Functional studies of the otrB gene from Streptomyces rimosus
Oxytetracyline, (OTC), is a secondary metabolite antibiotic produced by the actinomycete Streptomyces rimosus. All of the structural genes for OTC synthesis are clustered on the S. rimosus chromosome and flanked by resistance determinants, otrA and otrB. OtrA mediates resistance through non-covalent modification of the bacterial ribosome whilst otrB, the subject of this thesis, acts by efflux of the drug from the cell. At the outset of this thesis otrB had previously been cloned and sequenced. During the course of the work some discrepancies with previously published data were found, parts of the gene were then re-sequenced and a revised otrB sequence submitted to the NCBI database. The OtrB protein is a transmembrane protein belonging the Major facilitator Superfamily (MFS). Sequence comparison with other members of the family shows OtrB to be part of a subfamily of antibiotic transporters and multi drug resistance proteins made up from 14 transmembrane helices (6+2+6) arrangement. OtrB contains many conserved motifs typical of the subfamily and of the MFS. otrB was cloned into E. coli and expressed. The functional activity of the cloned gene was assessed by growth on various concentrations of OTC. Substrate specificity was investigated using TET and CTC in the growth media. Isolation of OtrB from E. coli as a polyhistidine fusion was attempted, reasons why this was not successful are discussed. Tetracycline exporters from Gram-negative bacteria generally contain 12 transmembrane helices (6+6). The relevance of the two "extra" helices present in OtrB was investigated by the construction and expression in E. coli, of a deletion mutant in which the two putative central helices were absent.