Genetic Evidence for Distinct Functions of Peptidoglycan Endopeptidases in Escherichia coli

Abstract

Peptidoglycan (PG) is an essential component of the bacterial exoskeleton that plays a pivotal role in the maintenance of cell shape and resistance to cell lysis under high turgor pressures. The synthesis and degradation of PG must be tightly regulated during bacterial cell elongation and division. Unlike enzymes involved in PG synthesis, PG hydrolases show high redundancy in many bacteria including Escherichia coli. In this study, we showed that PG endopeptidases have distinct roles in cell growth and division. Phenotypic analysis of mutants lacking one of seven PG endopeptidases identified a MepM-specific phenotype, salt sensitivity, and a MepS-specific phenotype, EDTA sensitivity. Complementation test in each phenotype showed that the phenotype of the mepM mutant was restored only by MepM, whereas the phenotype of the mepS mutant was restored by MepS or by overexpression of MepH, PbpG, or MepM. These distinct phenotypes depend on both the specific localizations and specific domains of MepM and MepS. Finally, using the identified phenotypes, we revealed that MepM and MepH were genetically associated with both penicillin-binding protein 1a (PBP1a) and PBP1b, whereas MepS and PbpG were genetically associated with only PBP1b. Notably, a defect in PBP1a or PBP1b phenocopied the mepM mutant, suggesting the importance of MepM on PG synthesis. Therefore, our results indicate that each PG endopeptidase plays a distinct role in cell growth and division, depending on its distinct domains and cellular localizations.

Distinct Amino Acid Availability-Dependent Regulatory Mechanisms of MepS and MepM Levels in Escherichia coli

“…This result is consistent with data from a previous report (Singh et al, 2012). In a previous study, we found a salt-sensitive phenotype in the mepM mutant (Park et al, 2020). A similar reversion of the phenotype was detected in the salt-sensitive phenotype of the mepM mutant (Figure 1C); the salt-sensitive phenotype of the mepM mutant was not observed in the M9 minimal medium.…”

“…In a previous study, we found an EDTA-sensitive phenotype in the mepS mutant (Park et al, 2020). This phenotype was restored by the overexpression of MepH, PbpG, and MepM, as well as MepS.…”

“…Distinct roles of PG endopeptidases were also reported in Lactobacillus plantarum (Duchene et al, 2019). Deletion of PG endopeptidase MepM caused salt sensitivity, which was also observed in the mutant defective for PBP1a or PBP1b (Park et al, 2020). Deletion of PG endopeptidase MepS resulted in EDTA sensitivity and the growth defect on the NA medium at high temperature (Singh et al, 2012;Park et al, 2020).…”

“…Deletion of PG endopeptidase MepM caused salt sensitivity, which was also observed in the mutant defective for PBP1a or PBP1b (Park et al, 2020). Deletion of PG endopeptidase MepS resulted in EDTA sensitivity and the growth defect on the NA medium at high temperature (Singh et al, 2012;Park et al, 2020). Depletion of MepM and MepS was synthetically lethal in the LB medium, and depletion of MepM, MepS, and MepH was synthetically lethal in the minimal medium (Singh et al, 2012).…”

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

“…In some cases, apparently redundant PG enzymes can be distinguished by differences in substrate specificity (10,11), subcellular localization (12)(13)(14), and interaction partners (13,(15)(16)(17)(18). Additionally, recent work from our group and others revealed the activity of certain "redundant" PG synthases and autolysins changes based on the physiochemical properties of the growth medium (19)(20)(21)(22)(23)(24)(25). We discovered two semi-redundant E. coli PG synthases, PBP1a and PBP1b, are preferentially required for PG integrity in opposing pH environments, in part due to pH-dependent differences in enzymatic activity (21).…”