Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

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dc.contributor.advisor O'Leary, Niall D.
dc.contributor.advisor Dobson, Alan D. W.
dc.contributor.author Ryan, William James
dc.date.accessioned 2013-01-22T10:28:35Z
dc.date.available 2014-01-22T05:00:05Z
dc.date.issued 2013-01
dc.date.submitted 2013-01
dc.identifier.citation Ryan, W.J. 2013. Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3. PhD Thesis, University College Cork. en
dc.identifier.uri http://hdl.handle.net/10468/904
dc.description.abstract The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions. en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher University College Cork en
dc.rights © 2013, William J. Ryan en
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/ en
dc.subject gacS en
dc.subject ClpX en
dc.subject Global regulators en
dc.subject.lcsh Biopolymers en
dc.subject.lcsh Pseudomonas en
dc.title Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3 en
dc.type Doctoral thesis en
dc.type.qualificationlevel Doctoral en
dc.type.qualificationname PhD (Science) en
dc.internal.availability Full text available en
dc.check.info Access to pages 166 – 204 is restricted for 12 months by request of the author. Full text will be available from 22 January 2014. en
dc.description.version Accepted Version en
dc.contributor.funder Environmental Protection Agency en
dc.description.status Not peer reviewed en
dc.internal.school Microbiology en
dc.internal.school Environmental Research Institute en


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