Autoregulation ofswrAAand Motility inBacillus subtilis

Abstract

We demonstrate that transcription of the gene swrAA, required for swarming migration in Bacillus subtilis, is driven by two promoters: a sigD-dependent promoter and a putative sigA-dependent promoter, which is inactive during growth in liquid Luria-Bertani medium and becomes active in the presence of the phosphorylated form of the response regulator DegU or on semisolid surfaces. Since sigD transcription is enhanced by SwrAA, this finding reveals that swrA expression is controlled by a positive feedback loop. We also demonstrate that the positive action of SwrAA in swimming and swarming motility is prevented in strains carrying a deletion of the two-component system degS-degU and that this effect is independent of swrAA transcription. Therefore, both DegU and SwrAA must be present to achieve full motility in B. subtilis.A wild-type copy of the swrAA gene is necessary for swarming motility in both undomesticated and laboratory strains of Bacillus subtilis (3,12). Laboratory strains (e.g., 168) that carry an sfp 0 allele and a frameshift mutation in the swrAA gene have a nonswarmer (Swr Ϫ ) phenotype (3,12,13,14,31). The role played by swrAA in swarming is to enhance transcription of the operon fla/che, which contains sigD, the gene coding for the alternative sigma factor D , as well as genes necessary for flagellum biosynthesis and chemotaxis (14). This notwithstanding, SwrAA does not resemble a DNA binding protein and does not show any particular feature by in silico analysis, nor does it display any similarity to characterized entries in protein databases, hampering the elucidation of its mechanism of action.In order to gain insights into the biological role that it plays in the activation of the swarming behavior, our efforts were concentrated on the expression profile of the swrA dicistronic operon which contains swrAA (3).The pleiotropic effects on the synthesis of degradative enzymes, competence, sporulation, and motility of mutations in the two-component system DegS-DegU have been extensively described previously (16,23). In particular, motility is negatively affected by degS(Hy) and degU(Hy) mutations. These mutations increase the half-life of the phosphorylated form of DegU (DegUϳP) (1,6,23). In contrast, the necessity of a low level of DegUϳP, for motility in general (36) and for swarming in particular (15, 37), has been pointed out for undomesticated and laboratory strains, but its role has yet to be established. Albeit in previous reports swrAA was never identified as a DegU-regulated gene (18, 26), it has been recently shown that swrAA transcription is negatively affected by deletions of degU or degS (15).Here we demonstrate that swrAA has two promoters: a Ddependent promoter, active in planktonic growth, and a putative A -dependent promoter triggered by DegUϳP, in concordance with published results (15). Furthermore, we show that in the absence of the functional alleles of either DegU or SwrAA B. subtilis is unable to fully swim as well as to swarm (12, 37). Although DegU activates swrAA transcripti...

SwrAA activates poly-γ-glutamate synthesis in addition to swarming in Bacillus subtilis

“…The swrAA 2 motility phenotype could be suppressed by a fla/che promoter upmutation, and microarray experiments indicated that SwrAA enhances expression of the fla/che operon, acting on the s A -dependent promoter P fla/che(A) (Kearns & Losick, 2005). Several lines of evidence point to a cooperation between SwrAA and DegU~P in motility (Kobayashi, 2007;Verhamme et al, 2007;Calvio et al, 2008) and here we demonstrate not only that the two proteins must cooperate to drive c-PGA production, but also that such an effect is achieved independently of the activation of the fla/che promoter.…”

“…Swimming and swarming assays were performed as already described (Calvio et al, 2008). Motility was scored as positive when cells colonized the entire 8.5 cm plate in 13 h at 30 uC.…”

“…Laboratory strains carry a single nucleotide insertion in the coding region of swrAA leading to premature product truncation (swrAA 2 ) (Kearns et al, 2004). In a laboratory strain, complementation experiments with a wild-type (wt) copy of the gene resulted in a significant improvement in both swimming capacity and ability to swarm on semi-solid surfaces treated with surfactin (Calvio et al, 2005(Calvio et al, , 2008, biasing cells into a s D -ON state (Kearns & Losick, 2005). The swrAA 2 motility phenotype could be suppressed by a fla/che promoter upmutation, and microarray experiments indicated that SwrAA enhances expression of the fla/che operon, acting on the s A -dependent promoter P fla/che(A) (Kearns & Losick, 2005).…”

“…Bacterial strains and primers used in this study are listed in Tables 1 and 2, respectively. Our swrAA + PB5249 strain is a spontaneous derivative of JH642 (Calvio et al, 2005), while PB5370 is a marker-less strain derived from PB5249 carrying the swrAA 2 gene (Calvio et al, 2008).…”

“…Transformants were selected for resistance to kanamycin and spectinomycin and checked for the conservation of the swrAA + marker. For the construction of degU32(Hy) strains carrying the mutant swrA promoters, strains PB5393, PB5394 and PB5395 (Calvio et al, 2008) were transformed with pLoxSpec/degSU(Hy), producing strains PB5438, PB5439 and PB5440, respectively.…”