Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): autoflex

ORGANISM(S): Pseudomonas Aeruginosa Pao1

SUBMITTER: Emeline Bouffartigues

LAB HEAD: Prof Marc Feuilloley

PROVIDER: PXD021299 | Pride | 2020-10-05

REPOSITORIES: Pride

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Publications

The Temperature-Regulation of <i>Pseudomonas aeruginosa cmaX-cfrX-cmpX</i> Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX.

Bouffartigues Emeline E Si Hadj Mohand Ishac I Maillot Olivier O Tortuel Damien D Omnes Jordane J David Audrey A Tahrioui Ali A Duchesne Rachel R Azuama Cecil Onyedikachi CO Nusser Michael M Brenner-Weiss Gerald G Bazire Alexis A Connil Nathalie N Orange Nicole N Feuilloley Marc G J MGJ Lesouhaitier Olivier O Dufour Alain A Cornelis Pierre P Chevalier Sylvie S

Frontiers in microbiology 20201021

<i>Pseudomonas aeruginosa</i> is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functi ...[more]

PMID: 33193206

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Project description:A core component of the M-NM-1-proteobacterial general stress response is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed M-NM-1-proteobacterial genome sequences contain two or more copies of genes encoding M-OM-^CEcfG-like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti-sigma factor (NepR) and two-component response regulator (PhyR). So far, the widespread occurrence of additional, non-canonical M-OM-^CEcfG copies has not satisfactorily been explained. This work explores the hierarchical relation between Rhizobium etli M-OM-^CEcfG1 and M-OM-^CEcfG2, canonical and non-canonical M-OM-^CEcfG proteins, respectively. Contrary to reports in other species, we find that M-OM-^CEcfG1 and M-OM-^CEcfG2 act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. M-OM-^CEcfG1 drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of M-OM-^CEcfG2, accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel M-OM-^CEcfG targets. We propose a modified model for general stress response regulation in R. etli, in which M-OM-^CEcfG1 and M-OM-^CEcfG2 function largely independently. Based on a phylogenetic analysis and considering the prevalence of M-NM-1-proteobacterial genomes with multiple M-OM-^CEcfG copies, this model may also be applicable to numerous other species. In this study, we investigate to which degree R. etli EcfG1 and EcfG2 regulation is interdependent. Furthermore, we demonstrate that both sigma factors control distinct regulons and pinpoint unique EcfG2 target genes. We also identify non-coding RNAs (ncRNAs) as novel targets of EcfG1 and EcfG2 and show that expression of at least one of these ncRNAs is under direct EcfG control. The presence of ncRNAs in the regulon of an EcfG type sigma factor has not been described previously and adds an extra level of complexity to the regulation of the general stress response in Alpha-proteobacteria. Moreover, considering the widespread existence of Alpha-proteobacterial genomes with multiple EcfG copies, the here presented results not only lead to a better understanding of the general stress response in R. etli, but also contribute to a conceptual paradigm for general stress response regulation by multiple EcfG proteins in Alpha-proteobacteria. Comparative transcriptome analyses were carried out on four samples: the parental strain (WT) and mutants in either sigma factor gene ecfG1, sigma factor gene ecfG2, and both genes combined.

Project description:Background: Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome-wide mRNA expression changes in PCa cells in response to Runx2. Results: We engineered a C4-2B PCa sub-line called C4-2B/Rx2dox, in which doxycycline (Dox) treatment stimulates Runx2 expression from very low levels to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer-associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal. Conclusions: The effects of Runx2 in C4-2B/Rx2dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways. C4-2B/Rx2dox cells were subjected to microarray gene expression analysis after one and two days of treatment with either Dox or vehicle in biological quadruplicates (a total of 16 samples).

Dataset Information

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

Publications

The Temperature-Regulation of <i>Pseudomonas aeruginosa cmaX-cfrX-cmpX</i> Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX.

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