Gene position in a long operon governs motility development in <i>Bacillus subtilis</i>

Cozy, Loralyn M.; Kearns, Daniel B.

doi:10.1111/j.1365-2958.2010.07112.x

Cited by 65 publications

(109 citation statements)

References 71 publications

Supporting

Mentioning

109

Contrasting

Order By: Relevance

“…These cells therefore appear identical to B. subtilis chains that previously were first identified as a distinct subpopulation present in liquid cultures. In such cells, the production and activity of the transcription factor s D is switched off, resulting in the absence of hag expression (flagellin) and of the expression of certain autolysins encoded by lytABC Chen et al, 2009;Cozy & Kearns, 2010).…”

Section: Resultsmentioning

confidence: 99%

“…These chain forms appeared identical to the subpopulation of chaining cells in cultures of B. subtilis observed at the centre of swarms on rich medium (Kearns & Losick, 2003Julkowska et al, 2005). The origin of these cells has been shown in liquid cultures to be dependent upon a bistable switch Cozy & Kearns, 2010), which ultimately regulates the level of the transcription factor s D , leading to repression of flagellar and autolysin genes in the chain forms. The SinI/SinR and Slr regulators also play a critical role in this switch, involving the upregulation of extracellular matrix production (Chu et al, 2006(Chu et al, , 2008Kobayashi, 2008;Chai et al, 2009; see also Chen et al, 2009), as we also found here.…”

Section: Long-chain Formsmentioning

confidence: 99%

See 1 more Smart Citation

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

et al. 2011

View full text Add to dashboard Cite

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyperbranched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp + (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30-40 %), it is dispensable at high humidity. Dendritic migration (flagella-and surfactin-dependent) of strains 168 sfp + swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (singlecell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication. INTRODUCTIONThe formation of bacterial communities, such as Bacillus subtilis colonies, was shown recently, while this work was in progress, to occur through a developmental-like program, associated with the presence of different cell types (see Vlamakis et al., 2008;Lemon et al., 2008;Ló pez & Kolter, 2010). Another form of community swarming migration over a surface, apparently occurring in thin fluid films, requires flagella, a surfactant and the production of specialized swarmer cells ( , 2008). Two types of swarming-like migration by B. subtilis have been described. On Luria broth (LB) agar at low humidity and 37 u C (primarily analysed with nondomesticated strains such as 3610), migration proceeds as an expanding confluent field, headed by small groups of bacteria, apparently with little change in individual size (Kearns & Losick, 2003;Julkowska et al., 2004). In contrast, we have described the...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Long-chain Formsmentioning

confidence: 99%

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

et al. 2011

View full text Add to dashboard Cite

show abstract

“…To generate the transcriptional fusion of P comGA to mCherry, a fragment containing the comGA promoter was amplified by using 3610 as a template and primer pair 1124/1514 and was digested with EcoRI and NheI. The digested fragment was ligated into the EcoRI and NheI sites of pDP195 (27) containing a spectinomycin cassette to create pKB111. pKB111 was integrated into PY79 at the ectopic site thrC.…”

