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CsrT Represents a New Class of csrA-Like Regulatory Genes Associated with Integrative Conjugative Elements of Legionella pneumophila.

ABSTRACT:

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Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA. Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution of csrA regulators to this class of mobile genetic elements, we analyzed here the activity of the csrA paralog encoded on Legionella pneumophila ICE-?ox. Deletion of this gene, which we name csrT, had no observed effect under laboratory conditions. However, ectopic expression of csrT abrogated the protection to hydrogen peroxide and macrophage degradation that ICE-?ox confers to L. pneumophila. When ectopically expressed, csrT also repressed L. pneumophila flagellin production and motility, a function similar to the core genome's canonical csrA. Moreover, csrT restored the repression of motility to csrA mutants of Bacillus subtilis, a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinherited csrA-type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expanding L. pneumophila versatility.

Importance

ICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within the Legionella genus also carry paralogs of the essential life cycle regulator csrA. It is striking that the csrA loci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To investigate whether ICE-encoded csrA paralogs are bona fide regulators, we analyzed ICE-?ox as a model system. When expressed ectopically, its csrA paralog inhibited multiple ICE-?ox phenotypes, as well as the motility of not only Legionella but also Bacillus subtilis. Accordingly, we predict that CsrA regulators equip legionellae ICEs to promote their spread via dedicated type IV secretion systems.

SUBMITTER: Abbott ZD

PROVIDER: S-EPMC4719454 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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csrT Represents a New Class of csrA-Like Regulatory Genes Associated with Integrative Conjugative Elements of Legionella pneumophila.

Abbott Zachary D ZD Flynn Kaitlin J KJ Byrne Brenda G BG Mukherjee Sampriti S Kearns Daniel B DB Swanson Michele S MS

Journal of bacteriology 20151123 3

<h4>Unlabelled</h4>Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA. Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediat ...[more]

PMID: 26598366

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Project description:ABSTRACT Integrative conjugative elements (ICEs) are mobile blocks of DNA that can contribute to bacterial evolution by self-directed transmission of advantageous traits. Here, we analyze the activity of a putative 65-kb ICE harbored by Legionella pneumophila using molecular genetics, conjugation assays, a phenotype microarray screen, and macrophage infections. The element transferred to a naive L. pneumophila strain, integrated site-specifically, and conferred increased resistance to oxacillin, penicillin, hydrogen peroxide, and bleach. Furthermore, the element increased survival of L. pneumophila within restrictive mouse macrophages. In particular, this ICE protects L. pneumophila from phagocyte oxidase activity, since mutation of the macrophage NADPH oxidase eliminated the fitness difference between strains that carried and those that lacked the mobile element. Renamed ICE-?ox (for ?-lactam antibiotics and oxidative stress), this transposable element is predicted to contribute to the emergence of L. pneumophila strains that are more fit in natural and engineered water systems and in macrophages. IMPORTANCE Bacteria evolve rapidly by acquiring new traits via horizontal gene transfer. Integrative conjugative elements (ICEs) are mobile blocks of DNA that encode the machinery necessary to spread among bacterial populations. ICEs transfer antibiotic resistance and other bacterial survival factors as cargo genes carried within the element. Here, we show that Legionella pneumophila, the causative agent of Legionnaires' disease, carries ICE-?ox, which enhances the resistance of this opportunistic pathogen to bleach and ?-lactam antibiotics. Moreover, L. pneumophila strains encoding ICE-?ox are more resistant to macrophages that carry phagocyte oxidase. Accordingly, ICE-?ox is predicted to increase the fitness of L. pneumophila in natural and engineered waters and in humans. To our knowledge, this is the first description of an ICE that confers oxidative stress resistance to a nosocomial pathogen.

Project description:UnlabelledCritical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments. Legionella pneumophila occupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA. A striking feature of the legionellae surveyed is that each of 14 strains encodes 4 to 7 csrA-like genes, candidate regulators of distinct fitness traits. Here we focus on the one csrA paralog (lpg1593) that, like the canonical csrA, is conserved in all 14 strains surveyed. Phenotypic analysis revealed that long-term survival in tap water is promoted by the lpg1593 locus, which we name csrR (for "CsrA-similar protein for resilience"). As predicted by its GGA motif, csrR mRNA was bound directly by the canonical CsrA protein, as judged by electromobility shift and RNA-footprinting assays. Furthermore, CsrA repressed translation of csrR mRNA in vivo, as determined by analysis of csrR-gfp reporters, csrR mRNA stability in the presence and absence of csrA expression, and mutation of the CsrA binding site identified on the csrR mRNA. Thus, CsrA not only governs the transition from replication to transmission but also represses translation of its paralog csrR when nutrients are available. We propose that, during prolonged starvation, relief of CsrA repression permits CsrR protein to coordinate L. pneumophila's switch to a cell type that is resilient in water supplies.ImportancePersistence of L. pneumophila in water systems is a public health risk, and yet there is little understanding of the genetic determinants that equip this opportunistic pathogen to adapt to and survive in natural or engineered water systems. A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well. Our finding that every sequenced L. pneumophila strain carries several csrA paralogs-including two common to all isolates--indicates that the legionellae exploit CsrA regulatory switches for multiple purposes. Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenic L. pneumophila.