Downloaded from www.microbiologyresearch.org by IP: 54.237.109.29 On: Thu, 09 Feb 2017 10:41:07 Influence of heterologous MreB proteins on cell morphology of Bacillus subtilis Kathrin Schirner and Jeff Errington Correspondence Jeff Errington jeff.errington@newcastle.ac.uk Institute for Cell and Molecular Biosciences, Newcastle University, Medical School, Framlington Place, Newcastle Upon Tyne NE2 4HH, UK Received 14 May 2009 Revised 17 July 2009 Accepted 23 July 2009 The prokaryotic cytoskeletal protein MreB is thought to govern cell shape by positioning the cell wall synthetic apparatus at growth sites in the cell. In rod-shaped bacteria it forms helical filaments that run around the periphery of the rod during elongation. Gram-positive bacteria often contain more than one mreB gene. Bacillus subtilis has three mreB-like genes, mreB, mbl and mreBH, the first two of which have been shown to be essential under normal growth conditions. Expression of an mreB homologue from the closely related organism Bacillus licheniformis did not have any effect on cell growth or morphology. In contrast, expression of mreB from the phylogenetically more distant bacterium Clostridium perfringens produced shape defects and ultimately cell death, due to disruption of the endogenous MreB cytoskeleton. However, expression of either mreB B. licheniformis (mreB Bl ) or mreB C. perfringens (mreB Cp ) was sufficient to confer a rod shape to B. subtilis deleted for the three mreB isologues, supporting the idea that the three proteins have largely redundant functions in cell morphogenesis. Expression of mreBCD Bl could fully compensate for the loss of mreBCD in B. subtilis and led to the formation of rod- shaped cells. In contrast, expression of mreBCD Cp was not sufficient to confer a rod shape to B. subtilis DmreBCD, indicating that a complex of these three cell shape determinants is not enough for cell morphogenesis of B. subtilis. INTRODUCTION For a long time, the presence of cytoskeletal elements has been considered a characteristic eukaryotic trait. However, in the last decade it has emerged that prokaryotes also have proteins with cytoskeletal function. One large group of cytoskeletal elements are the actin homologues, repre- sented mainly by the MreB proteins in bacteria. Homologues of MreB have been found in most species with complex, i.e. non-spherical, cell morphology (Bork et al., 1992; Carballido-Lo ´ pez, 2006b; van den Ent et al., 2001). Gram-negative organisms tend to have one mreB gene, usually in an operon with mreC and mreD genes, whilst Gram-positive bacteria often have multiple mreB isologues, but only one of these normally occurs in an operon with mreCD. Bacillus subtilis encodes three actin-like proteins called MreB, MreBH and Mbl. The mreB gene is defined by its position in an operon with mreC and mreD, whilst the other two genes are organized in monocistronic units. The single mutants of any of the three mreB genes are viable if cultured in medium supplemented with high concentra- tions of Mg 2+ : for mreB and mbl mutants, a concentration of approximately 3 mM is required, but the mreBH mutant only needs 100 mM Mg 2+ to grow (Carballido-Lo ´ pez et al., 2006; Formstone & Errington, 2005; Schirner & Errington, 2009). All three single mutants have a defect in cell morphology. mreB mutant cells have an increased diameter and are straight, whereas mbl mutants are also wider but are characteristically twisted. mreBH mutant cells are straight but thinner than the wild-type. MreB, Mbl and MreBH co-localize, forming one helical structure along the long axis of the cell underneath the cytoplasmic membrane (Carballido-Lo ´ pez et al., 2006; Jones et al., 2001). It has been suggested that they have a role in cell shape determination by organizing the peptidoglycan synthetic machinery at the cell surface (Carballido-Lo ´ pez, 2006). This is consistent with observations that peptidoglycan synthesis occurs in a similar helical pattern (Daniel & Errington, 2003; Hayhurst et al., 2008; Tiyanont et al., 2006a; Kawai et al., 2009a). MreC and MreD have a role in cell elongation, and interaction studies suggest that they are in the same morphogenic pathway as MreB (Figge et al., 2004; Jones et al., 2001; Kruse et al., 2005; Leaver & Errington, 2005; Lee & Stewart, 2003; Rogers et al., 1970; Wachi et al., 1989). Viability of mreC and mreD deletion strains is restricted to conditions in which the growth medium is supplemented with high concentrations of Mg 2+ , similar to mreB mutants, but additionally these strains require an osmoprotectant, such as sucrose, to prevent cell lysis; the mutant cells completely lose their rod shape and grow as Abbreviations: NA, nutrient agar; PAB, Difco Antibiotic Medium 3; YFP, yellow fluorescent protein. Microbiology (2009), 155, 3611–3621 DOI 10.1099/mic.0.030692-0 030692 G 2009 SGM Printed in Great Britain 3611