The proposed research aims to study the structural, regulatory and metabolic features responsible for the extremely diverse xenobiotic catabolism of Rhodococcus. The genetics and biochemistry of naphthalene catabolism will be investigated by: ( 1 ) direct DNA cloning, sequencing and site-specific mutagenesis of genes, operon(s) and regulatory regions involved in naphthalene catabolism using innovative mobilizable cosmid vectors that Dr. Archer has developed for Nocardioform Actinomycetes which permit functional screening for entire catabolic pathways in the genetically compatible, but catabolically naive host Corynebacterium glutamicum; (2) biochemical studies of purified and/or overexpressed enzyme substrate ranges and kinetics coupled with crossfeeding experiments of mutants carrying specific gene knock-outs will be performed to investigate pathway specificity, carbon flux and cross talk between polyaromatic and monoaromatic catabolic reactions. Enzyme data will be integrated to produce a model for naphthalene catabolic flux. The protein structural data derived will be further analysed using computer aided "homology mapping" techniques to locate functional important regions within key enzymes. Analysis of gene-regulatory elements will enhance our understanding of Gram-positive promoter structure and gene regulation under oligotrophy.
Training content (objective, benefit and expected impact) The genetic and biochemical proposed research will augment my D.E.A./MSc training as a biochemical engineer. Additionally it will provide a thorough education in microbial genetics. On gaining a PhD, I intend to continue my research career, most probably in an applied research institute or company. Links with industry / industrial relevance (22) Dr. Archer has developed very strong industrial research programme developing pollution biosensors (funding over 220,000 ECU since 1992). The results of the proposed research, particularly with respect to genetic regulation, will be exploited in collaboration with Prior Scientific Instruments UK, an SME located near Cambridge.