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Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases.

ABSTRACT: Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg²⁺ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg²⁺ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.

SUBMITTER: Tesson B

PROVIDER: S-EPMC8782873 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:By chance, we discovered a window of extracellular magnesium (Mg2+) availability that modulates the division frequency of Bacillus subtilis without affecting its growth rate. In this window, cells grown with excess Mg2+ produce shorter cells than do those grown in unsupplemented medium. The Mg2+-responsive adjustment in cell length occurs in both rich and minimal media as well as in domesticated and undomesticated strains. Of other divalent cations tested, manganese (Mn2+) and zinc (Zn2+) also resulted in cell shortening, but this occurred only at concentrations that affected growth. Cell length decreased proportionally with increasing Mg2+ from 0.2 mM to 4.0 mM, with little or no detectable change being observed in labile, intracellular Mg2+, based on a riboswitch reporter. Cells grown in excess Mg2+ had fewer nucleoids and possessed more FtsZ-rings per unit cell length, consistent with the increased division frequency. Remarkably, when shifting cells from unsupplemented to supplemented medium, more than half of the cell length decrease occurred in the first 10 min, consistent with rapid division onset. Relative to unsupplemented cells, cells growing at steady-state with excess Mg2+ showed an enhanced expression of a large number of SigB-regulated genes and the activation of the Fur, MntR, and Zur regulons. Thus, by manipulating the availability of one nutrient, we were able to uncouple the growth rate from the division frequency and identify transcriptional changes that suggest that cell division is accompanied by the general stress response and an enhanced demand to sequester and/or increase the uptake of iron, Mn2+, and Zn2+. IMPORTANCE The signals that cells use to trigger cell division are unknown. Although division is often considered intrinsic to the cell cycle, microorganisms can continue to grow and repeat rounds of DNA replication without dividing, indicating that cycles of division can be skipped. Here, we show that by manipulating a single nutrient, namely, Mg2+, cell division can be uncoupled from the growth rate. This finding can be applied to investigate the nature of the cell division signal(s).

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Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases.

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