Robin is a PhD student in the Murrell lab at the UEA. His project is studying the bacterial metabolism of isoprene, primarily in Rhodococcus sp. AD45 and Variovorax species. This project is funded by a European Research Council Advanced Grant Studentship.
Prof J Colin Murrell
Dr Jonathan Todd
MRes Molecular Microbiology, BSc (Hons) Microbiology, Nottingham Trent University
Project Description: Metabolism of Isoprene by an environmental Variovorax
With global emissions of isoprene estimated at 600 Tg per year, it is becoming increasingly important to understand the biogeochemical cycling of this volatile organic compound (VOC). Soil-dwelling bacteria are known to act as a sink for biogenic isoprene, mitigating the release of isoprene from trees. The metabolic pathway responsible for the breakdown of isoprene by bacteria is still poorly understood. The initial conversion of isoprene to epoxyisoprene is conducted by an isoprene monooxygenase, a step which has been studied in Rhodococcus sp. AD45. This Gram-positive estuarine isolate has served as the workhorse for isoprene research.
Variovorax sp. WS11, a Gram-negative bacterium isolated from willow soil, has been found to possess an isoprene monooxygenase gene cluster very similar to that of Rhodococcus sp. AD45. Variovorax sp. WS11 grows using isoprene, and its genome sequence indicates a range of metabolic versatility inherent in Variovorax sp. Variovorax sp. WS11 can utilise a range of sugars, carboxylic acids, and alkenes as the sole sources of carbon and energy. Genetics systems developed in the closely-related Variovorax paradoxus EPS have bene optimised for WS11, providing a platform for various genetics approaches.
A key aim of my project is to study the regulation and expression of isoprene metabolism genes in strain WS11. By analysing isoA expression and isoprene uptake rate in WS11, I have identified indication of isoA by isoprene and catabolite repression of isoprene metabolism by sugars. I aim to study expression and regulation of isoprene metabolism further through a range of molecular techniques, including LacZ reporter fusions of isoprene-metabolic genes. In the long term I aim to characterise the individual components of isoprene metabolism in WS11, particularly the steps subsequent to isoprene epoxidation. It is assumed that isoGHIJ encode the enzymes responsible for these steps. Variovorax sp. WS11 may be a much more tractable platform for characterising these genes than Rhodococcus sp. AD45.
My previous research has covered a variety of fields. My undergraduate research project focused on the role of DDX5, an RNA helicase linked to expression of chemotherapy targets, in chemotherapy resistance in breast cancer. My project focused on screening CRISPR/Cas9-edited breast cancer cell lines for viable DDX5-knockout cells. This was approached primarily through Western blotting and immunofluorescence to visualise changes in DDX5 expression. At postgraduate level, I studied the role of outer membrane vesicles in the antimicrobial resistance of Helicobacter pylori. I demonstrated that OMV increased the resistance of H. pylori to amoxicillin, a key component of the recommended H. pylori treatment, and LL-37, a cationic antimicrobial compound produced by the gastric mucosa.
Growth of microbes on isoprene, physiology, biochemistry, molecular genetics. PCR, cloning, sequencing, genomics, qPCR, transcriptomics, mutagenesis, Western blotting, Immunofluorescence.
European Research Council Advanced Grant awarded to Prof Colin Murrell