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FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila

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Abstract

In Legionella pneumophila, the regulation of the flagellum and the expression of virulence traits are linked. FleQ, RpoN and FliA are the major regulators of the flagellar regulon. We demonstrated here that all three regulatory proteins mentioned (FleQ, RpoN and FliA) are necessary for full in vivo fitness of L. pneumophila strains Corby and Paris. In this study, we clarified the role of FleQ for fliA expression from the level of mRNA toward protein translation. FleQ enhanced fliA expression, but FleQ and RpoN were not necessary for basal expression. In addition, we identified the initiation site of fliA in L. pneumophila and found a putative σ70 promoter element localized upstream. The initiation site was not influenced in the ΔfleQ or ΔrpoN mutant strain. We demonstrated that there is no significant difference in the regulation of fliA between strains Corby and Paris, but the FleQ-dependent induction of fliA transcription in the exponential phase is stronger in strain Paris than in strain Corby. In addition, we showed for the first time the presence of a straight hook at the pole of the non-flagellated ΔfliA and ΔfliD mutant strains by electron microscopy, indicating the presence of an intact basal body in these strains.

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Acknowledgments

We would like to thank Ursula Erikli for careful reading of the manuscript. This work received financial support from the Robert Koch-Institute and by grant HE2854/5-1 + 2 from the Deutsche Forschungsgemeinschaft DFG (Bonn, Germany) to K.H.

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Authors and Affiliations

  1. Cellular Interactions of Bacterial Pathogens, Centre for Biological Security, Division 2 (ZBS2), Robert Koch-Institute, Nordufer 20, 13353, Berlin, Germany

    Tino Schulz, Kerstin Rydzewski, Eva Schunder & Klaus Heuner

  2. Centre for Biological Security, Division 4 (ZBS4), Robert Koch-Institute, Berlin, Germany

    Gudrun Holland & Norbert Bannert

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  1. Tino Schulz
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  2. Kerstin Rydzewski
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  3. Eva Schunder
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  6. Klaus Heuner
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Correspondence to Klaus Heuner.

Additional information

Communicated by S. Suerbaum.

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203_2012_833_MOESM1_ESM.pdf

Online Resource 1 Reverse transcription (RT-)PCR analysis of L. pneumophila Corby. Bacteria were grown in AYE medium and samples were taken during different growth phases and prepared for RT-PCR as well as Western blot analysis. For RT-PCR experiments, whole-cell RNA was isolated and equal amounts (10 ng) were used for amplification. For all samples, RT-PCR was performed at (a) 24 cycles, (b) 27 cycles or (c) 30 cycles, respectively. Results were carried out in at least two independent experiments. Abbreviations: E, exponential growth phase; LE, late exponential growth phase; PE, post-exponential growth phase; S, stationary growth phase (PDF 285 kb)

203_2012_833_MOESM2_ESM.pdf

Online Resource 2 Determination of the transcriptional start site of fliA via 5′RACE experiments – raw data. RNA from late exponentially grown L. pneumophila Corby wild type (LpC wt; a, b), LpC ΔfleQ (a, c) and LpC ΔrpoN (a, d) mutant strains was transcribed in cDNA, polyA-tailed and amplified. (a) All RNA samples contained a control RNA to verify the 5′RACE reaction via PCR. cDNA synthesis (K1; primers: neo2/rev and neo3/for; product: 157 bp), cDNA purification (K2; primers: neo2/rev and neo3/for; product: 157 bp) and d(A)-tailing reaction (K3; primers: neo2/rev and oligo d(T)-anchor; product: 293 bp) were verified with primers specific to the control RNA, respectively. The PCR product (R; primers: oligo d(T)-anchor and FliA_RACE_5R; product: ~ 500 bp) was sequenced using the anti-sense primer FliA_RACE_6R. The identified transcriptional start site is indicated (+1; b, c, d) (PDF 278 kb)

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Schulz, T., Rydzewski, K., Schunder, E. et al. FliA expression analysis and influence of the regulatory proteins RpoN, FleQ and FliA on virulence and in vivo fitness in Legionella pneumophila . Arch Microbiol 194, 977–989 (2012). https://doi.org/10.1007/s00203-012-0833-y

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