The Alternative Sigma Factor AlgT RepressesPseudomonas aeruginosaFlagellum Biosynthesis by Inhibiting Expression offleQ

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Abstract
Pseudomonas aeruginosa poses a serious risk in individuals suffering from cystic fibrosis (CF). Strains colonizing the CF lung are generally motile but frequently convert to a nonmotile phenotype as the disease progresses. In many cases, this is coordinately regulated with the overproduction of the exopolysaccharide alginate. Both the expression of alginate (mucoidy) and the loss of flagellum synthesis may provide the bacterium with a selective advantage in the CF lung. Previously published data showed that the regulation of alginate production and flagellum biosynthesis in the CF isolate FRD1 is inversely controlled by the alternative sigma factor AlgT. In this study, we observed that in CF isolates, the mucoid and the nonmotile phenotypes occur predominantly together. Using microarrays, we compared the transcriptomes of isogenic AlgT+ and AlgT− P. aeruginosa and discovered that AlgT significantly downregulated the majority of flagellar genes. A pronounced inhibitory effect was observed in several genes essential for proper flagellum expression, including fleQ, which encodes an essential flagellar regulator. The microarray data were confirmed by reverse transcriptase PCR analysis and promoter fusion assays in isogenic AlgT+ and AlgT− strains. Transmission electron microscopy, motility assays, and Western blots showed that ectopic expression of FleQ in mucoid, nonmotile CF isolates restored flagellum biosynthesis and motility. Together, these data show that AlgT mediates the negative control of flagellum expression by inhibiting the expression of the flagellar regulator fleQ.