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Ph.D Research

The metabolism of dimethylsulfide in the Bacteria

The research I conducted during my Ph.D concerned the various ways in which Bacteria can metabolise the volatile organosulfur compound dimethylsulfide (DMS). This page provides a brief summary of my Ph.D, highlighting key areas of research that I carried out at the University of Warwick under the supervision of Professor J Colin Murrell and Dr Hendrik Schäfer.

Introduction

Dimethylsulfide (DMS) is a toxic, corrosive, flammable volatile organosulfur compound (VOSC) that has been implicated in playing key roles in climate control (in the so-called CLAW hypothesis) and in the biogeochemical cycling of sulfur. Whilst DMS is present in a wide range of environments, plants and animals (it is responsible for the smell of cabbage and sweetcorn, for example), it is thought to be of greatest importance in the oceans, which is where my research centred. DMS is produced by dying phytoplankton and algae and, whilst a proportion of it is volatilised into the lower atmosphere, where it plays a role in assisting the formation of clouds, over 80% of DMS formed in the oceans is thought to be degraded by Bacteria. Very little had been determined with respect to the biochemistry, physiology and ecology of Bacteria that can metabolise DMS prior to my Ph.D and I completed work in a number of different Bacteria, isolated from both the marine environment and from soils. These organisms used different pathways of DMS metabolism and I have demonstrated a number of environmental sinks for DMS during the course of my research.

DMS Monooxygenase from Hyphomicrobium sulfonivorans

One of the key enzymes thought to be involved in DMS metabolism was identified in 1981 - DMS monooxygenase - in a member of the Alphaproteobacteria isolated from soil - Hyphomicrobium sp. S. This organism has long since been lost from laboratory cultures and so my studies into the enzyme were undertaken using a closely-related species - Hyphomicrobium sulfonivorans, which was isolated from garden soil by Dr Ann P Wood and Dr Elena Borodina at King's College London. By using SDS-PAGE to separate proteins contained within H. sulfonivorans when grown on DMS or on methanol as the sole carbon source, I was able to identify two polypeptides that were induced during growth on DMS. NH2-terminal sequencing of these polypeptides was performed and they were shown to be related to the subunits of a family of monooxygenase enzymes - the flavin-dependent monooxygenases, which are also found in Vibrio spp., Pseudomonas spp. and Rhodococcus spp. Using gel filtration chromatography and hydrophobic interaction chromatography, I was able to purify the enzyme and demonstrate that it oxidised DMS to formaldehyde and methanethiol at the expense of reduced flavin mononucleotide (FMNH2). An NADH-dependent FMN-oxidoreductase was found to be coupled to the enzyme in vivo, regenerating the FMNH2 at the expense of NADH.

DMS Metabolism in Methylophaga thiooxidans sp. nov.

Methylophaga thiooxidans is a marine methylotrophic member of the Gammaproteobacteria that was isolated by Dr Hendrik Schäfer at the University of Warwick from mixed phytoplankton cultures using DMS as the sole carbon source. The organism was shown to have a rapid rate of growth on DMS and was shown to be closely related to the dominant DMS-oxidising Bacteria identified in seawater using stable-isotope probing with [13C2]-DMS. Using physiological and chemical methods, I elucidated the pathway of DMS metabolism in M. thiooxidans and showed that, surprisingly, the organism used an internal form of chemolithoheterotrophy - oxidising DMS first to thiosulfate (S2O32-) and then to tetrathionate (S4O62-), obtaining electrons from this terminal oxidation step which I found was directly coupled to ATP synthesis.

DMS Metabolism in Sagittula Stellata

Sagittula stellata is a heterotrophic marine member of the Gammaproteobacteria that had previously been shown to oxidise DMS to dimethylsulfoxide (DMSO) during growth on glucose, though the mechanism or purpose of this oxidation remained unknown. Using chemostat culture on fructose or succinate with or without DMS as an additional substrate, I found that the molar growth yield (Y) was increased in the presence of DMS by up to 15%. I demonstrated that the oxidation of DMS to DMSO is coupled to ATP synthesis and that DMS is most likely to be brought into S. stellata cells by active transport.

Sampling at Movile Cave

Rich Boden

Rich dot Boden at warwick dot ac dot uk

D14 School of Life Sciences
University of Warwick
Coventry
CV4 7AL
Lab: (024) 7652 2557

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