DNA sequencing and characterization of a chloramphenicol resistance gene from Streptomyces venezuelae.
Date
1993
Authors
Mosher, Roy Henry.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
A 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 $\sp1$H-NMR and $\sp $C-NMR spectra of the purified product showed the substance to be related to chloramphenicol but, with an electron-withdrawing group attached to carbon-1$\sp\prime$. $\sp{31}$P-NMR and mass spectroscopy established that the compound was chloramphenicol-1$\sp\prime$-phosphate. The results strongly suggest that orf-3 encodes a chloramphenicol kinase activity that detoxifies chloramphenicol by O-phosphorylation at carbon-1$\sp\prime$. In a bioassay using the chloramphenicol-hypersensitive Micrococcus luteus, chloramphenicol-1$\sp\prime$-phosphate showed negligible antibiotic activity when compared to chloramphenicol.
Thesis (Ph.D.)--Dalhousie University (Canada), 1993.
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 $\sp1$H-NMR and $\sp $C-NMR spectra of the purified product showed the substance to be related to chloramphenicol but, with an electron-withdrawing group attached to carbon-1$\sp\prime$. $\sp{31}$P-NMR and mass spectroscopy established that the compound was chloramphenicol-1$\sp\prime$-phosphate. The results strongly suggest that orf-3 encodes a chloramphenicol kinase activity that detoxifies chloramphenicol by O-phosphorylation at carbon-1$\sp\prime$. In a bioassay using the chloramphenicol-hypersensitive Micrococcus luteus, chloramphenicol-1$\sp\prime$-phosphate showed negligible antibiotic activity when compared to chloramphenicol.
Thesis (Ph.D.)--Dalhousie University (Canada), 1993.
Keywords
Biology, Molecular., Biology, Microbiology.