Epr plays a key role in DegU-mediated swarming motility of Bacillus subtilis

Epr, a minor extracellular protease, is involved in the swarming motility of Bacillus subtilis . It does so by providing essential signals required for swarming. It has also been demonstrated that DegU is required for swarming and that it occurs at very low levels of DegU∼P and is inhibited at high levels of DegU∼P. In this study, we show that maximal epr expression is observed at very low concentrations of DegU∼P, whereas it is repressed at high DegU∼P. A parallel effect of DegU∼P levels on swarming motility is also observed, where very low levels of DegU∼P support swarming and excessive DegU∼P abolishes swarming. We further demonstrate that the defect of swarming motility in a degU ⁻ strain can be rescued, albeit incompletely, by increased expression of an exogenous epr gene. We also show that an additional extracellular factor(s), apart from epr , regulated by DegU, is required for robust swarming.     

Enhanced Control of Cucumber Wilt Disease by Bacillus amyloliquefaciens SQR9 by Altering the Regulation of Its DegU Phosphorylation

Bacillus amyloliquefaciens strain SQR9, isolated from the cucumber rhizosphere, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion through efficient root colonization. In the Gram-positive bacterium Bacillus, the response regulator DegU regulates genetic competence, swarming motility, biofilm formation, complex colony architecture, and protease production. In this study, we report that stepwise phosphorylation of DegU in B. amyloliquefaciens SQR9 can influence biocontrol activity by coordinating multicellular behavior and regulating the synthesis of antibiotics. Results from in vitro and in situ experiments and quantitative PCR (qPCR) studies demonstrate the following: (i) that the lowest level of phosphorylated DegU (DegU∼P) (the degQ mutation) impairs complex colony architecture, biofilm formation, colonization activities, and biocontrol efficiency of Fusarium wilt disease but increases the production of macrolactin and bacillaene, and (ii) that increasing the level of DegU∼P by degQ and degSU overexpression significantly improves complex colony architecture, biofilm formation, colonization activities, production of the antibiotics bacillomycin D and difficidin, and efficiency of biocontrol of Fusarium wilt disease. The results offer a new strategy to enhance the biocontrol efficacy of Bacillus amyloliquefaciens SQR9.     

DegU-phosphate activates expression of the anti-sigma factor FlgM in Bacillus subtilis

The bacterial flagellum is a complex molecular machine that is assembled by more than 30 proteins and is rotated to propel cells either through liquids or over solid surfaces. Flagellar gene expression is extensively regulated to co-ordinate flagellar assembly in both space and time. In Bacillus subtilis, the proteins of unknown function, SwrA and SwrB, and the alternative sigma factor σ(D) are required to activate expression of the flagellar filament protein, flagellin. Here we determine that in the absence of SwrA and SwrB, the phosphorylated form of the response regulator DegU inhibits σ(D) -dependent gene expression indirectly by binding to the P(flgM) promoter region and activating expression of the anti-sigma factor FlgM. We further demonstrate that DegU-P-dependent activation of FlgM is essential to inhibit flagellin expression when flagellar basal body assembly is disrupted. Regulation of FlgM is poorly understood outside of Salmonella, and differential control of FlgM expression may be a common means of coupling flagellin expression to flagellar assembly.     

Closely linked genetic loci required for swarm cell differentiation and multicellular migration by Proteus mirabilis

The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper-flagellated filaments able to undergo repeated cycles of co-ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm-defective mutants lacked flagella and were non-motile non-swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non-swarming, MNS), or were motile and able to form swarm cells but displayed aberrant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed-field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2 mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112 kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and a second within a neighbouring 95 kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).     

Bacillus subtilis two-component system sensory kinase DegS is regulated by serine phosphorylation in its input domain

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity. The phosphorylation state of the response regulator DegU also does not confer a straightforward "on/off" response; it is fine-tuned and at different levels triggers different sub-regulons. Here we describe serine phosphorylation of the DegS sensing domain, which stimulates its kinase activity. We demonstrate that DegS phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number of physiological assays focused on different processes regulated by DegU, DegS S76D phosphomimetic mutant behaved like a strain with intermediate levels of DegU phosphorylation, whereas DegS S76A behaved like a strain with lower levels of DegU phophorylation. These findings suggest a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system.     

