2b). On the other hand, although the motA and motB mutants produced flagella, they were still unable to move because MotA and MotB formed a proton channel that transferred proton-motive force to drive the flagella (Asai et al., 2003); either motA or motB gene mutations resulted in the production of nonfunctional flagella (Figs 2b and 3c). These data demonstrate that the swarming of C. freundii is dependent on functional flagella, as in other swarming bacteria (Kearns, 2010). The largest gene cluster identified in our study is involved in the synthesis of lipopolysaccharide. Altogether, 13 mutants were isolated,

of which six mutated genes –wzx, rfaL, rfbX, rfaJ/CKO_05084, rfaJ/CKO_05086, and rfaG– were identified. The swarming ability of these mutants was dramatically decreased (two of them are shown in Fig. 3g and h as examples). As observed directly LDE225 supplier under inverted microscope, only a few bacterial cells were actively motile in the swarming colonies of these mutants and these were mainly distributed at the edges. In the central region, most cells formed aggregates that scarcely moved (Videos S2 and S3). In contrast, Nutlin-3a cell line in wild-type colonies, all swarming cells were actively motile (cells in the edge of colonies were less active) and no aggregation was observed (Video S1). The hydrophilicity

of these mutants was decreased compared with the wild type (Fig. S2), which could have led to the aggregation. In a previous study, many transposon swarming mutants isolated in Salmonella enterica serovar Typhimurium have been shown to have mutations in the lipopolysaccharide biosynthetic pathway (Toguchi et al., 2000). The authors suggested that

the O antigen directly or indirectly improved the surface wettability required for swarm colony expansion. Our observation showed that the polysaccharide structure on the cell surface had important role not only in overcoming Bcl-w friction between bacterial cells and media surface, but also in reducing intercellular interaction. The poorly motile aggregates formed with bacteria on the agar surface because of the O antigen defects could account for the defective swarming in addition to the decreased wettability of the agar surface. rcsC and rcsD mutants were identified in this study, and both mutants displayed defective swarming behavior (Fig. 3a and b). The products of rcsC and rcsD, together with RcsB, constitute the regulator of the capsule synthesis (Rcs) phosphorelay system. The regulator RcsB is activated by the transfer of a phosphate group from its cognate sensor, RcsC, through a histidine-containing phosphotransmitter (Hpt) domain intermediate called RcsD (previously called YojN; Takeda et al., 2001). The Rcs system has been implicated in the regulation of bacterial responses to osmotic and other kinds of membrane stress, growth at low temperatures in the presence of glucose and zinc, and growth on solid surfaces (Carballes et al.