Control of flagellar gene regulation in Legionella pneumophila and its relation to growth phase

Abstract

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.

FIG. 1.

The infectivity of the L. pneumophila ΔfleQ, ΔfleR, ΔrpoN, and ΔfliA mutant strains is reduced in MH-S macrophages. To test the contributions of the flagellar regulon to the infectivity in macrophages, MH-S monolayers were incubated for 2 h after a mild centrifugation, with each strain at an MOI of 0.3. Cell-associated viable L. pneumophila bacteria were enumerated as CFU and expressed as the mean percentage of the initial microbial inoculum recovered ± SD, calculated from six to nine independent experiments. The bars represent percentages of viable and cell-associated L. pneumophila (infectivity). PE, postexponetial growth phase; E, exponential growth phase. Asterisks indicate statistically significant differences (*, P < 0.001 by a two-tailed Student's t test) in comparison to WT PE-phase L. pneumophila.

FIG. 2.

Expression of flagellin and FleQ of L. pneumophila WT strain Paris; the rpoN, fleQ, fleR, and fliA mutant strains; and the complemented fleQ mutant strain. FleQ and FlaA were visualized by Western blot analysis of whole-cell lysates from liquid cultures grown to an OD of 4.2 (PE phase) using anti-FlaA (A) and anti-FleQ (B) antisera. Lane 1, wild-type (Wt) strain Paris; lane 2, L. pneumophila strain Paris ΔrpoN mutant; lane 3, L. pneumophila strain Paris ΔfleQ mutant; lane 4, L. pneumophila strain Paris ΔfleR mutant; lane 5, L. pneumophila strain Paris ΔfliA mutant; and lane 6, L. pneumophila strain Paris ΔfleQ mutant complemented (compl.) with native FleQ.

FIG. 3.

Primer extension-mediated mapping of the transcriptional start site of the fleS and flgB genes. Shown are reference sequencing reactions (lanes T, G, C, and A) and primer extension of L. pneumophila WT RNA harvested in the postexponential growth phase (lane PE). The sequence of the coding strand, encompassing the 3′ end of the extension product (*), is shown to the right. (A) Sequence of the upstream region of fleS indicating the start sites of the mRNA, as determined in the gel to the left, the RpoN and sigma 70 binding sites, and the primer used for primer extension. (B) Sequence of the upstream region of flgB indicating the start sites of the mRNA, as determined in the gels to the left, and the RpoN and sigma 70 binding sites.

FIG. 4.

Model for transcriptional regulation of the various flagellar genes (class I to IV) in L. pneumophila. “?” denotes an unknown factor or factors. FleQ is probably controlled by the σ⁷⁰ factor. Together with RpoN, FleQ then enhances flagellar class II gene transcription. FleQ independent from RpoN enhances flagellar class III and IV gene transcription, including that of fliA, encoding the σ²⁸ factor. FliA then regulates the expression of the flagellar class III genes flgMN and flhB′ and the class IV genes flaAG, fliDS, and motY to complete the flagellum. FleR and RpoN seem to be responsible for a negative feedback loop on flagellar genes, possibly involving letE.