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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 σ 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 σ factor SigX revealing its role in complex virulence-associated phenotypes in P. aeruginosa.

ORGANISM(S): Pseudomonas aeruginosa UCBPP-PA14

PROVIDER: GSE50937 | GEO | 2013/11/11

SECONDARY ACCESSION(S): PRJNA219396

REPOSITORIES: GEO

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

Project description: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

2013-11-11 | E-GEOD-50937 | biostudies-arrayexpress

SigX ECF sigma factor deletion mutant expression profile in Pseudomonas aeruginosa in M9 minimal medium (M9G)

2014-01-01 | GSE51076 | GEO

SigX ECF sigma factor deletion mutant expression profile in Pseudomonas aeruginosa in M9 minimal medium (M9G)

Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa. To better understand the cellular function of SigX, a deletion mutant of the sigX gene (PAOSX) was generated and its expression profile was compared with parental strain Pseudomonas aeruginosa H103. To this end, H103 and a sigX mutant were cultured in M9G, in which their growth are similar. Three independant biological replicate were taken for the RNA extraction and hybridization on affymetrix array in the middle of the exponential growth phase.

2014-01-01 | E-GEOD-51076 | biostudies-arrayexpress

The temperature-regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX operon reveals an intriguing molecular network involving the sigma factors AlgU and SigX

Project description:Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notablydue to its large number of transcription regulators. The extracytoplasmic sigma factor (ECF sigma AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative sigma factor. The SigX ECF sigma emerged as a major regulator involved in the envelope stress response via membrane remodelling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECF sigma SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis.

2020-10-05 | PXD021299 | Pride

Cyclic diguanylate regulates virulence factor genes via multiple riboswitches in Clostridium difficile

Project description:The c-di-GMP regulon in C. difficile

2018-09-20 | GSE120198 | GEO

Phenotypic and integrated analysis of a comprehensive Pseudomonas aeruginosa PAO1 library of mutants lacking cyclic-di-GMP-related genes

Project description:Pseudomonas aeruginosa is a Gram-negative bacterium able to survive and adapt in a multitude of niches as well as thriving within many different hosts. This versatility lies within a large genome of ca 6Mbp and a tight control in the expression of thousands of genes. Among the regulatory mechanisms widely spread in bacteria, cyclic-di-GMP signaling is one which influences all levels of control. It is made by diguanylate cyclases and broken by phosphodiesterases while the intracellular level of this molecule drives phenotypic responses. The signaling involve modification of enzymes or proteins function upon c-di-GMP binding, including modifying the activity of regulators which in turn will impact the transcriptome. In P. aeruginosa, there are ca 40 genes encoding putative DGC or PDE. The combined activity of those enzymes should reflect the c-di-GMP concentration while specific phenotypic output could be correlated to a given set of dgc/pde. This notion of specificity has been addressed in several studies and different strains of P. aeruginosa. Here, we engineered a mutant library for the 41 individual dgc/pde genes in P. aeruginosa PAO1. In most cases, we observed a significant to slight variation in the global c-di-GMP pool, with essentially an impact on biofilm phenotypes including initial attachment and maturation, while very little is observed on motility which differs from previous studies. We observe that minor changes in c-di-GMP level have a drastic phenotypic impact thus supporting the idea of local vs global c-di-GMP pool. Our RNA-seq analysis indicates that all PAO1 dgc/pde genes are expressed in both planktonic and biofilm growth conditions and our work suggests that c-di-GMP networks owe to be reconstructed in one single strain and cannot be built by cross-comparison with studies in other strains.

