Role of RpoS and MutS in phase variation of Pseudomonas sp. PCL1171 Daan van den Broek, Thomas F. C. Chin-A-Woeng,3 Guido V. Bloemberg and Ben J. J. Lugtenberg Correspondence Ben J. J. Lugtenberg Lugtenberg@rulbim.leidenuniv.nl Institute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Received 22 November 2004 Revised 14 January 2005 Accepted 19 January 2005 Pseudomonas sp. strain PCL1171 undergoes reversible colony phase variation between opaque phase I and translucent phase II colonies, which is dependent on spontaneous mutations in the regulatory genes gacA and gacS. Mutation of the mutS gene and constitutive expression of rpoS increases the frequency at which gac mutants appear 1000- and 10-fold, respectively. Experiments were designed to study the relationship between gacS, rpoS and mutS. These studies showed that (i) a functional gac system is required for the expression of rpoS, (ii) RpoS suppresses the expression of mutS and therefore increases the frequency of gac mutants, and (iii) upon mutation of rpoS and gacS, the expression of mutS is increased. Mutation of gacS abolishes suppression of mutS expression in stationary growth, suggesting that additional gac-dependent factors are involved in this suppression. In conclusion, inefficient mutation repair via MutS, of which the expression is influenced by gacA/S itself and by rpoS in combination with other factors, contributes to the high frequency of mutations accumulating in gacA/S. The role of RpoS in the growth advantage of a gac mutant was analysed, and mutation of rpoS only reduced the length of the lag phase, but did not affect the growth rate, suggesting a role for both RpoS and a reduction of metabolic load in the growth advantage of a gac mutant. INTRODUCTION Phase variation is a process of reversible high-frequency phenotypic switching that is mediated by mutations, reorganizations or other modifications of DNA. This pro- cess is used by several bacterial species to generate popula- tion diversity, which in turn increases bacterial fitness and plays a role in niche adaptation (Saunders et al., 2003). Phase variation in Pseudomonas sp. PCL1171 is dependent on the accumulation and subsequent removal of muta- tions in the gacA and gacS genes (van den Broek et al., 2005). These genes affect the production of secondary metabolites, such as a lipopeptide with antifungal activity, a biosurfactant, and the exo-enzymes chitinase, lipase and protease (van den Broek et al., 2003). The gacA/gacS two-component regulatory system consists of a membrane- bound sensor kinase, GacS, and a response regulator, GacA. The latter protein belongs to the FixJ family of transcrip- tional regulators (Laville et al., 1992; Rich et al., 1994). The gac system regulates secondary metabolism and the pro- duction of exo-enzymes (Hrabak & Willis, 1993; Laville et al., 1992; Schmidli-Sacherer et al., 1997; Reimmann et al., 1997). In several Pseudomonas species, the gacA and gacS genes are subject to spontaneous mutation, and gac mutants appear for example in nutrient-rich liquid medium (Bull et al., 2001; Duffy & Defago, 1995; van den Broek et al., 2003) and on plant roots (Sanchez-Contreras et al., 2002; Chancey et al., 2002; Schmidt-Eisenlohr et al., 2003; Achouak et al., 2004). In PCL1171, spontaneous mutation of the gacA/S system is the basis for phase variation. Information on factors influencing the introduction of mutations in the gac genes of Pseudomonas as part of a phase-variation mechanism or by phenotypic selection are scarce. Previously, we have reported that MutS-dependent mis- match repair affects phase variation (van den Broek et al., 2003, 2005). MutS is involved in the methyl-directed recognition of DNA mismatches related to replication, which include base mismatches and small insertion and deletion mispairs (Modrich, 1991). The expression of mismatch repair systems can be negatively influenced by stress or growth-limiting conditions, resulting in increased genetic and population diversity (Kivisaar, 2003). For example, in Escherichia coli, mutS is negatively regulated by the general stress-response sigma factor RpoS (Tsui et al., 1997). In Pseudomonas, the regulatory link between RpoS and MutS and the role of these proteins in bacterial phase variation have not been studied. To identify and understand genetic factors which influence the high Abbreviation: RT-PCR, real-time PCR. 3Present address: LUMC, Molecular Cell Biology, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. 0002-7777 G 2005 SGM Printed in Great Britain 1403 Microbiology (2005), 151, 1403–1408 DOI 10.1099/mic.0.27777-0