title: GacS-dependent regulation of polyhydroxyalkanoate synthesis in Pseudomonas putida CA-3.
abstract: To date, limited reports are available on the regulatory systems exerting control over bacterial synthesis of the biodegradable polyester group known as polyhydroxyalkanoates (PHAs). In this study, we performed random mini-Tn5 mutagenesis of the Pseudomonas putida CA-3 genome and screened transconjugants on nitrogen-limited medium for reduced PHA accumulation phenotypes. Disruption of a GacS sensor kinase in one such mutant was found to eliminate medium-chain-length PHA production in Pseudomonas putida CA-3. Recombinant expression of wild-type gacS from a pBBRgacS vector fully restored PHA accumulation capacity in the mutant strain. PCR-based screening of the P. putida CA-3 genome identified gene homologues of the GacS/GacA-rsm small RNA (sRNA) regulatory cascade with 96% similarity to published P. putida genomes. However, reverse transcription-PCR (RT-PCR) analyses revealed active transcription of the rsmY and rsmZ sRNAs in gacS-disrupted P. putida CA-3, which is atypical of the commonly reported Gac/Rsm regulatory cascade. Quantitative real-time RT-PCR analyses of the phaC1 synthase responsible for polymer formation in P. putida CA-3 indicated no statistically significant difference in transcript levels between the wild-type and gacS-disrupted strains. Subsequently, SDS-PAGE protein analyses of these strains identified posttranscriptional control of phaC1 synthase as a key aspect in the regulation of PHA synthesis by P. putida CA-3.
authors.affiliation: Environmental Research Institute, University College Cork, Cork, Ireland.
authors.forename: William J
authors.initials: WJ
authors.lastname: Ryan
authors.forename: Niall D
authors.initials: ND
authors.lastname: O'Leary
authors.forename: Mark
authors.initials: M
authors.lastname: O'Mahony
authors.forename: Alan D W
authors.initials: AD
authors.lastname: Dobson
mesh_terms: D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D054813:Polyhydroxyalkanoates / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D054813:Polyhydroxyalkanoates; D014157:Transcription Factors; D011494:Protein Kinases