find that SanA functions independently of its paralog, ElyC, known to regulate the synthesis of enterobacterial common antigen (ECA), a surface polysaccharide found in the cell envelopes of most enteric bacteria. Overall, our results suggest a role for SanA in the maintenance of optimal PG synthesis, providing evidence for the existence of an additional layer of regulation in Gram-negative cell envelope biogenesis.

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the ‘space-making activity’ is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently cross-linked mesh-like polymer made up of several glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli , the ‘space-making activity’ is known to be achieved by cleavage of cross-links between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in making space for cell wall expansion in E. coli . We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential cross-link specific endopeptidases, MepS, MepM and MepH. Further detailed genetic and biochemical analysis revealed that MltD works in conjunction with cross-link specific endopeptidases to expand the PG sacculus. Interestingly, we find that cellular MltD levels are stringently controlled by two independent regulatory pathways. MltD undergoes regulated proteolysis by NlpI-Prc, a periplasmic adaptor-protease complex that specifically degrades two of the elongation-specific endopeptidases, MepS and MepH. In addition, MltD levels are post-transcriptionally controlled by RpoS, a stationary-phase specific sigma factor. Overall, our results show that coordinated cleavage of the glycan strands and the peptide cross-bridges facilitates the opening of the PG mesh for successful expansion of the cell wall during growth of a bacterium. AUTHOR SUMMARY: Most bacteria are protected by a cell wall made up of peptidoglycan (PG), a mesh-like large polymer. PG consists of several linear glycan strands interlinked through short peptide chains to form a continuous meshwork around the bacterial cytoplasmic membrane. Because PG tightly encases the cytoplasmic membrane, the growth of a bacterial cell is coupled to the expansion of PG requiring the activity of hydrolytic enzymes that cleave PG cross-links to make space for incorporation of new PG material. ln E. coli , a set of redundant cross-link specific endopeptidases are known to be crucial for expansion of PG. In this study, we show that cleavage of the glycan polymers by MltD, a glycan cleaving enzyme compensates the absence of cross-link cleavage and contributes to the expansion of PG. Overall, our work shows a previously unknown role of glycan hydrolases in cell wall expansion identifying these as potential targets for development of cell wall-specific antimicrobial agents.

Most bacteria are surrounded by a protective exoskeleton called peptidoglycan (PG), an extensively cross-linked mesh-like macromolecule. In bacteria, such as Escherichia coli , the cross-links in the PG are of two types: a major fraction is of 4-3 type whereas a minor fraction is of 3-3 type.

Bacterial cell wall contains peptidoglycan (PG) to protect the cells from turgor and environmental stress. PG consists of polymeric glycans cross-linked with each other by short peptide chains and forms an elastic mesh-like sacculus around the cytoplasmic membrane. Bacteria encode a plethora of PG hydrolytic enzymes of diverse specificity playing crucial roles in growth, division, or turnover of PG. In Escherichia coli, the cross-link-specific endopeptidases, MepS, -M, and -H, facilitate the enlargement of PG sacculus during cell elongation, whereas LytM-domain factors, EnvC and NlpD activate the division-specific amidases, AmiA, -B, and -C to facilitate the cell separation. In a screen to isolate additional factors involved in PG enlargement, we identified actS (encoding a LytM paralog, formerly ygeR) as its overexpression compensated the loss of elongation-specific endopeptidase, MepS. The overexpression of ActS resulted in the generation of partly denuded glycan strands in PG sacculi, indicating that ActS is either an amidase or an activator of amidase(s). The detailed genetic and biochemical analyses established that ActS is not a PG hydrolase, but an activator of the division-specific amidase, AmiC. However, interestingly, the suppression of the mepS growth defects by actS is not mediated through AmiC. The domain-deletion experiments confirmed the requirement of the N-terminal LysM domain of ActS for the activation of AmiC, but not for the alleviation of growth defects in mepS mutants, indicating that ActS performs two distinctive PG metabolic functions. Altogether our results suggest that in addition to activating the division-specific amidase, AmiC, ActS modulates yet another pathway that remains to be identified.