Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity

ABSTRACT: We analyzed a deletion mutant of the ECF M-OM-^C factor SigX and applied mRNA profiling to define the SigX dependent regulon in P. aeruginosa in response to low osmolarity medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and c-di-GMP signaling. In accordance, a sigX deletion mutant exhibited an altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative M-OM-^C factor SigX revealing its role in complex virulence-associated phenotypes in P. aeruginosa. Transcriptome profiling of the SigX deletion mutant and overexpressing wildtype complemented by ChIP-seq

ORGANISM(S): Pseudomonas aeruginosa UCBPP-PA14

SUBMITTER: Denitsa Eckweiler

PROVIDER: E-GEOD-50937 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity.

Blanka Andrea A Schulz Sebastian S Eckweiler Denitsa D Franke Raimo R Bielecka Agata A Nicolai Tanja T Casilag Fiordiligie F Düvel Juliane J Abraham Wolf-Rainer WR Kaever Volkhard V Häussler Susanne S

Journal of bacteriology 20131101 2

Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative ...[more]

PMID: 24187091

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Project description:Bacterial lifestyles are dictated by the conditions encountered during colonization of a specific ecological niche or host. A wide range of environmental stimuli triggers sensory modules, that modify activity of proteins, or/and initiate a transfer of information in complex regulatory networks, which modulate gene expression, and therefore adaptation. The transition between planktonic and biofilm growth is dependent on the homeostasis of the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclase (DGC) activity favor biofilm while low levels maintained by phosphodiesterase (PDE) enzymes encourage planktonic style or biofilm dispersal. In Pseudomonas aeruginosa over 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual enzymes possessing both canonical DGC and PDE motifs, i.e. GGDEF and EAL, respectively. It has been previously reported that the deletion of the PDE/DGC dual enzyme PA0285, one of 5 c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms and causes elevated c-di-GMP levels in P. aeruginosa. Here we confirm that this role is conserved in various P. aeruginosa (PAO1, PA14 and PAK) and Pseudomonas putida strains. Deletion of PA0285 has the highest impact during the early stage of surface colonization, and RNA-seq analysis in biofilm context indicates that it may be associated with upregulation of cupA fimbrial genes. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds the respective GTP (GGDEF) and c-di-GMP (EAL) substrates but only exhibits PDE activity in vitro. Both GGDEF and EAL domains are important for PA0285 PDE activity in vivo as suggested by site-directed mutants in each of the key motifs. Complementation of the PA0285 mutant strain with the C-terminal GGDEF/EAL portion in trans is not as effective as with the full-length gene suggesting that the transmembrane region and PAS domains contained in PA0285’s N-terminal portion influence its PDE activity in vivo. We speculate that the transmembrane portion or/and PAS domains of PA0285 impede its PDE activity in vivo through modulating the conformation of the enzyme, as shown for P. aeruginosa RbdA, in response to an unknown stimulus.

Dataset Information

Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity

Publications

Identification of the alternative sigma factor SigX regulon and its implications for Pseudomonas aeruginosa pathogenicity.

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