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Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3

BACKGROUND: Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobi...

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Autores principales: O' Leary, Niall D, O' Mahony, Mark M, Dobson, Alan DW
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224230/
https://www.ncbi.nlm.nih.gov/pubmed/21995721
http://dx.doi.org/10.1186/1471-2180-11-229
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author O' Leary, Niall D
O' Mahony, Mark M
Dobson, Alan DW
author_facet O' Leary, Niall D
O' Mahony, Mark M
Dobson, Alan DW
author_sort O' Leary, Niall D
collection PubMed
description BACKGROUND: Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. RESULTS: One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ(54 )consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. CONCLUSIONS: In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads.
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spelling pubmed-32242302011-11-27 Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3 O' Leary, Niall D O' Mahony, Mark M Dobson, Alan DW BMC Microbiol Research Article BACKGROUND: Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. RESULTS: One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ(54 )consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by primer extension analysis. Comparative analyses of genomes encoding phenylacetyl CoA, (PACoA), catabolic operons identified a common association among styrene degradation linked PACoA catabolons in Pseudomonas species studied to date. CONCLUSIONS: In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon paaL gene, encoding a transport protein essential for phenylacetic acid utilisation in P. putida CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading Pseudomonads. BioMed Central 2011-10-13 /pmc/articles/PMC3224230/ /pubmed/21995721 http://dx.doi.org/10.1186/1471-2180-11-229 Text en Copyright ©2011 O' Leary et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
O' Leary, Niall D
O' Mahony, Mark M
Dobson, Alan DW
Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title_full Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title_fullStr Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title_full_unstemmed Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title_short Regulation of phenylacetic acid uptake is σ(54 )dependent in Pseudomonas putida CA-3
title_sort regulation of phenylacetic acid uptake is σ(54 )dependent in pseudomonas putida ca-3
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224230/
https://www.ncbi.nlm.nih.gov/pubmed/21995721
http://dx.doi.org/10.1186/1471-2180-11-229
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