Swarming motility in undomesticated Bacillus subtilis

Swarming motility was identified and characterized in an undomesticated strain of Bacillus subtilis. Rapid surface migration was preceded by a cell density-dependent lag period, which could be eliminated if actively swarming cells were used as the inoculum. The leading edge of the swarm was characterized by multicellular rafts of highly flagellated cells. Flagellum biosynthesis and surfactant production were required for swarming. Swarming was not found in any of several standard laboratory strains. Laboratory strains are characteristically unable to produce surfactant, but such a strain remained unable to swarm even when surfactant was provided by extracellular complementation. We conclude that robust swarming is a feature of undomesticated B. subtilis and that this behaviour has been lost or attenuated in laboratory strains through the accumulation of multiple genetic defects.     

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.     

The Role of SwrA,DegU and PD3 in fla/che Expression in B. subtilis

In B. subtilis swarming and robust swimming motility require the positive trigger of SwrA on fla/che operon expression. Despite having an essential and specific activity, how SwrA executes this task has remained elusive thus far. We demonstrate here that SwrA acts at the main σ^A-dependent fla/che promoter P_A(fla/che) through DegU. Electrophoretic mobility shift assays (EMSA) reveal that SwrA forms a complex with the phosphorylated form of DegU (DegU~P) at P_A(fla/che) while it is unable to do so with either unphosphorylated DegU or the DegU32(Hy) mutant protein. Motility assays show that a highly phosphorylated DegU is not detrimental for flagellar motility provided that SwrA is present; however, DegU~P represses P_A(fla/che) in the absence of SwrA. Overall, our data support a model in which DegU~P is a dual regulator, acting either as a repressor when alone or as a positive regulator of P_A(fla/che) when combined with SwrA. Finally, we demonstrate that the σ^D-dependent P_D3(fla/che) promoter plays an important role in motility, representing a contingent feedback loop necessary to maintain basal motility when swrA is switched to the non-functional swrA ^- status.     

RssAB signaling coordinates early development of surface multicellularity in Serratia marcescens

Bacteria can coordinate several multicellular behaviors in response to environmental changes. Among these, swarming and biofilm formation have attracted significant attention for their correlation with bacterial pathogenicity. However, little is known about when and where the signaling occurs to trigger either swarming or biofilm formation. We have previously identified an RssAB two-component system involved in the regulation of swarming motility and biofilm formation in Serratia marcescens. Here we monitored the RssAB signaling status within single cells by tracing the location of the translational fusion protein EGFP-RssB following development of swarming or biofilm formation. RssAB signaling is specifically activated before surface migration in swarming development and during the early stage of biofilm formation. The activation results in the release of RssB from its cognate inner membrane sensor kinase, RssA, to the cytoplasm where the downstream gene promoters are located. Such dynamic localization of RssB requires phosphorylation of this regulator. By revealing the temporal activation of RssAB signaling following development of surface multicellular behavior, our findings contribute to an improved understanding of how bacteria coordinate their lifestyle on a surface.     

Response regulator DegU of Listeria monocytogenes regulates the expression of flagella-specific genes

An isogenic mutant of Listeria monocytogenes EGD with a deletion of the response regulator gene degU showed a lack of motility due to the absence of flagella. In the present study, we used two-dimensional gel electrophoresis, mass-spectrometry and microarray analyses to identify the listerial genes that depend on DegU for expression. We found that the two L. monocytogenes operons encoding flagella-specific genes and the monocistronically transcribed flaA gene are positively regulated by DegU at 24 degrees C, but are not expressed at 37 degrees C.     

Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

AIMS: (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(furanone) of the marine alga Delisea pulchra was synthesized, and its inhibition of swarming motility and biofilm formation of Bacillus subtilis was investigated. METHODS AND RESULTS: Furanone was found to inhibit both the growth of B. subtilis and its swarming motility in a concentration-dependent way. In addition, as shown by confocal scanning laser microscopy, furanone inhibited the biofilm formation of B. subtilis. At 40 microg ml(-1), furanone decreased the biofilm thickness by 25%, decreased the number of water channels, and reduced the percentage of live cells by 63%. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Natural furanone has potential for controlling the multicellular behaviour of Gram-positive bacteria.     

Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.