The alternative sigma factor AlgT represses Pseudomonas aeruginosa flagellum biosynthesis by inhibiting expression of fleQ

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.

FIG. 1.

Flagellin expression of mucoid P. aeruginosa CF isolates. A representative Western blot of whole-cell lysates was derived from P. aeruginosa strains. Lane 1, control for flagellin A serotype; lane 2, control for flagellin B serotype; lane 3, fliC mutant WFPA50; lanes 4 to 14, mucoid CF isolates. The results indicated that 70% of the strains tested were mucoid and lacked flagellin.

FIG. 2.

AlgT inhibits flagellum biosynthesis. Whole-cell lysates of mucoid, nonmotile P. aeruginosa CF isolates and their isogenic algT mutants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined for flagellin expression by Western blotting with flagellin B antiserum. Lane 1, PAO1 (flagellin B control); lanes 2 and 3, mucoid, nonmotile CF isolate 1, AlgT⁺ and AlgT⁻, respectively; lanes 4 and 5, mucoid, nonmotile CF isolate 2, AlgT⁺ and AlgT⁻, respectively; lanes 6 and 7, mucoid, nonmotile CF isolate 3, AlgT⁺ and AlgT⁻, respectively. As a loading control, a second gel was simultaneously prepared and processed by Coomassie blue staining and contained equivalent amounts of total protein.

FIG. 3.

Organization of P. aeruginosa flagellar genes and the effect of AlgT on their expression. Microarray analysis comparing transcriptomes of isogenic AlgT⁺ and AlgT⁻ P. aeruginosa strains was performed. Significantly lower levels of mRNA were observed for the majority of flagellar genes. fleQ, which encodes the flagellar master switch, was identified as the highest target of AlgT within the flagellar hierarchy. Arrows indicate the direction of transcription of the flagellar operons.

FIG. 4.

RT-PCR comparing flagellar gene expression in isogenic AlgT⁺ and AlgT⁻ P. aeruginosa strains. RT-PCR was performed on representatives of the four classes of flagellar genes to compare their mRNA levels in isogenic AlgT⁺ and AlgT⁻ P. aeruginosa strains. The RT-PCRs were separated on an agarose gel, and band intensities were compared using a Kodak Image Station 2000RT system. omlA was included as constitutive control (34). Shown are the averages of four independent experiments and standard deviations. Differences (n-fold) in mRNA levels for both microarray and RT-PCRs are indicated.

FIG. 5.

AlgT inhibits fleQ expression. fleQ::lacZ fusions were integrated into the chromosomes of mucoid, nonmotile P. aeruginosa CF isolates and their isogenic algT mutants at the neutral attB site. Promoter activity was measured with β-galactosidase assays using ONPG as a substrate and determined as amounts of ONPG hydrolyzed min⁻¹ as a function of cell density. omlA::lacZ fusions were included as a constitutive control (34). Shown are the averages of four independent experiments and standard deviations.

FIG. 6.

Inhibition of fleQ by AlgT is sufficient for the loss of flagellum expression. (A) Western blot of whole-cell lysates. Lane 1, type B flagellin; lane 2, FRD1; lane 3, FRD2700 (FRD1 with fleQ under the control of an arabinose-inducible promoter) with no arabinose; lanes 4 to 7, FRD2700 plus arabinose (lane 4, 0.1% arabinose; lane 5, 0.5% arabinose; lane 6, 1.0% arabinose; lane 7, 2.5% arabinose). (B) Motility assay on 0.3% agar. (I) PAO1 (motile control); (II) FRD1 (nonmotile control); (III) FRD2700 with no arabinose; (IV) FRD2700 plus 0.1% arabinose. (C) TEM of negatively stained P. aeruginosa. (I) FRD2700 with no arabinose; (II) FRD2700 plus 0.1% arabinose; (IV) FRD2700 plus 2.5% arabinose.