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Title: Genetic analysis of DMSP metabolism in the marine Roseobacter clade
Author: Kirkwood, Mark
ISNI:       0000 0004 2736 148X
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2012
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Genetic, biochemical, bioinformatic and molecular approaches were used to analyse microbial catabolism of dimethylsulfoniopropionate (DMSP), an abundant anti-stress compound made by marine phytoplankton. Members of the Roseobacter clade of marine α-proteobacteria may catabolise DMSP by two different routes; demethylation to form methylmercaptopropionate (MMPA), and cleavage by DMSP-lyases, yielding volatile dimethylsulfide (DMS) plus acrylate. The DMSP-lyase, DddP, was purified from Roseovarius nubinhibens ISM and characterised in vitro. Nuclear magnetic resonance spectroscopy and gas chromatography confirmed bona fide DMSP lyase activity and mutation of predicted active-site residues abolished DMS production. DddP was also detected in the fungal coral pathogen Aspergillus sydowii, likely acquired from bacteria by inter-Domain horizontal-gene-transfer. A new DMSP-lyase, DddW, was identified in another Roseobacter species, Ruegeria pomeroyi DSS-3, initially by microarray-based demonstrations that transcription of dddW was induced in cells grown with DMSP. An adjacent gene encoded the cognate transcriptional regulator. Escherichia coli cells that over-expressed DddW cleaved DMSP into DMS plus acrylate. Thus, Ruegeria pomeroyi has three DMSP-lyases, with DddP and DddQ being known already; mutational analyses showed that all three contributed to its DMSP-dependent DMS (Ddd+) phenotype. Moran’s laboratory had shown that the DMSP demethylase was encoded by R. pomeroyi dmdA. I unveiled intimate links between the demethylation and the cleavage pathway(s). A key player is acuI, which is co-transcribed with dmdA, both genes being induced by DMSP and, more markedly, the DMSP-catabolite, acrylate. Furthermore, AcuI- mutants failed to grow on acrylate as sole carbon source and were more sensitive to its toxic effects. AcuI- mutants failed to grow on DMSP so, surprisingly, Ruegeria likely uses lyase pathway(s) to grow on this compound. A potential regulatory gene, transcribed divergently from dmdA, was also identified. The microarray also, wholly unexpectedly, revealed a suite of cox genes involved in carbon monoxide oxidation that was up-regulated in response to DMS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available