Nucleotide sequence analysis and expression of a tetracycline-resistance gene from Campylobacter jejuni

Abstract

Resistance to tetracycline in the microaerophilic Gram-negative bacterium Campylobacter jejuni is plasmid-mediated. A 6.9-kb HindIII DNA fragment containing the tetracycline-resistance (TcR) gene (designated tetO) from the C. jejuni conjugative plasmid pUA466 was cloned into pUC8, and the resultant plasmid pUOA1 was used to transform Escherichia coli to Tc resistance. The tetO gene was localized at a 2.0-kb region comprising 0.2-kb and 1.8-kb HincII fragments, and the nucleotide sequences were determined. The protein coding region of tetO contained a 1911-bp open reading frame which corresponded to a 72.3-kDa protein. Upstream from the start codon were hexanucleotides that resembled the canonical sequences found at the -10 region, -35 region and the ribosome-binding site of the prokaryotic promoter. The tetO gene product was expressed utilizing an E. coli-derived in vitro transcription/translation system. The polypeptide had an apparent Mr of 68,000. Comparison of the amino acid sequences of TetO to those of TetM (derived from the Gram-positive Streptococcus pneumoniae) revealed 76% homology. Hydrophilicity plot analyses of TetO and TetM proteins provided almost identical profiles. These results clearly support our earlier [Taylor et al., J. Bacteriol. 169 (1987) 2984-2989] suggestion that TcR determinants found in Gram-positive bacteria and in C. jejuni may have a common ancestry.