Section: Methodsmentioning

confidence: 99%

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

Konkol¹,

Blair²,

Kearns³

2013

Journal of Bacteriology

200

239

View full text Add to dashboard Cite

Natural competence is a process by which bacteria construct a membrane-associated machine for the uptake and integration of exogenous DNA. Many bacteria harbor genes for the DNA uptake machinery and yet are recalcitrant to DNA uptake for unknown reasons. For example, domesticated laboratory strains of Bacillus subtilis are renowned for high-frequency natural transformation, but the ancestral B. subtilis strain NCIB3610 is poorly competent. Here we find that endogenous plasmid pBS32 encodes a small protein, ComI, that inhibits transformation in the 3610 strain. ComI is a single-pass trans-membrane protein that appears to functionally inhibit the competence DNA uptake machinery. Functional inhibition of transformation may be common, and abolishing such inhibitors could be the key to permitting convenient genetic manipulation of a variety of industrially and medically relevant bacteria. Laboratory strains of Bacillus subtilis are powerful model genetic systems due to their rapid growth as dispersed cells and their ability to take up and incorporate exogenous DNA by natural competence (1, 2). The ancestral B. subtilis strain NCIB3610 (also known as 3610), however, retains many biological properties that were genetically bred out of the laboratory derivatives, including but not limited to floating pellicle biofilms, colonies of complex architecture, synthesis of an extracellular polysaccharide capsule, synthesis of a poly-␥-glutamate slime layer, synthesis of polyketide antimicrobials, synthesis of a nonribosomally synthesized lipopeptide surfactant, swarming and sliding surface motilities, and a large extrachromosomally maintained plasmid (3-9). Unfortunately, studies of the 3610 strain are hampered due to the fact that it is poorly competent, thus making genetic manipulation inconvenient (10).The induction of natural competence in laboratory strains is complex (11). During the transition to stationary phase, two parallel quorum-sensing systems activate genes that enhance the accumulation of the transcription factor ComK (2,(12)(13)(14). ComK becomes active in only a subpopulation of cells and directs expression of a regulon that includes approximately 20 gene products necessary for the construction of the competence machinery, a membrane-associated complex necessary for the uptake of exogenous DNA (11,(15)(16)(17). For cells that synthesize the competence machinery, exogenous double-stranded DNA binds to the cell surface, and single-stranded DNA (ssDNA) is then actively imported and recombined into the chromosome (1,(18)(19)(20). Why ancestral B. subtilis strain 3610 is poorly transformable is unknown.Here we determine that curing the 84-kb endogenous plasmid, here named pBS32, from the ancestral B. subtilis strain results in a 100-fold increase in transformability. We find that pBS32 encodes a small protein called ComI that appears to antagonize transformation by interfering with the competence machinery within the membrane. Functional inhibition of the competence machinery may be a confounding factor that prevent...

show abstract

“…The third region of this flagellar operon, F3 (flgB [CD0272], bp 309272 to 333302) resembles the large 27-kb fla-che operon of Bacillus subtilis (9). In B. subtilis, the genes are cotranscribed from a common promoter under the control of RNA polymerase and the vegetative 70 homolog A .…”

Section: Generation Of Flagellar Mutant Strainsmentioning

confidence: 99%

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

et al. 2012

View full text Add to dashboard Cite

We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. Clostridium difficile is a Gram-positive spore-forming bacillus which is recognized to be the major cause of nosocomial diarrhea associated with antibiotic therapy (35). The incidence of C. difficile infection has been rapidly increasing in both Europe and North America, and this increase in infections has been associated with a significantly high mortality rate (2, 35). The broad spectrum of diseases caused by C. difficile, which range from antibiotic-associated diarrhea to the potentially lethal, pseudomembranous colitis, has been shown to depend on the level of toxin produced (1), and this production is recognized as a critical determinant of pathogenicity. Following antibiotic therapy when the microbiota of the gastrointestinal tract is disrupted, infection by C. difficile is mediated by spores which germinate in the gut, followed by vegetative cell proliferation and the subsequent secretion of the two major virulence factors, the Rho glucosylating toxins TcdA and TcdB.The toxin-encoding genes (tcdA and tcdB) are localized to a 19.6-kb pathogenicity locus (PaLoc) which includes three other accessory genes, tcdR, tcdE and tcdC (43). TcdR is an alternative sigma factor required for transcription of the two toxin genes; TcdE has been described to be a putative holin-like protein involved in toxin secretion, although this role has recently been a source of debate (17, 32); and TcdC is an anti-sigma factor that negatively regulates tcdR-dependent transcription (28, 29). In addition, four other regulators of toxin synthesis have recently been identified: CepA (3), CodY ...

show abstract

Gene position in a long operon governs motility development in Bacillus subtilis

Cited by 65 publications

References 71 publications

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

Modulation of Toxin Production by the Flagellar Regulon in Clostridium difficile

Contact Info

Product

Resources

About