Dataset Information

Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665T.

ABSTRACT: Pantoea ananatis LMG 2665T synthesizes and utilizes acyl homoserine lactones (AHLs) for signalling. The complete set of genes regulated by the EanI/R quorum sensing (QS) system in this strain is still not fully known. In this study, RNA-sequencing (RNA-seq) was used to identify the EanI/R regulon in LMG 2665T. Pairwise comparisons of LMG 2665T in the absence of AHLs (Optical density (OD)600 = 0.2) and in the presence of AHLs (OD600 = 0.5) were performed. Additionally, pairwise comparisons of LMG 2665T and its QS mutant at OD600 = 0.5 were undertaken. In total, 608 genes were differentially expressed between LMG 2665T at OD600 = 0.5 versus the same strain at OD600 = 0.2 and 701 genes were differentially expressed between LMG 2665T versus its QS mutant at OD600 = 0.5. A total of 196 genes were commonly differentially expressed between the two approaches. These constituted approximately 4.5% of the whole transcriptome under the experimental conditions used in this study. The RNA-seq data was validated by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Genes found to be regulated by EanI/R QS were those coding for redox sensing, metabolism, flagella formation, flagella dependent motility, cell adhesion, biofilm formation, regulators, transport, chemotaxis, methyl accepting proteins, membrane proteins, cell wall synthesis, stress response and a large number of hypothetical proteins. The results of this study give insight into the genes that are regulated by the EanI/R system in LMG 2665T. Functional characterization of the QS regulated genes in LMG 2665T could assist in the formulation of control strategies for this plant pathogen.

SUBMITTER: Sibanda S

PROVIDER: S-EPMC5867869 | biostudies-other | 2018 Mar

REPOSITORIES: biostudies-other

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Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665T.

Sibanda Siphathele S Kwenda Stanford S Tanui Collins K CK Shyntum Divine Y DY Coutinho Teresa A TA Moleleki Lucy N LN

Genes 20180307 3

Pantoea ananatis LMG 2665T synthesizes and utilizes acyl homoserine lactones (AHLs) for signalling. The complete set of genes regulated by the EanI/R quorum sensing (QS) system in this strain is still not fully known. In this study, RNA-sequencing (RNA-seq) was used to identify the EanI/R regulon in LMG 2665T. Pairwise comparisons of LMG 2665T in the absence of AHLs (Optical density (OD)600 = 0.2) and in the presence of AHLs (OD600 = ...[more]

PMID: 29518982

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Project description:Quorum-sensing regulation of gene expression in Pseudomonas aeruginosa is complex. Two interconnected acyl-homoserine lactone (acyl-HSL) signal-receptor pairs, 3-oxo-dodecanoyl-HSL-LasR and butanoyl-HSL-RhlR, regulate more than 300 genes. The induction of most of the genes is delayed during growth of P. aeruginosa in complex medium, cannot be advanced by addition of exogenous signal, and requires additional regulatory components. Many of these late genes can be induced by addition of signals early by using specific media conditions. While several factors super-regulate the quorum receptors, others may co-regulate target promoters or may affect expression posttranscriptionally.To better understand the contributions of super-regulation and co-regulation to quorum-sensing gene expression, and to better understand the general structure of the quorum sensing network, we ectopically expressed the two receptors (in the presence of their cognate signals) and another component that affects quorum sensing, the stationary phase sigma factor RpoS, early in growth. We determined the effect on target gene expression by microarray and real-time PCR analysis. Our results show that many target genes (e.g. lasB and hcnABC) are directly responsive to receptor protein levels. Most genes (e.g. lasA, lecA, and phnAB), however, are not significantly affected, although at least some of these genes are directly regulated by quorum sensing. The majority of promoters advanced by RhlR appeared to be regulated directly, which allowed us to build a RhlR consensus sequence.The direct responsiveness of many quorum sensing target genes to receptor protein levels early in growth confirms the role of super-regulation in quorum sensing gene expression. The observation that the induction of most target genes is not affected by signal or receptor protein levels indicates that either target promoters are co-regulated by other transcription factors, or that expression is controlled posttranscriptionally. This architecture permits the integration of multiple signaling pathways resulting in quorum responses that require a "quorum" but are otherwise highly adaptable and receptive to environmental conditions.

Project description:In the opportunistic pathogen Pseudomonas aeruginosa, many virulence traits are finely regulated by quorum sensing (QS), an intercellular communication system that allows the cells of a population to coordinate gene expression in response to cell density. The key aspects underlying the functionality of the complex regulatory network governing QS in P. aeruginosa are still poorly understood, including the interplay between the effector protein PqsE and the transcriptional regulator RhlR in controlling the QS regulon. Different studies have focused on the characterization of PqsE- and RhlR-controlled genes in genetic backgrounds in which RhlR activity can be modulated by PqsE and pqsE expression is controlled by RhlR, thus hampering identification of the distinct regulons controlled by PqsE and RhlR. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of pqsE and/or rhlR was generated and validated. Transcriptomic analyses performed on this genetic background allowed us to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination. Transcriptomic data were validated via reverse transcription-quantitative PCR (RT-qPCR) and transcriptional fusions. Overall, our results showed that PqsE has a negligible effect on the P. aeruginosa transcriptome in the absence of RhlR, and that multiple RhlR subregulons exist with distinct dependency on PqsE. Overall, this study contributes to untangling the regulatory link between the pqs and rhl QS systems mediated by PqsE and RhlR and clarifying the impact of these QS elements on the P. aeruginosa transcriptome. IMPORTANCE The ability of Pseudomonas aeruginosa to cause difficult-to-treat infections relies on its capacity to fine-tune the expression of multiple virulence traits via the las, rhl, and pqs QS systems. Both the pqs effector protein PqsE and the rhl transcriptional regulator RhlR are required for full production of key virulence factors in vitro and pathogenicity in vivo. While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available to allow clear discrimination of the PqsE and RhlR regulons. The data produced in this study demonstrate that PqsE mainly impacts the P. aeruginosa transcriptome via an RhlR-dependent pathway and splits the RhlR regulon into PqsE-dependent and PqsE-independent subregulons. Besides contributing to untangling of the complex QS network of P. aeruginosa, our data confirm that both PqsE and RhlR are suitable targets for the development of antivirulence drugs.

Dataset Information

Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665T.

Publications

Transcriptome Profiling Reveals the EanI/R Quorum Sensing Regulon in Pantoea Ananatis LMG 2665<sup>T</sup>.

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