Molecular analysis of an oxytetracycline resistance gene from Streptomyces rimosus

Doyle, Deirdre (1987) Molecular analysis of an oxytetracycline resistance gene from Streptomyces rimosus. PhD thesis, University of Glasgow.

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Abstract

An oxytetracycline (OTC) resistance gene, otcl. has been cloned previously from Streptomvces rimosus 15883, a commercial producer of the antibiotic. This gene confers protection to the hosts ribosomes from arrest of translation by OTC. The otcl gene has been sequenced and its transcriptional regulation pattern has been analysed. The otcl structural gene encodes a polypeptide of 71.329KD. The predicted amino acid (a. a.) sequence is highly homologous with the tetM polypeptide, from a Streptococcal transposon Tn1545. The tetM polypeptide also protects the protein synthesing machinery from the action of tetracycline. The first 130 amino acids of each polypeptide are 67% homologous and smaller regions of extensive homology are dispersed throughout the sequences. The amino terminal end of elongation factor Tu (EF-Tu) from E. coli is also homologous. It exhibits 36% . homology and if chemically equivalent amino acids are considered, 48% homology to the otcl polypeptide. This homology includes the characterised GTP binding site of EF-Tu. The conservation of a. a. between otcl. tetM and EF-Tu suggests that the amino terminal end of otcl has a similar function to the amino terminal end of the EF-Tu. Possibly the otcl polypeptide acts as an alternative EF-Tu in S. rimosus which protects protein synthesis from the inhibitory effects of oxytetracycline. Two transcriptional start sites have been identified for the otcl structural gene. Transcription initiates 129bp (otclp1) and 339-344bp (otclp2) upstream of the translational initiation codon. otclp1 resembles an E. coli type consensus promoter and otcIp2 has not been sequenced it is external to the cloned DNA. Both transcripts were identified by S1 mapping with RNA from S. rimosus. Six closely clustered divergent transcriptional start sites (the otcpA1-6 promoters) have been identified with RNA from S. lividans TK24 containing a clone of the region 5' to the otcl structural gene and 11% of the coding region. These promoters overlap the otclp1 start site and the transcript overlaps the otcZ coding region (one of the OTC production genes) which is transcribed in the same direction as otcl. Promoter activity has been recorded from promoter probe constructs of this region but the divergent transcriptional activity is repressed in constructs which contain 45% of the otcl coding sequence. The transcriptional start sites of otcpA1-6 have not been identified with RNA from S. rimosus which is consistent with the promoter probe data. In promoter probe constructs otclp1 is partially repressed by promoter activity from otcpA1-6. otcpA1 is very similar to a group of streptomycete promoters which are probably transcribed by core RNA polymerase in association with sigma factor 49. The otcpA1-5 promoters show no homology to any recognized promoter sequence. The transcriptional complexity of this region indicates several levels of regulation i.e. the number and variety of promoter signals, repression of the divergent transcript by sequences within the coding region of otcl (possibly the otcl polypeptide itself) and partial repression of otclp1 by the transcriptional activity of overlapping promoters otcpA1-6. The function of the divergent transcript is unknown but it is possible that it regulates the expression of otcZ. This regulation might be mediated by an anti-sense RNA or if the transcript is translated, by the polypeptide. Such control of the expression of an OTC biosynthetic gene would ensure that a cell was expressing otcl and therefore resistant to OTC before the onset of OTC production.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology, Genetics
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-76670
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 13:56
Last Modified: 19 Nov 2019 13:56
URI: http://theses.gla.ac.uk/id/eprint/76670

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