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Rewiring of a sigma factor regulatory network in P. aeruginosa by a naturally occurring single nucleotide polymorphism

ABSTRACT: Transcriptional regulatory networks represent an important organizational element in the bacterial cell where signals from the cell state and the outside environment are integrated in terms of activation and inhibition of gene transcription. How these networks evolve is not well understood, and the effects of natural occurring polymorphisms within network components are often not known. Here, we systematically investigate the functional consequences of an amino acid substitution in the sigma factor protein RpoN in the opportunitic pathogen Pseudomonas aeruginosa. While RpoN is known to be important for virulence gene expression in P. aeruginosa, the particular amino acid substitution is important for ecological success of the bacteria in cystic fibrosis infections. We use Chromatin Immunoprecipitation coupled to deep sequencing (ChIP-seq), transcriptional profiling as well as in vitro protein-DNA interaction studies and show that the rpoN mutation results in reduced connections to only parts of the RpoN controlled regulatory network. On the other hand, the mutation results in activation of other sigma factor regulons. Finally, our results also show that the rpoN mutation results in a rewiring of the RpoN network in terms of increased connectivity and positive regulatory effect on a virulence associated locus. This molecular pleiotropy could not have been predicted from in silico analyses alone, and this work thus underlines the importance of achieving a molecular understanding of the effects of polymorphisms in regulator network components

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE81573 | GEO | 2019/05/01

REPOSITORIES: GEO

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RNA-seq for RpoN

Project description:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, most notably those with cystic fibrosis or whose immune systems are compromised. As a global regulator, RpoN has been characterized to control a group of virulence-related factors and quorum sensing (QS) genes in P. aeruginosa, but its detailed molecular regulatory mechanism is largely elusive. To further gain insights into the direct targets of RpoN in vivo, this study focused on identifying the potential targets of RpoN directly regulating QS system and T6SS. We performed a chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-seq) assay that identified 1, 068 binding sites of RpoN, including mostly metabolic genes, a group of genes in QS (lasI, rhlI and pqsR) and type VI secretion system (T6SS) (hcpA and hcpB). The direct regulation of these genes by RpoN has been biochemically and genetically verified. Results revealed that, on one hand, the deletion of rpoN resulted in reduced production of pyocyanin, motility and proteolytic activity. On the other hand, the production of rhamnolipids and biofilm formation was higher in the RpoN mutant than in the wild-type. In sum, the results indicated that RpoN had direct and profound effects on QS and T6SS, which provides new cues to better understand the detailed regulatory networks of virulence.

2018-06-30 | GSE110445 | GEO

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Project description:Alginate overproduction by P. aeruginosa, also known as mucoidy is associated with chronic endobronchial infections in cystic fibrosis (CF). Alginate biosynthesis in this bacterium is initiated by the extracytoplasmic function sigma factor (σ22, AlgU/T). In the wild type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered by the anti-sigma factor MucA that inhibits alginate production. However, the degradation of MucA by activated intramembrane proteases AlgW and/or MucP can lead to the conversion from nonmucoid strains to mucoid. Previously we reported that the absence of the sensor kinase KinB in PAO1 causes the initiation of AlgW-dependent proteolysis of MucA resulting in alginate overproduction. In the kinB mutant this activation requires alternate sigma factor RpoN (σ54). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant, RpoN controlled the expression of approximately 20% of the genome. Besides alginate biosynthesis and regulator genes such as AlgW, KinB, in concert with RpoN, also control a large number of genes including: those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, mice exhibited better survival when challenged with the kinB mutant than wt PAO1. Together, these data strongly suggest that KinB controls virulence factors important for acute pneumonia and conversion to mucoidy.