2022-05-23 | GSE203348 | GEO

Use of transcriptome data to study the c-di-GMP signaling network in Pseudomonas aeruginosa clinical isolates

Project description:In the Pseudomonas aeruginosa type strain PA14, 40 genes are known to encode for diguanylate cyclases (DGCs) and/or phosphodiesterases (PDEs), which modulate the intracellular pool of the nucleotide second messenger c-di-GMP. While in general, high levels of c-di-GMP drive the switch from highly motile phenotypes towards a sessile lifestyle, the different c-di-GMP modulating enzymes are responsible for smaller and in parts non-overlapping phenotypes. In this study, we sought to utilize previously recorded P. aeruginosa gene expression datasets on 414 clinical isolates to uncover transcriptional changes as a result of a high expression of genes encoding diguanylate cyclases. We demonstrate that the selection of sub-groups of clinical isolates with high versus low expression of regulatory genes served the identification of their downstream regulons. Thus, analyzing transcriptional profiles of clinical isolates with variant expression of diguanylate cyclase genes might provide a unique opportunity to bypass the problem that for many c-di-GMP modulating enzymes it is not known under which conditions their expression is activated. However, because the high c-di-GMP associated phenotypes were rapidly lost in the clinical isolates, we were unable to confirm diguanylate specific gene regulation. It is possible that the elevated c-di-GMP concentrations observed in the clinical P. aeruginosa isolates were due to a memory response, in which the bacteria maintain a certain level of c-di-GMP even after being removed from the conditions that induced the production of c-di-GMP. Further studies would be needed to determine the specific mechanisms underlying this memory response in P. aeruginosa.

2023-08-21 | GSE233207 | GEO

Analysis of sigma factor-dependent gene expression in Pseudomonas aeruginosa by RNA sequencing

Project description:Sigma factors are master regulators of bacterial transcription which direct gene expression of specific subsets of genes. In particular, alternative sigma factors are well-known to be key players of bacterial adaptation to changing environments. To elucidate the regulatory network of sigma factors in P. aeruginosa, an integrative approach including sigma factor-dependent mRNA profiling was performed to define the primary regulon of each sigma factor.

2015-02-12 | GSE54998 | GEO

ChIP-seq study of sigma factors in Pseudomonas aeruginosa

Project description:Sigma factors are master regulators of bacterial transcription which direct gene expression of specific subsets of genes. In particular, alternative sigma factors are well-known to be key players of bacterial adaptation to changing environments. To elucidate the regulatory network of sigma factors in P. aeruginosa, an integrative approach including ChIP-seq of 11 polyhistidine-tag sigma factors was performed to define the primary regulon of each sigma factor.

2015-02-12 | GSE54997 | GEO

The scr circuit in Vibrio parahaemolyticus: output targets and transcriptional regulation by a c-di-GMP responsive protein

Project description:Vibrio parahaemolyticus scr genes modulate expression of gene sets pertinent to swarming and biofilm formation. They do so by affecting the level of the second messenger c-di-GMP. Here we explore the extent of this regulation by comparing the transcriptomes of a scrABC mutant and its wild-type parental strain. The scope of transcriptional effects modulated by c-di-GMP includes ~100 genes that are positively and negatively regulated. An elevated cellular level of c-di-GMP represses the surface sensing regulon including the genes encoding the lateral flagellar and type three secretion systems while inducing expression of genes encoding cell surface molecules and capsular polysaccharide. Expression of a few transcriptional regulators was also affected, and here we describe the role of one, CpsQ. CpsQ is one of four V. parahaemolyticus homologs in the CsgD/VpsT family of regulators, members of which have been implicated in c-di-GMP signaling. Mutations in cpsQ, like defects in another previously identified capsule regulator, cpsR, suppress the sticky phenotype of scr mutants. By using a combination of mutant and reporter analyses in Vibrio and E. coli, CpsQ is shown to be the direct positive regulator of cpsA transcription and its cpsA-activating ability is found to be responsive to the cellular level of c-di-GMP. Unexpectedly, we find that a low level of this nucleotide diminishes the stability of CpsQ. The molecular interplay in this signaling circuit is further defined by demonstrating that CpsQ is epistatic to CpsS, a negative regulator of capsule. CpsR activates cpsQ, and CpsQ can also regulate its own transcription. Wildtype Vibrio parahaemolyticus (LM5674) and scrABC mutant (LM6567) were grown on rich mediim agar plates and gene expression profiles were compared.

2011-12-31 | E-GEOD-30508 | biostudies-arrayexpress

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