Browsing by Author "Hart, Stephen Lewis"

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    Characterization of pigment production by Escherichia Coli containing a cloned Rhodococcus gene
    (1991) Hart, Stephen Lewis; Woods, David R
    The screening of a Rhodococcus gene bank in Escherichia coli lead to the discovery of a pigment-producing clone. Initial studies revealed a blue component and a pink component in bacterial pigment preparations and suggested that they were different from the prodigiosins and actinorhodins produced by other actinomycetes. This dissertation represents a further analysis of the genetics and biochemistry of pigment production, and the development of an insertional-inactivation cloning vector utilizing the Rhodococcus pigment gene. The DNA of the pigment-producing clone was analysed by restriction mapping and sequenced. A 'single Rhodococcus gene of 1.1 kbp was found to be responsible for pigment production in E. coli. This gene had a putative ribosome binding site and coded for an enzyme of Mr 42,560. Deletion analysis and in vitro transcription-translation experiments supported the hypothesis that pigment production in E. coli was due to a single enzyme. The gene and gene product did not show any similarity when compared with nucleotide and protein sequences in computer data bases. Evidence was obtained from bacterial studies that the pigment pathway involved the conversion of tryptophan to indole by tryptophanase of E. coli and then to the pigment by the action of the cloned Rhodococcus gene. The solubility properties of the pigment, TLC analysis and NMR and visible spectrophotometry supported the hypothesis that the blue pigment was indigo and that the red pigment was indirubin, an isomer of indigo. An insertional-inactivation cloning vector, pSLH8, was developed containing the pigment gene as a marker. The advantages of this vector were that it produced pigmented colonies on LB agar plates without any further requirement for expensive substrates such as X-Gal ,which is required by the pUC series of vectors, and it is capable of being used in any E. coli strain whereas the pUC plasmids require special mutant strains for the detection of recombinants.