MreB proteins are bacterial actin homologs involved in cell morphogenesis and various other cellular processes. However, the effector proteins used by MreBs remain largely unknown. Bacillus subtilis has three MreB isoforms. Mbl and possibly MreB have previously been shown to be implicated in cell wall synthesis. We have now found that the third isoform, MreBH, colocalizes with the two other MreB isoforms in B. subtilis and also has an important role in cell morphogenesis. MreBH can physically interact with a cell wall hydrolase, LytE, and is required for its helical pattern of extracellular localization. Moreover, lytE and mreBH mutants exhibit similar cell-wall-related defects. We propose that controlled elongation of rod-shaped B. subtilis depends on the coordination of cell wall synthesis and hydrolysis in helical tracts defined by MreB proteins. Our data also suggest that physical interactions with intracellular actin bundles can influence the later localization pattern of extracellular effectors.

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.

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB In most bacteria, the rigid cell wall (CW) is responsible for providing shape and structural integrity to the cell. The thick CW of gram-positive bacteria is a multilayered structure composed predominantly of peptidoglycan (PG) (also called murein) and anionic polymers, particularly teichoic acids (TA) (for recent reviews, see references 5, 47, and 54). The highly cross-linked PG polymer (poly-N-acetylglucosamine and Nacetylmuramic acid) network is an essential determinant of cell shape and is responsible for protection from the cellular turgor pressure. Many roles for TA have been proposed, including cell shape maintenance (61, 68), resistance to antimicrobial peptides (1, 35, 36), biofilm formation (27), acid tolerance (9) and, efficient release of secreted proteins into the culture medium (49).TA are either covalently bound to the PG (wall TA [WTA]) or anchored to the cytoplasmic membrane (lipo-TA). In the gram-positive model organism Bacillus subtilis, WTA is present in quantities roughly equal to those of PG and constitutes the major class of anionic polymers (26). The type of WTA polymer varies between strains. In B. subtilis 168, the major WTA consists of a poly-glycerol-phosphate [poly-(Gro-P)] chain of 45 to 60 subunits and a "PG linkage unit" of N-acetylglucosamine-␤-(1-4)-N-acetylmannosamine (GlcNAc-ManNAc). WTA is covalently linked to the PG through a phosphodiester bond between the anomeric carbon of GlcNAc in the PG linkage unit and the 6-hydroxyl of MurNAc in the PG chain.In B. subtilis 168 the genes responsible for WTA synthesis are tagABDEFGHO and mnaA (Fig.

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