Molecular ecology of marine isoprene degradation

Isoprene is an atmospheric trace gas whose emissions to the atmosphere are roughly equal to that of methane. It is highly reactive and has the potential to affect climate through a variety of interactions in the atmosphere, including the formation of ozone. In the marine environment alone, algae produce up to 11 Tg y-1 of isoprene. To date, little is known about its degradation by microbes in the marine environment. In this project, isoprene-degrading bacteria from a range of marine sites were characterised and Illumina sequencing was used to mine the genomes of isoprene-degrading strains related to Gordonia polyisoprenivorans and Mycobacterium hodleri, isolated from the Colne Estuary, Essex. From these genomes, we retrieved novel sequences encoding isoprene monooxygenase, previously identified in a terrestrial Rhodococcus species. This information allowed the design of specific PCR primer sets for the isoA gene, encoding the alpha subunit of isoprene monooxygenase, to retrieve isoprene-specific genes from environmental samples. The primers amplify isoA from a wide range of marine genera. A database of isoA sequences from extant isoprene degraders and isoA sequences retrieved by PCR from DNA from a variety of different marine environments was created. The data obtained demonstrated that isoprene monooxygenase genes are widespread in the marine environment. Other work focused on the physiology of isoprene-degrading bacteria, particularly the marine isolate Gordonia polyisoprenivorans. SDS-PAGE, oxygen electrode assays and RT-PCR were also used to investigate the regulation of soluble diiron centre monooxygenases in this organism, and showed that two separate, inducible monooxygenase enzyme systems exist in this organism and are responsible for the oxidation of isoprene and propane. DNA-Stable Isotope Probing revealed that members of the genera Rhodococcus, Mycobacterium, Gordonia and Microbacterium are active isoprene degraders in the Colne Estuary