The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant

Abstract

Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the 28 class of alternative factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila 28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.Legionella pneumophila, a ubiquitous microorganism inhabiting freshwater biotopes and man-made water systems, is also a pathogen of humans causing severe pneumonia termed Legionnaires' disease. In the environment, the bacteria are able to replicate intracellularly in amoebae and other protozoa (14,39,46). Legionella infection occurs after inhalation of aerosolized bacteria. The bacteria enter the human lungs, where they are capable of invading and proliferating in alveolar macrophages and blood monocytes.A growing number of virulence factors elaborated by L. pneumophila contribute to the pathogenicity of the organism. These include a number of genetic loci that have been implicated in being important for intracellular growth (for a review, see reference 6). The icm locus is essential for the intracellular multiplication of L. pneumophila within human macrophages and is also required for virulence in guinea pigs (5, 30). Adjacent to icm is dot, a gene essential for intracellular replication and proper trafficking of host organelles during macrophage infection (3-5). Several surface-associated cellular components have been implicated in virulence. The Mip (macrophage infectivity potentiator) protein contributes to intracellular survival of L. pneumophila, and invasion experiments using a null mutation in mip revealed that Mip is required for early survival processes of L. pneumophila in eukaryotic cells (9, 10, 47). Convincing evidence suggests that the major outer membrane protein (encoded by ompS) is involved in the uptake of bacteria by the host cell (22). The major outer membr...

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

“…flaA) (Albert-Weissenberger et al 2010;Brüggemann et al 2006;Jacobi et al 2004;Heuner and Albert-Weissenberger 2008). It is known that L. pneumophila rpoN, fleQ, fliA and flaA mutant strains are non-flagellated and non-motile (Heuner et al 1995;Heuner et al 2002;Heuner et al 1997;Jacobi et al 2004;Molofsky et al 2005). Conflicting data suggested different regulation mechanisms in strains Corby and Paris for fliA expression.…”

Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes

“…As most of the genome of L. pneumophila has already been sequenced (http://genome3.cpmc.columbia .edu/ϳlegion/index.html), we looked for all the genes that have homology to known sigma factors and found that L. pneumophila contains homologs to at least six sigma factors (RpoD, RpoH, RpoF, RpoE, RpoS, and RpoN). The promoter sequence recognized by two of these sigma factors in L. pneumophila is already known (2,22). RpoH and RpoF recognize sequences similar to the ones recognized by their E. coli homologs (CTTGAAA [11 to 16]CCCATnT for RpoH [n ϭ A, G, T, or C] and TAAA [15]GCCGATAA for RpoF).…”

Flagellum of Legionella pneumophila Positively Affects the Early Phase of Infection of Eukaryotic Host Cells

“…Previous reports suggest that the expression of flagella is temperature regulated, since it is repressed at temperatures higher than 37°C (36). Recently, we demonstrated that the expression of the flaA gene is regulated at the transcriptional level (24) by the alternative 28 factor (FliA) and probably by FlaR, a regulator of the LysR family (25). Moreover, the complex flagellum expression and assembly seems to be coordinatively regulated with other virulence-associated traits (6,16,38,40).…”