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.

Keywords: LytM domain; MepH; MepM; MepS; NlpC/P60 domain; endopeptidase; peptidoglycan; peptidoglycan hydrolase.

FIGURE 1

Distinct phenotypes of PG endopeptidase mutants. The wild-type and PG endopeptidase mutant cells were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl or 1 mM EDTA as indicated.

FIGURE 2

Complementation analysis of the phenotypes of the mepM and mepS mutants. (A) The wild-type, mepM mutant, and mepM mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl and the indicated concentrations of arabinose. (B) The wild-type, mepS mutant, and mepS mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 1 mM EDTA and the indicated concentrations of arabinose.

FIGURE 3

The importance of the localization of MepM and MepS on their function. (A) The wild-type, mepM mutant, and mepM mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl and the indicated concentrations of arabinose. (B) The wild-type, mepS mutant, and mepS mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 1 mM EDTA and the indicated concentrations of arabinose.

FIGURE 4

Distinct roles of MepM and MepS are not due to their unique localization. (A) The wild-type, mepM mutant, and mepM mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl and the indicated concentrations of arabinose. (B) The wild-type, mepS mutant, and mepS mutant cells harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 1 mM EDTA and the indicated concentrations of arabinose.

FIGURE 5

Distinct effects of MepM, MepS, MepH, and PbpG on PBP1a and PBP1b. (A) The effect of MepM on PBP1a and PBP1b. The cells of the indicated strains were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or an LB plate containing 1% arabinose. (B) Salt sensitivity of the mrcA, mrcB, lpoA, and lpoB mutants. The cells of the indicated strains were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl or 1 mM EDTA as indicated. (C) The effect of MepS on PBP1b. The cells of the indicated strains were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or an LB plate containing 1 mM EDTA and 1% arabinose. (D) The effects of MepH and PbpG on PBP1a and PBP1b. The cells of the indicated strains were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or an LB plate containing 1 mM EDTA and 1% arabinose.

FIGURE 6

The importance of MepM in adaptation to salt stress. (A) The effect of simultaneous overexpression of MepS, MepH, and PbpG on salt sensitivity of the mepM mutant. The cells of the wild-type, mepM mutant, and mepM mutant harboring the indicated plasmids were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 mM NaCl and the indicated concentrations of arabinose. (B) The effect of the mepS mepH double or mepS mepH pbpG triple deletion on adaptation to salt stress. The wild-type and the indicated mutant cells were serially diluted from 10⁸ to 10⁴ cells/mL in 10-fold steps and spotted onto an LB plate or LB plates containing 750 or 800 mM NaCl as indicated.

FIGURE 7

The model for the distinct roles of MepM, MepS, MepH, and PbpG. MepM and MepS are located in the inner membrane and outer membrane, respectively, whereas MepH and PbpG are located in the periplasm. MepM is a transmembrane protein, whereas MepS is a lipoprotein. MepM is genetically associated with both PBP1a and PBP1b, and MepH supports its function. MepS is genetically associated with PBP1b, and PbpG supports its function. The width of arrows indicates the degree of the effects of endopeptidases on PBP1a or PBP1b.