Cloning the smell of the seaside - molecular genetics of dimethyl sulphide production by bacteria

We've all been to the seaside and we've all been told by a knowing parent to 'breathe in that ozone', because it's 'good for you'. Well, firstly, it's not ozone and second, it's not terribly good for you. That distinctive aroma is, in fact another gas, called dimethyl sulphide (DMS) and it has been known since 1971 that it is hugely important, with some 30 million tons of it being liberated into the air, world wide, every year. And once in the atmosphere it has other major effects, being the 'seed' that sets off cloud formation over the oceans. Indeed, it has been proposed that the production of this molecule is on such a scale that it has major effects on the world's climate. Yet, despite all this, we have absolutely no idea of how, at a molecular level, this process occurs. This is all the more surprising since we have known for some time that many marine bacteria, some of which are easy to grow in the laboratory, can liberate DMS if supplied with the key precursor molecule, called Dimethylsulphiopropionate - DMSP for short. Not a compound one reads about every day, yet there are over two billion tonnes of it in the world's oceans, seas and seashores. That's the weight, give or take, of another seaside symbol, the Blackpool Tower - 70,000 times over. Amazing. This DMSP molecule is used by the great masses of marine plant life - seaweeds and microscopic plankton - as a buffer, or osmo-protectant, against the saltiness of the sea. When these plants die, some of the DMSP that escapes from them is used as food by some marine bacteria and, when they do so, they convert some of it to the DMS gas in the process. We recently isolated one such DMSP-consuming bacterium from the Norfolk coast and used various molecular techniques to get our hands on some of the genes that are involved. By looking at their sequences, we can guess what the genes might be doing and, so far, it looks as if the mechanisms are very different from those hypothetical ones that had been proposed before. We also saw that very similar genes exist in some other, very unexpected, types of bacteria, such as those that live, symbiotically, on the roots of land plants. So the extent of DMS production by bacteria may be far wider and varied than we had thought. We now hope to get a much deeper understanding on this process, at least in 'our' strain. We want to identify and characterize all the enzymes that are involved and we want to know how the pathway is regulated - we already know that these bacteria are not stupid, since they only switch on their systems for degrading the DMSP if the compound is present in their environment. Once we know what is happening with this Norfolk strain, it should be fairly straightforward to find out if other types of marine bacteria that eat DMSP do so in the same way. So, for the first time, we are close to getting a real insight into the molecular details of this pathway, allowing us to amuse, fascinate and educate our friends the next time we go to Great Yarmouth and somebody asks about the delicate scent of rotting seaweed that drifts up from the golden sands.

We will undertake molecular genetic analyses of the catabolism of Dimethylsulphiopropionate (DMSP) in the marine gamma-proteobacterium Marinomonas that can grow on DMSP as sole C source. This work will be greatly aided by access to the genome sequence of the strain, being done at the Joint Genome Institute, thanks to a grant to AWBJ from the U. S. Dept of Energy. We already identified some of the (dpc) genes for DMSP catabolism and showed that they are regulated, in response to DMSP and to CO2. The planned work involves: 1. Isolation and charactersiation of insertion mutations, to identify the full complement of Marinomonas genes involved in DMSP catabolism. This will be done by targetted insertions into genes already suspected of being involved in the process and by random transposon mutagenesis of the Marinomonas genome. 2. Attempts to introduce DMSP-catabolising activity to other bacteria on cloned DNA fragments of the Marinomonas genome. 3. Use of proteomics to determine the range of gene products whose abundance is affected by exposure of Marinomonas to DMSP and CO2. 4. Use of sophisticated analytical procedures, including the use of labelled DMSP, to deduce the pathway and intermediates in DMSP breakdown in Marinomonas. This will exploit the mutants that are defective at different stages in the pathway. 5. Identification of the cis-acting regulatory sequences that are recognised by the DpcR regulatory protein in response to DMSP and/or CO2. 6. Mutational dissection of the regions of DpcR protein that respond to the two different co-inducers, DMSP and CO2. 7. Comparison of the molecular processes involved in DMSP breakdown in Marinomonas with those in an unusual strain of Rhizobium and in the root-colonising species Burkholderia ambifaria, the analyses of these two species to be done in parallel studies in our laboratory. 8. Analysis and annotation of the Marinomonas genome sequence, suitable for publication.