DNA sequencing and characterization of a chloramphenicol resistance gene from Streptomyces venezuelae.

摘要

chloramphenicol resistance determinant from S. venezuelae ISP5230 genomic DNA, originally cloned as a 6.5-kb SstI-SstI fragment in the recombinant plasmid pJV3, was localized by in vitro deletion to a 2.4-kb KpnI-SstI DNA fragment in the recombinant plasmid pJV4. When subcloned in the phagemid vectors pTZ18R and pTZ19R, neither the 2.4-kb nor 6.5-kb fragment conferred chloramphenicol resistance on Escherichia coli.;DNA sequencing and nucleotide sequence analysis of the 2.4-kb fragment, predicted the presence of three complete and one incomplete open reading frames. The largest open reading frame, orf-4, encoded a hydrophobic polypeptide that showed significant sequence similarity to the putative chloramphenicol-efflux proteins of Streptomyces lividans and Rhodococcus fascians. Located immediately downstream of orf-4 was an open reading frame (orf-3) encoding a polypeptide (Orf3) that showed limited but significant sequence similarity to a number of proteins that required nucleotide co-factors. The discovery of a conserved ATP/GTP-binding site motif near the amino-terminus of Orf3, suggested a biological process such as a phosphotransferase reaction requiring a high-energy co-factor. A comparison of the derived amino acid sequences for orf-1 and orf-2 with those in current databases showed no significant similarities.;Streptomyces lividans transformants RM3 and RM4, carrying plasmids pJV3 and pJV4 respectively, rapidly metabolized chloramphenicol to one predominant product. Structure analysis based on