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The genetic analysis of the soil actinomycete <i>Rhodococcus </i>has been hampered by a lack of genetic tools. In recent years methods for gene cloning by gain of function into an <i>E. coli</i> or <i>Rhodococcus</i> host have been established. Methods for cloning <i>Rhodococcus</i> genes (particularly into <i>E. coli</i>) are fraught with difficulties, due to restriction/methylation of DNA, integration and ineffectual gene expression in the host. The establishment of a gene disruption system would overcome these difficulties and allow selection of useful phenotypes by loss of function. In this work a recently developed <i>in vitro</i> Tn5-based mutagenesis system was adapted for use of <i>Rhodococcus</i>. Electroporation protocols generating sufficient numbers of transformants were established and a random knockout library was constructed in a <i>Rhodococcus</i> type-strain. Part of this work involved investigations of <i>Rhodococcus</i> cell envelope ultrastructure and the use of growth supplements to aid transformation. Library coverage was investigated by the identification and sequencing of a number of amino acid auxotrophs. The Tn5-based system was applied to a wild-type soil <i>Rhodococcus </i>isolate and a random knockout library was constructed. A number of mutants unable to grow in the presence of toluene and benzene were isolated. A number of transposon delivery vectors based on either Tn5 or IS<i>903</i> were constructed and problems of transposant selection overcome. For the purposes of construction the sequencing and analysis of two <i>Rhodococcus </i>plasmid replicons was carried out. The IS<i>903</i>-based vector although fully functional in <i>E. coli</i> failed to transpose in <i>Rhodococcus </i>and the possible reasons are discussed. Preliminary characterisation of a putative inducible promoter from <i>Rhodococcus </i>was carried out and the use of reporter genes <i>yfp </i>and <i>luxAB</i> established. The replicative Tn5 delivery vector was adapted to include the promoter/regulator to drive transposase expression however this vector was subjected to deletion in the <i>Rhodococcus </i>host.

<i>Serratia marcescens </i>274 contains a prodigiosin biosynthetic gene cluster, termed the <i>pig</i> cluster. The <i>pig</i> cluster contains 14 <i>pig</i> genes, <i>pigA</i> to <i>N</i> that were cloned on the cosmid pPIG4. The pPIG4 cosmid was able to direct the synthesis of prodigiosin in <i>Escherichia coli</i>. This is the first example of reconstitution of prodigiosin synthesis in this host. pPIG4 also encoded production of pigment in a biosynthetic mutant of <i>Serratia </i>sp. 39006, though not in <i>Erwinia carotovora</i> subsp. <i>carotovora. </i>The pigments from <i>Serratia </i>sp. 39006 and <i>S. marcescens </i>274 were purified and analysed using ES-MS. The <i>pig </i>genes, <i>pigA</i> to <i>N</i>, were sequenced, as were the genes flanking the cluster: <i>cueR </i>5’ of <i>pigA</i> and <i>copA </i>3’ of <i>pigN. </i>The <i>pig</i> gene cluster is arranged similarly to the <i>Serratia </i>sp. 39006 <i>pig</i> cluster, with <i>pigABCDEFGHJKLMN </i>all in one direction of transcription, suggesting an operon. Two striking differences between the <i>Serratia </i>sp. 39006 and the <i>S. marcescens </i>274 <i>pig </i>clusters are that (1) the <i>Serratia</i> sp. 39006 <i>pig </i>cluster contains an extra gene, <i>pigO</i>, the product of which shows low similarity to a VirR related protein and (2) the <i>S. marcescens </i>274 <i>pig</i> cluster is flanked by <i>cueR</i> and <i>copA</i> homologues. These genes, which encode a regulator and a copper transporter respectively, are typically adjacent and divergently transcribed in other bacteria. The <i>Serratia </i>sp. 39006 and <i>S. marcescens </i>274 <i>pig</i> genes encode proteins that are from 52 to 85% similar to each other. The <i>pig</i> genes also show similarity to genes found in the <i>red </i>cluster of <i>Streptomyces coelicolor</i>. The <i>red</i> cluster encodes 23 proteins that direct the synthesis of undecylprodigiosin. At least 12 of the <i>red</i> genes have homologues among the <i>pig</i> genes. Red and Pig homologues share between 23 to 43% similarity with each other. The order and orientation of the <i>red</i> genes compared to the <i>pig</i> genes is completely different, indicating gene rearrangement if these two clusters arose by divergent evolution.