Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis.
We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor sigma(28) (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the sigma(28) binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with sigma(28) were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by sigma(28) or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by sigma(28). Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other sigma-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other sigma-factors apart from sigma(28) may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.
Ding
Lisha
L
Graduate School of the Chinese Academy of Sciences, Beijing 100049, China.|Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan 430071, China.
Wang
Yao
Y
Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan 430071, China.
Hu
Yangbo
Y
Graduate School of the Chinese Academy of Sciences, Beijing 100049, China.|Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan 430071, China.
Atkinson
Steve
S
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Williams
Paul
P
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Chen
Shiyun
S
Wuhan Institute of Virology, The Chinese Academy of Sciences, Wuhan 430071, China.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017124:Conserved Sequence / Q000502:physiology; D029968:Escherichia coli Proteins / Q000378:metabolism; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D005408:Flagellin; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000378:metabolism; D014158:Transcription, Genetic; D015011:Yersinia pseudotuberculosis / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C064971:FliA protein, Bacteria; C516095:FliC protein, E coli; D012808:Sigma Factor; D005408:Flagellin; C071266:FlgM protein, Bacteria