Hydrolysis of peptidoglycan is modulated by amidation of meso‐diaminopimelic acid and Mg²⁺ in Bacillus subtilis

Abstract

SummaryThe ability of excess Mg2+ to compensate the absence of cell wall related genes in Bacillus subtilis has been known for a long time, but the mechanism has remained obscure. Here, we show that the rigidity of wild‐type cells remains unaffected with excess Mg2+, but the proportion of amidated meso‐diaminopimelic (mDAP) acid in their peptidoglycan (PG) is significantly reduced. We identify the amidotransferase AsnB as responsible for mDAP amidation and show that the gene encoding it is essential without added Mg2+. Growth without excess Mg2+ causes ΔasnB mutant cells to deform and ultimately lyse. In cell regions with deformations, PG insertion is orderly and indistinguishable from the wild‐type. However, PG degradation is unevenly distributed along the sidewalls. Furthermore, ΔasnB mutant cells exhibit increased sensitivity to antibiotics targeting the cell wall. These results suggest that absence of amidated mDAP causes a lethal deregulation of PG hydrolysis that can be inhibited by increased levels of Mg2+. Consistently, we find that Mg2+ inhibits autolysis of wild‐type cells. We suggest that Mg2+ helps to maintain the balance between PG synthesis and hydrolysis in cell wall mutants where this balance is perturbed in favor of increased degradation.

Flotillin mediated membrane fluidity controls peptidoglycan synthesis and MreB movement

“…S4 ). SMM has a higher Mg 2+ concentration, which is known to rescue various cell shape mutations by inhibition of cell wall hydrolysis 37 . However, the increase in Mg 2+ was not sufficient to explain the reversal of phenotype as cells grown on LB supplemented with Mg 2+ (6 mM, concentration in SMM, or 20 mM) still displayed the elongated phenotype with delocalized peptidoglycan synthesis (Fig.…”

“…Strikingly, none of the mutant strains had an apparent phenotype when cultivated in Spizizen’s minimal medium (SMM, Figure 2B ), and peptidoglycan synthesis and lipid dyes were no longer accumulating at division sites in the Δ floAT strain ( Figure 2 - figure supplement 2 ). SMM has a higher Mg 2+ concentration, which is known to rescue various cell shape mutations by inhibition of cell wall hydrolysis 39 . However, the increase in Mg 2+ was not sufficient to explain the reversal of phenotype as cells grown on LB supplemented with Mg 2+ (6 mM, concentration in SMM, or 20 mM) still displayed the elongated phenotype with delocalized peptidoglycan synthesis (Figure 2 - figure supplement 3) .…”

AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

“…In B. subtilis, the ΔasnB mutant was more sensitive to lysozyme and to antibiotics targeting different steps of cell-wall synthesis [5]. We therefore tested whether the C. difficile asnB mutant was more sensitive to these compounds.…”

“…More recently, meso-DAP amidation of PG was shown to impact the activity of l,d-transpeptidases in Enterococcus faecium and M. tuberculosis and to be required for growth of these species [4]. The amidotransferase AsnB from Bacillus subtilis was also shown to be responsible for meso-DAP amidation [5]. In Lactococcus lactis, asnH encodes the asparagine synthetase involved in the amidation of PG d-Asp residues [6] and the absence of this modification increased the cell susceptibility to endogenous autolysins, lysozyme and nisin.…”

Recent advances in understanding how rod-like bacteria stably maintain their cell shapes

“…Thus, the regulation of cell-wall hydrolases is highly important for the maintenance of rod-like shape. Accordingly, variations in hydrolase activity through substrate modification 102 or through changes of hydrolase abundance lead to changes in cell shape 103 . Thus, it is conceivable that the rate of cell-surface expansion is regulated through hydrolase activity while cell-wall synthesis would be responsible merely for maintaining cell-wall integrity.…”