2012-03-01 | GSE35248 | GEO

Comparison between Borrelia burgdorferi wild type strain and cognate rpoS, rpoN and rrp2 mutants

Project description:The RpoN-RpoS alternative sigma factor pathway is essential for key adaptive responses by Borrelia burgdorferi, particularly those involved in the infection of a mammalian host. A putative response regulator, Rrp2, ostensibly is required for activation of the RpoN-dependent transcription of rpoS. However, questions remain regarding the extent to which the three major constituents of this pathway (Rrp2, RpoN, and RpoS) act interdependently. To assess the functional interplay between Rrp2, RpoN, and RpoS, we employed microarray analyses to compare gene expression levels in rrp2, rpoN, or rpoS mutants of parental strain 297. We identified 98 genes that were similarly regulated by Rrp2, RpoN and RpoS, and an additional 47 genes were determined to be likely regulated by this pathway. The substantial overlap between genes regulated by RpoS and RpoN provides compelling evidence that these two alternative sigma factors form a congruous pathway and that RpoN regulates B. burgdorferi gene expression through RpoS. Although several known B. burgdorferi virulence determinants were regulated by the RpoN-RpoS pathway, a defined function has yet to be ascribed to most of the genes substantially regulated by Rrp2, RpoN and RpoS, therefore providing additional avenues for future research into the genetic determinants contributing to virulence expression by B. burgdorferi. Keywords: gene profiling

2008-09-09 | GSE11284 | GEO

Expression of RpoN molecular roadblock in Pseudomonas aeruginosa PAO1 in rich media

Project description:The bacterial transcription factor RpoN regulates an extensive network of genes whose products are involved in diverse biological functions. We constructed a small peptide termed the RpoN molecular roadblock, which binds to and blocks transcription from RpoN promoters. This RpoN molecular roadblock can be used in any bacterium to obtain information on the RpoN regulon. We expressed the RpoN molecular roadblock in P. aeruginosa PAO1 and used microarrays to identify genes that were differentially transcribed due to the RpoN molecular roadblock. The RpoN molecular roadblock was expressed in P. aeruginosa PAO1 in mid-exponential phase in rich media. Total RNA was isolated and prepped for Affymetrix GeneChips.

2012-02-09 | E-GEOD-35632 | biostudies-arrayexpress

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

2012-03-01 | E-GEOD-35248 | biostudies-arrayexpress

Expression of RpoN molecular roadblock in Pseudomonas aeruginosa PAO1 in rich media

2012-02-09 | GSE35632 | GEO

RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins

Project description:Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and compete with microbial competitors. These T6SS machines can be loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the three main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control of the regulator RsmA in repressing most known T6SS-related genes. Moreover, it is noticeable that a sigma factor activator (SFA) protein is encoded in each of H2- and H3-T6SS clusters, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins that usually work in concert with the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of all H2-T6SS-linked VgrG and effector arsenal to enable very effective interbacterial killing. Such RpoN-dependent control is exerted both directly and indirectly. The function of the SFA protein is specific since the H3-T6SS associated Sfa3 cannot complement loss of Sfa2. Our study further delineates the multiple regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely to respond and adapt to a range of environmental conditions that a versatile organism like P. aeruginosa would encounter.

2022-02-23 | GSE185398 | GEO

Comparative RNA-seq analysis of the transcriptome of the Pseudomonas aeruginosa PAO1 wild-type strain with that of the ΔvreR mutant

Project description:Bacterial gene expression is controlled by modifying the promoter affinity of the RNA polymerase (RNAP). This control occurs through the substitution of the RNAP σ subunit and by the interaction with transcription factors. The pathogen Pseudomonas aeruginosa contains several σ factors, including σVreI. This protein belongs to the extracytoplasmic function group of the σ70 family. Expression and activity of σECFs are tightly regulated and only occur in response to specific signals. σVreI is encoded within the vreAIR gene cluster. The expression of this cluster occurs in response to inorganic phosphate (Pi) limitation. σVreI activity is modulated by VreR anti-sigma factor, which keeps σVreI inactive. In response to a still unidentified host signal, VreR is proteolytically degraded and σVreI released and activated. σVreI interacts then with the RNAP and targets expression of the σVreI regulon, which includes virulence genes that increase P. aeruginosa pathogenicity. In this work, we compared the transcriptome of the P. aeruginosa PAO1 wild-type strain with that of the ΔvreR mutant upon bacterial growth in Pi starvation. Forty-six transcripts were more abundant in the ΔvreR mutant than in the PAO1 wild-type strain, and nine were less abundant, including the vreR transcript.

2019-03-13 | GSE122253 | 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.

2014-01-01 | GSE51076 | GEO

The RpoN Regulon of Escherichia coli O157:H7

Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR.

2009-08-08 | GSE17467 | GEO

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