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.

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage 29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the 29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the 29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.Bacillus subtilis ͉ phage 29 G enes of the mreB family encode homologues of eukaryotic actin (1, 2) that form a cytoskeleton in most non-spherical bacteria (3-6). MreB proteins form filamentous structures following a helical path around the inner surface of the cytoplasmic membrane (1). These actin-like filaments are continuously remodelled during cell-cycle progression (7-11). Evidence is accumulating that the bacterial MreB cytoskeleton plays key roles in several important cellular processes such as cell shape determination, chromosome segregation, and cell polarity (1,3,8,(12)(13)(14)(15)(16). Whereas Gram-negative bacteria have a single mreB gene, Gram-positive bacteria often have multiple mreB homologues. Bacillus subtilis encodes 3 MreB isoforms: MreB, Mbl,.For decades, evidence has been provided that replication of phage DNA, like that of other prokaryotic genomes, occurs at the cytoplasmic membrane (for review see 20). However, little is known about the proteins or their organization in membraneassociated replication of viral genomes in bacteria. Phages 29 and SPP1 infect the Gram-positive bacterium B. subtilis, and phage PRD1 infects the Gram-negative bacterium Escherichia coli. Whereas PRD1 and 29 use the protein-primed mechanism of DNA replication, phage SPP1 replicates its DNA initially via the theta mode and later via a rolling circle mode [reviewed in (21)]. Here we show a key role for the MreB cytoskeleton in phages replicating by different modes in the distantly related bacteria E. coli and B. subtilis. Thus, the efficiency of replication of phage PRD1, and that of phages SPP1 and 29, is severely affected in the absence of an intact cytoskeleton.The underlying mechanism by which the cytoskeleton leads to efficient phage DNA replication was analyzed in detail for B. subtilis phage 29, whose DNA replication has been well characterized in vitro. The 29 genome consists of a linear doublestranded DNA (dsDNA) with a terminal protein (TP) covalently linked at each 5Ј end that is the primer for the initiation of phage DNA replication. Hence, initiation of 29 DNA replication occurs via a so-called protein-prim...

A decade ago, two breakthrough descriptions were reported: 1) the first helix-like protein localization pattern of MreB and its paralog Mbl in Bacillus subtilis and 2) the crystal structure of Thermotoga maritima MreB1, which was remarkably similar to that of actin. These discoveries strongly stimulated the field of bacterial development, leading to the identification of many new cytoskeletal proteins (1) and the publication of many studies describing the helical patterns of protein, DNA and even lipid domains. However, today, new breakthroughs are shaking up what had become a dogma. Instead of helical structures, MreBs appear to form discrete patches that move circumferentially around the cell, questioning the idea of MreB cables forming an actin-like cytoskeleton. Furthermore, increasing evidence of biochemical properties that are unlike the properties of actin suggest that the molecular behavior of MreB proteins may be different. The aim of this review is to summarize the current knowledge of the so-called "actin-like" MreB cytoskeleton through a discussion of the model Gram-positive bacterium B. subtilis and the most recent findings in this rapidly evolving research field.