Biochemical and genetic diversity of a critical step in the sulphur cycle - molecular studies of bacterial dimethyl sulphide production

The gas dimethyl sulphide (DMS for short) does strange things to animals. Tiny shrimp-like crustaceans, some seabirds and even harbour seals get excited by it, since its aroma is a sign of nearby food. And, for the general populace, it is a much-loved memento of days by the beach, being part of the evocative smell of the seaside. But DMS is much more important than that. It represents the major route for transfer of sulphur from the oceans to land and, as such, is a critical part of the natural sulphur cycle, in which living organisms transfer sulphur from one form to another. You may not know it, but many of the molecules in your body contain sulphur and without it we cannot survive. DMS is also important for our environment because when it gets into the air, it is oxidised to form other compounds that act as 'seeds' for cloud formation over the oceans, just as flecks of dust set off crystallisation in crystal gardens. This may affect our climate, by reducing the amounts of light that reaches Earth's surface, perhaps even causing 'global diming'. Furthermore, its oxidation contributes significantly to the phenomenon of acid rain. DMS is made in huge amounts in the oceans - around 300 million tons each year. It is generated by microbes that use another molecule, with a ridiculously long name - dimethylsulphoniopropionate, (DMSP) - as a food source. This DMSP is made by seaweeds and the masses of plankton in the oceans, helping them to survive the stresses of life at sea. When they die, the DMSP is liberated, and is then eaten by some bacteria and fungi, and some of these produce the DMS as a by-product. Other bacteria are more genteel - they devour DMSP in a very different way, which does not produce this microbial sulphurous burp. Given the importance of DMS, it is quite astonishing that today we know so little about genetic basis of its prodution. However, scientists at the University of East Anglia have recently made an important breakthrough by identifying the bacterial genes that are involved in making the DMS. In doing this, they have uncovered at least three completely different ways in which bacteria can make this gas. Not only that, but in the natural environment, these genes can be transferred among very different marine bacteria. Even more remarkably, they were transferred from some bacteria to completely different life forms, such as fungi. And, some of these enzymes, and the organisms that contain them, are of types that had never been suspected of being involved in this important process. Other researchers, at the University of Georgia, have done related work on the genes that are important for the pathway that does not make DMS. Using that information, and their own findings, the UEA group have found one marine bacterium - called Roseovarius nubinhibens - which is a real glutton for DMSP. It has two, possibly three, different ways to make DMS and, as if that were not enough, it can also break down DMSP by the other pathway that does not make DMS. The UEA group now wants to look at the properties of the enzymes that make DMS in this species and to see how a single bacterium decides which of its different pathways it should use under different circumstances - the concentration of the DMSP that is available, the acidity of the medium, the cell density and so on. By learning about these features on these newly discovered systems, we will be much better placed to understand how and why bacteria choose to break down the DMSP in one way, rather than another, and why some make lots of the important gas, but others only make a little. This knowledge will provide great new insights into the natural cycling of one of the most important elements for life on earth.

We have recently identified the genes for four different DMSP lyases, each of whose products cleaves DMSP to acrylate and DMS. We plan to determine the major features of these enzymes, termed DddP, DddL, DddLA and DddLB, which belong to at least two totally distinct protein families. This will involve purifying each of these proteins and characterising kinetically their capacities to catalyse the DMSP to DMS conversion, as well as their interactions with related molecules (e.g. glycine betaine) and potential inhibitors. Since these enzymes are predicted to be metallo-proteins, the metal contents on isolation will be analysed and the effects of metals and chelators on their activities will be investigated. Site-directed mutagenesis will be used to identify regions that are critical for their activity. Mutants in the marine bacterium Roseovarius dddP, dddLA, dddLB and dmdA genes (the last of which encodes DMSP demethylase) will be made by insertional mutagenesis and the effect on DMSP catabolism via the demethylase and the lyase pathways will be tested. The effects of environmental parameters (such as DMSP concentration, growth phase and nutritional status) on ddd and dmdA gene expression will be investigated. The role of a likely regulatory gene on the expression of the ddd/dmdA genes will also be examined, and by using labelled substrates, the identities and amounts of catabolites made by the different Ddd and DmdA systems will be determined.