Regulation of phenylacetic acid uptake is σ⁵⁴dependent in <it>Pseudomonas putida </it>CA-3

• Main Authors: O' Mahony Mark M, O' Leary Niall D, Dobson Alan DW
• Format: Article
• Language: English
• Published: BMC 2011-10-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/11/229

<p>Abstract</p> <p>Background</p> <p>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 <it>Pseudomonas putida </it>CA-3 was subjected to random mini-Tn<it>5 </it>mutagenesis and mutants screened for altered styrene/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences.</p> <p>Results</p> <p>One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn<it>5 </it>insertion in the <it>rpoN </it>gene encoding σ54. Complementation of the D7 mutant with the wild type <it>rpoN </it>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 <it>P. putida </it>CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne <it>paaL </it>in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the <it>paaL </it>promoter from <it>P. putida </it>CA-3 revealed two σ⁵⁴consensus 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 <it>Pseudomonas </it>species studied to date.</p> <p>Conclusions</p> <p>In summary, this is the first study to report RpoN dependent transcriptional activation of the PACoA catabolon <it>paaL </it>gene, encoding a transport protein essential for phenylacetic acid utilisation in <it>P. putida </it>CA-3. Bioinformatic analysis is provided to suggest this regulatory link may be common among styrene degrading <it>Pseudomonads</it>.</p>