Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS

Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from i...

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Main Authors:	Daddaoua, Abdelali, Fillet, Sandy, Fernández, Matilde, Udaondo, Zulema, Krell, Tino, Ramos, Juan L.
Format:	Online
Language:	English
Published:	Public Library of Science 2012
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402500/

id	pubmed-3402500
recordtype	oai_dc
spelling	pubmed-34025002012-07-27 Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS Daddaoua, Abdelali Fillet, Sandy Fernández, Matilde Udaondo, Zulema Krell, Tino Ramos, Juan L. Research Article Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the Pgad and Pkgu for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of Pseudomonas aeruginosa. We show using DNaseI-footprint analysis that in P. aeruginosa PtxR binds to the -35 region of the PtoxA promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the Pkgu and Pgad promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. In vitro, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of Pkgu and Pgad promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the PtoxA promoter, or via the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator. Public Library of Science 2012-07-23 /pmc/articles/PMC3402500/ /pubmed/22844393 http://dx.doi.org/10.1371/journal.pone.0039390 Text en Daddaoua et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Daddaoua, Abdelali Fillet, Sandy Fernández, Matilde Udaondo, Zulema Krell, Tino Ramos, Juan L.
spellingShingle	Daddaoua, Abdelali Fillet, Sandy Fernández, Matilde Udaondo, Zulema Krell, Tino Ramos, Juan L. Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
author_facet	Daddaoua, Abdelali Fillet, Sandy Fernández, Matilde Udaondo, Zulema Krell, Tino Ramos, Juan L.
author_sort	Daddaoua, Abdelali
title	Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
title_short	Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
title_full	Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
title_fullStr	Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
title_full_unstemmed	Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS
title_sort	genes for carbon metabolism and the toxa virulence factor in pseudomonas aeruginosa are regulated through molecular interactions of ptxr and ptxs
description	Homologs of the transcriptional regulator PtxS are omnipresent in Pseudomonas, whereas PtxR homologues are exclusively found in human pathogenic Pseudomonas species. In all Pseudomonas sp., PtxS with 2-ketogluconate is the regulator of the gluconate degradation pathway and controls expression from its own promoter and also from the Pgad and Pkgu for the catabolic operons. There is evidence that PtxS and PtxR play a central role in the regulation of exotoxin A expression, a relevant primary virulence factor of Pseudomonas aeruginosa. We show using DNaseI-footprint analysis that in P. aeruginosa PtxR binds to the -35 region of the PtoxA promoter in front of the exotoxin A gene, whereas PtxS does not bind to this promoter. Bioinformatic and DNaseI-footprint analysis identified a PtxR binding site in the Pkgu and Pgad promoters that overlaps the -35 region, while the PtxS operator site is located 50 bp downstream from the PtxR site. In vitro, PtxS recognises PtxR with nanomolar affinity, but this interaction does not occur in the presence of 2-ketogluconate, the specific effector of PtxS. DNAaseI footprint assays of Pkgu and Pgad promoters with PtxS and PtxR showed a strong region of hyper-reactivity between both regulator binding sites, indicative of DNA distortion when both proteins are bound; however in the presence of 2-ketogluconate no protection was observed. We conclude that PtxS modulates PtxR activity in response to 2-ketogluconate by complex formation in solution in the case of the PtoxA promoter, or via the formation of a DNA loop as in the regulation of gluconate catabolic genes. Data suggest two different mechanisms of control exerted by the same regulator.
publisher	Public Library of Science
publishDate	2012
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402500/
_version_	1611544724437467136

Genes for Carbon Metabolism and the ToxA Virulence Factor in Pseudomonas aeruginosa Are Regulated through Molecular Interactions of PtxR and PtxS

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