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The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

ABSTRACT: Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila sigma28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.

SUBMITTER: Heuner K

PROVIDER: S-EPMC178656 | biostudies-other | 1997 Jan

REPOSITORIES: biostudies-other

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The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

Heuner K K Hacker J J Brand B C BC

Journal of bacteriology 19970101 1

Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been ...[more]

PMID: 8981975

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Project description:Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophilaL. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase- and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.

Project description:Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coliIMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE.

Dataset Information

The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

Publications

The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

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