Extended phenotype of an mreB-like mutant in Azospirillum brasilense Emanuele G. Biondi, 1 3 Francesca Marini, 2 Fabio Altieri, 3 Laura Bonzi, 4 Marco Bazzicalupo 1 and Maddalena del Gallo 5 Correspondence Maddalena del Gallo delgallo@aquila.infn.it/maddalena. delgallo@libero.it 1 Dip. Biologia Animale e Genetica, Universita ` di Firenze, 50125 Firenze, Italy 2 Dip. Agrobiologia e Agrochimica, Universita ` della Tuscia, 01100 Viterbo, Italy 3 Dip. Scienze Biochimiche, Universita ` ‘La Sapienza’, 00185 Roma, Italy 4 Dip. Biologia Vegetale, Universita ` di Firenze, 50100 Firenze, Italy 5 Dip. Biologia di Base e Applicata, Universita ` dell’Aquila, Via Vetoio, Coppito, 67100 L’Aquila, Italy Received 12 November 2003 Revised 18 December 2003 Accepted 16 April 2004 Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant. Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation. INTRODUCTION In rod-shaped bacteria, cell morphogenesis takes place during the cell cycle. Several genes are involved in the maintenance of shape in Escherichia coli and Bacillus subtilis and mutations of these genes result in a spherical shape. mreB is the first gene of the murein (mre) gene cluster, mapping at position 73?2 of the E. coli chromosome. mreB encodes a 37 kDa protein that shows significant sequence similarity with members of the Hsp70 superfamily (Bork et al., 1992). MreB polymerizes to form helical filamentous structures, which are thought to be homologous to eukaryotic actin filaments (Jones et al., 2001; van den Ent et al., 2001). Inactivation of MreB results in the formation of spherical cells. The mutation of mreB in E. coli results in an increase in FtsI activity. This in turn increases the concentration of PBP3, a penicillin-binding protein, leading to hyperseptation (Wachi & Matsuhashi, 1989). Recently, in Caulobacter crescentus, it has been hypothesized that MreB filament structure serves as an organizer for the complex of PB2-peptidoglycan synthesis (Figge et al., 2004). mreB homologues have been found in many prokaryotes, includ- ing Archaea (Costa & Anton, 1993; Abhayawardhane & Stewart, 1995; Burger et al., 2000), but are absent in constitutively round bacteria (Jones et al., 2001). Members of the genus Azospirillum are capable of nitrogen fixation under microaerophilic conditions in association with the roots of several grasses (Do ¨bereiner, 1991). The vibroid form of Azospirillum brasilense has a polar flagellum and is highly motile (Do ¨bereiner & Day, 1976; Tarrand et al., 1978). However, under certain environmental conditions, particularly when inside plant tissue, Azospirillum cells become round and non-motile, and are referred to as encapsulated or C-forms (Becking, 1985; Berg et al., 1979; Do ¨bereiner & Day, 1976), or as cysts (Sadasivan & Neyra, 1985, 1987). The cellular envelopes of these forms are thicker than those of vegetative cells (Murray & Moyles, 1987). Polymorphic forms of Azospirillum appear in response to different factors, such as medium composition (C/N ratio), pH, age of the culture, polysaccharide production and plant colonization (Bashan et al., 1991; Becking, 1985; Berg et al., 1980; Burdman et al., 2000, 2001; Sadasivan & Neyra, 1985, 1987; Tal & Okon, 1985). Azospirillum appears to form several different types of cyst-like cell: pleomorphic cyst-like forms associated with cultured sugarcane-callus tissue and with root colonization 3Present address: Bauer Center for Genomics Research, Harvard University, 7 Divinity Ave, 02138, Cambridge, MA, USA. The GenBank/EMBL/DDBJ accession number for the A. brasilense mreB-like gene sequence reported in this paper is AF438483. Abbreviations: CFW, Calcofluor White; PS, polysaccharide; SEM, scanning electron microscopy. 0002-6904 G 2004 SGM Printed in Great Britain 2465 Microbiology (2004), 150, 2465–2474 DOI 10.1099/mic.0.26904-0