Peer-Reviewed Journal Details
Mandatory Fields
Ryan, WJ;O'Leary, ND;O'Mahony, M;Dobson, ADW
2013
March
Applied and Environmental Microbiology
GacS-Dependent Regulation of Polyhydroxyalkanoate Synthesis in Pseudomonas putida CA-3
Validated
WOS: 18 ()
Optional Fields
SIGNAL-TRANSDUCTION PATHWAY MESSENGER-RNA RECOGNITION ESCHERICHIA-COLI AZOTOBACTER-VINELANDII PHENYLACETIC ACID GENETIC-ANALYSIS BIOSYNTHESIS EXPRESSION OLEOVORANS STYRENE
79
1795
1802
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.
WASHINGTON
0099-2240
10.1128/AEM.02962-12
Grant Details