A magnesium‐dependent mreB null mutant: implications for the role of mreB in Bacillus subtilis

Abstract

SummaryMreB shares a common prokaryotic ancestor with actin and is present in almost all rod-shaped bacteria. MreB proteins have been implicated in a range of important cell processes, including cell morphogenesis, chromosome segregation and cell polarity. The mreB gene frequently lies at the beginning of a cluster of genes, immediately upstream of the conserved mreC and mreD genes. RNA analysis showed that in Bacillus subtilis mreB is co-transcribed with mreC and that these genes form part of an operon under the control of a promoter(s) upstream of mreB . Construction of an in-frame deletion of mreB and its complementation by mreB + only, in trans , established that the gene is important for maintenance of cell width and cell viability under normal growth conditions, independent of polar effects on downstream genes. Remarkably, virtually normal growth was restored to the mreB null mutant in the presence of high concentrations of magnesium, especially when high concentrations of the osmoprotectant, sucrose were also present. Under these conditions, cells could be maintained in the complete absence of an mreB gene, with almost normal morphology. No detectable effect on chromosome segregation was evident in the mutant, nor was there an effect on the topology of nascent peptidoglycan insertion. A GFP-MreB fusion was used to look at the localization of MreB in live cells. The pattern of localization was similar to that previously described, but no tight linkage to nucleoid positioning was evident. Propagation of the mreB null mutant in the absence of magnesium and sucrose led to a progressive increase in cell width, culminating in cell lysis. Cell division was also perturbed but this effect may be secondary to the disturbance in cell width. These results suggest that the major role of MreB in B. subtilis lies in the control of cell diameter.

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

“…We found that Mg 2+ inhibited the lysis of the ΔasnB mutant, and it was also previously shown to prevent the lysis of Δ mreB mutants (Formstone and Errington, 2005). Furthermore, Mg 2+ inhibits cell lysis induced by diverse conditions in unrelated Gram‐negative microorganisms (Rayman and MacLeod, 1975; Leduc et al ., 1982; Gschwender and Hofschneider, 1969).…”

“…Hypotheses put forward to explain this effect include the idea that Mg 2+ may somehow rigidify the cell wall, or that it may affect the activity of cell wall associated enzymes, or both (Formstone and Errington, 2005). We have tested the effect of Mg 2+ on the rigidity of the cell wall of wild‐type B. subtilis cells using AFM and have found no effect.…”

“…The system involved in sidewall elongation is controlled by the Rod system, a system of morphogenetic proteins including the bacterial actin homologue MreB as well as MreC, MreD, RodA, RodZ and elongation‐specific PBPs (Eun et al ., 2015). One of the unexplained aspects of the Rod system in B. subtilis is that the lethality and/or morphological defects of the absence of some of its components can be overcome by adding Mg 2+ to the growth medium (Formstone and Errington, 2005). mreB and its paralog mbl are essential in standard laboratory conditions.…”

“…However, when growth media are supplemented with 5–25 mM Mg 2+ , mreB and mbl mutants grow and divide normally and assume a normal rod‐shaped morphology. When Mg 2+ is depleted, the morphological phenotype becomes manifest and they grow as deformed and ballooning cells before eventually lysing (Formstone and Errington, 2005; Chastanet and Carballido‐Lopez, 2012). Mg 2+ likewise suppresses the viability and/or morphological defects of several other cell wall related mutants (e.g.…”

“…Two main hypotheses have been put forth to explain it. It has been suggested that Mg 2+ may have a structural role in enhancing the rigidity of the cell wall or, alternatively (or simultaneously), a regulatory role by affecting the activity of cell‐wall embedded enzymes (Formstone and Errington, 2005). It has been known for several decades that Mg 2+ binds to cell walls of Gram‐positive bacteria (Beveridge and Murray, 1980; Heckels et al ., 1977).…”