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

ABSTRACT: 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 ?(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 ?(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.

SUBMITTER: Hsueh YH

PROVIDER: S-EPMC3193947 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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

Hsueh Yi-Huang YH Cozy Loralyn M LM Sham Lok-To LT Calvo Rebecca A RA Gutu Alina D AD Winkler Malcolm E ME Kearns Daniel B DB

Molecular microbiology 20110718 4

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 i ...[more]

PMID: 21736639

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Project description:FlgM, an antagonist of FliA (also known as σ28), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ28 to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliATd) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliATd. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliATd and prevents it from binding to the σ28-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliATd turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria. IMPORTANCE Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors. While the structure and function of PFs have been extensively studied in spirochetes, the molecular mechanism that regulates the PFs' morphogenesis and assembly is poorly understood. In this report, FlgM, an anti-σ28 factor, is identified and functionally characterized in the oral spirochete Treponema denticola. Our results show that FlgM regulates the number and polarity of PFs via a unique mechanism. Identification of FliA and FlgM in T. denticola sets a benchmark to investigate their roles in other spirochetes.

Project description:RNA polymerase sigma factor sigma(F) initiates the prespore-specific program of gene expression during Bacillus subtilis sporulation. sigma(F) governs transcription of spoIIIG, encoding the late prespore-specific regulator sigma(G). However, transcription of spoIIIG is delayed relative to other genes under the control of sigma(F), and after synthesis, sigma(G) is initially kept in an inactive form. Activation of sigma(G) requires the complete engulfment of the prespore by the mother cell and expression of the spoIIIA and spoIIIJ loci. We screened for random mutations in spoIIIG that bypassed the requirement for spoIIIA for the activation of sigma(G). We found a mutation (spoIIIGE156K) that resulted in an amino acid substitution at position 156, which is adjacent to the position of a mutation (E155K) previously shown to prevent interaction of SpoIIAB with sigma(G). Comparative modelling techniques and in vivo studies suggested that the spoIIIGE156K mutation interferes with the interaction of SpoIIAB with sigma(G). The sigma(GE156K) isoform restored sigma(G)-directed gene expression to spoIIIA mutant cells. However, expression of sspE-lacZ in the spoIIIA spoIIIGE156K double mutant was delayed relative to completion of the engulfment process and was not confined to the prespore. Rather, beta-galactosidase accumulated throughout the entire cell at late times in development. This suggests that the activity of sigma(GE156K) is still regulated in the prespore of a spoIIIA mutant, but not by SpoIIAB. In agreement with this suggestion, we also found that expression of spoIIIGE156K from the promoter for the early prespore-specific gene spoIIQ still resulted in sspE-lacZ induction at the normal time during sporulation, coincidently with completion of the engulfment process. In contrast, transcription of spoIIIGE156K, but not of the wild-type spoIIIG gene, from the mother cell-specific spoIID promoter permitted the rapid induction of sspE-lacZ expression. Together, the results suggest that SpoIIAB is either redundant or has no role in the regulation of sigma(G) in the prespore.

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Dataset Information

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

Publications

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

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