Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas
1Department of Medicine, Division of Infectious Diseases, University of Florida, Gainesville, Florida 32610, USA.
Abstract:
We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa. FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus. A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the sigma(28) binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor sigma(28) was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus sigma(54) recognition sites and one sigma(28) site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either sigma(28) or sigma(54). A transcript was detected by primer extension that was 8 bp downstream of the consensus sigma(28)-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both sigma(28)-dependent and -independent mechanisms of transcription exist.
Insights
The Pseudomonas aeruginosa flgM gene negatively regulates flagellin synthesis by inhibiting FliA activity. This finding reveals a unique control system for motility in this bacterium.
Area of Science:
- Microbiology
- Bacterial Physiology
- Molecular Biology
Background:
- Flagellar motility is crucial for bacterial pathogenesis and environmental adaptation.
- The flgM gene product, FlgM, is known to regulate flagellar synthesis in some bacteria.
- Pseudomonas aeruginosa is an opportunistic pathogen where motility plays a significant role.
Purpose of the Study:
- To functionally characterize the flgM gene in Pseudomonas aeruginosa.
- To elucidate the regulatory mechanism of flagellin synthesis controlled by FlgM.
- To investigate the interaction between FlgM and the sigma factor FliA.
Main Methods:
- Gene inactivation of flgM in P. aeruginosa.
- Construction and analysis of transcriptional fusions (fliC-lacZ, flgM-lacZ).
- Western blot analysis for flagellin detection.
- Yeast two-hybrid system for protein interaction studies.
- Primer extension to identify transcript start sites.
Main Results:
- Inactivation of flgM led to increased fliC promoter activity and flagellin levels.
- FlgM was shown to directly interact with the sigma-28 factor (FliA).
- The flgM promoter exhibited both sigma-28-dependent and -independent transcription.
- A novel regulatory system for flagellin synthesis was identified in P. aeruginosa.
Conclusions:
- FlgM negatively regulates flagellin synthesis in P. aeruginosa by inhibiting FliA activity.
- This regulatory mechanism differs from that in enteric bacteria and resembles systems in Vibrio cholerae.
- The findings contribute to understanding bacterial motility regulation and potential therapeutic targets.
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