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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Pseudomonas aeruginosa PAO1

PROVIDER: GSE155025 | GEO | 2020/12/23

REPOSITORIES: GEO

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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [ChIP-seq]

Project description:The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome. Although cells can tolerate the loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of prophage Pf4 and subsequent superinfection of the cell. In other bacteria, H-NS promotes cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase; however, it is not known if whether MvaT and MvaU function similarly. Here we utilize a parental strain that cannot be infected by Pf4 phage to define the collective MvaT and MvaU regulon and demonstrate that the combined loss of both MvaT and MvaU leads to increased intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ70 to genomic regions vacated by these proteins. Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and point to a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.

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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [RNA-seq]

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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

Project description:H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [RNA-seq]

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H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [ChIP-seq]

Project description:H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [ChIP-seq]

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Repression of phase-variable cup gene expression by H-NS-like proteins in Pseudomonas aeruginosa.

Project description:The cupA gene cluster of Pseudomonas aeruginosa encodes components of a putative fimbrial structure that enable this opportunistic human pathogen to form biofilms on abiotic surfaces. In P. aeruginosa, cupA gene expression is repressed by MvaT, a putative transcription regulator thought to belong to the H-NS family of nucleoid-associated proteins that typically function by repressing transcription. Here, we present evidence that MvaT controls phase-variable (ON/OFF) expression of the cupA fimbrial gene cluster. Using a directed proteomic approach, we show that MvaT associates with a related protein in P. aeruginosa called MvaU. Analysis with a bacterial two-hybrid system designed to facilitate the study of protein dimerization indicates that MvaT and MvaU can form both heteromeric and homomeric complexes, and that formation of these complexes is mediated through the N-terminal regions of MvaT and MvaU, both of which are predicted to adopt a coiled-coil conformation. We show further that, like MvaT, MvaU can repress phase-variable expression of the cupA gene cluster. Our findings suggest that fimbrial genes important for biofilm formation can be expressed in a phase-variable manner in P. aeruginosa, provide insight into the molecular mechanism of MvaT-dependent gene control, and lend further weight to the postulate that MvaT proteins are H-NS-like in nature.

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Project description:Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/?-10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

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