A molecular investigation of the otrB locus of Streptomyces rimosus
The gene cluster encoding production of oxytetracycline (OTC) by Streptomyces rimosus (the commercial producer) has been studied in this laboratory. The topic of this thesis was the region of the otc cluster including and upstream of the OTC-resistance gene otrB, which has been shown previously to be responsible for reduced accumulation of the antibiotic. The otrB gene was sequenced. The sequence revealed some discrepancies with previously-published data on tet347 (Reynes et al., 1988; Journal of General Microbiology 134: 585-598), an OTC-resistant determinant from another strain of S.rimosus. The deduced gene product of the otrB showed considerable identity with efflux proteins from other Gram-negative and Gram-positive bacteria. These proteins contain conserved functional motifs, and OtrB was analysed in this context. The transcriptional start of otrB was identified. Several short investigations were undertaken into the physiology of antibiotic production. (1) A transcriptional fusion vector (pIJ2843) using catechol oxygenase as a reporter was used to monitor the response of transcription of various regions of the otc cluster (cloned from a high-producing strain) to changes in external phosphate concentration. These data were compared in relation to antibiotic production by the wild-type strain. (2) A transposon mutagenesis strategy was used to attempt to generate novel mutations within the otc cluster. (3) The presence of genetically-engineered haemoglobin cloned into S.rimosus production strains was investigated. The relationship between expression of the recombinant protein, antibiotic production and the aeration of the S.rimosus cultures is discussed.