Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance (original) (raw)
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Microbiology-sgm, 1991
The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCSl, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.
Antimicrobial Agents and Chemotherapy, 1985
Bacteroides uniformis RYC3373 resistant to 64 micrograms of chloramphenicol per ml was isolated from a peritoneal pelvic abscess of a patient not previously treated with this drug. Chloramphenicol resistance was transferable at low frequency to a suitable Bacteroides fragilis recipient. The acquisition of resistance was linked to the presence of a 39.5-kilobase plasmid (pRYC3373), which was subsequently transferred to a secondary recipient. The transfer of Cm resistance occurred by a conjugation-like process. Donor and transconjugant strains produced chloramphenicol acetyltransferase constitutively. The Km for chloramphenicol was 40 microM, and its inactivation by 5-5'-dithiobis(2-nitrobenzoic acid) suggested its similarity to the type II enterobacterial enzymes encoded by different conjugative plasmids and also to a previously described enzyme of B. fragilis F47 and F48. The specific activity and the resistance level in pRYC3373-bearing strains were more than 10-fold higher tha...
Detection of a Novel Chloramphenicol Resistance Plasmid from “Equine”Staphylococcus sciuri
Journal of Veterinary Medicine Series B-infectious Diseases and Veterinary Public Health, 1990
A small chloramphenicol resistance (Cm®) plasmid of 4.65 kB could be detected in an “equine” Staphylococcus sciuri-culture. This plasmid, designated as pSC3, was identified by interspecific protoplast transformation. On the basis of restriction endonuclease analyses a detailed restriction map of pSC 3 could be constructed. This allowed structural comparisons of pSC 3 with Cm®-plasmids of other staphylococcal species from infections of humans and animals and identification of pSC 3 as a member of the pC221-family of staphylococcal Cm®-plasmids. The pSC3-plasmid encoded an inducible chloramphenicol acetyltransferase as confirmed by enzymatic assays. This enzyme could be demonstrated in cell-free lysates of Cm®-induced pSC 3-transformants.
Intraspecies transfer of a chloramphenicol-resistance plasmid of staphylococcal origin
2003
BACKGROUND & OBJECTIVES The emergence of antibiotic-resistant bacteria is a phenomenon of concern to the clinician as well as to the pharmaceutical industry, because it is the major cause of failure in the treatment of infectious diseases. The genetic exchange of plasmids containing antibiotic resistant determinants (R-plasmids) between organisms of the same or different species is believed to play a crucial role in the evolution of antibiotic resistant bacteria. Staphylococcus aureus is well known for its multi-drug resistance (MDR). This work was undertaken to study the intraspecies transfer of a chloramphenicol (C) resistance staphylococcal R-plasmid among different clinical isolates of S. aureus. METHODS From a MDR S. aureus MC524 strain, a small plasmid pMC524/MBM was isolated. Lysostaphin lysis and sucrose mediated detergent lysis were used for plasmid preparation. Agarose gel electrophoresis, transformation experiments, Southern blotting and hybridization were done. Restricti...
Antimicrobial Agents and Chemotherapy, 2000
The 16.5-kbp plasmid pSCFS1 from Staphylococcus sciuri mediated combined resistance to chloramphenicol and florfenicol. The gene responsible for this resistance property, cfr , was cloned and sequenced. The amino acid sequence of the Cfr protein revealed no homology to known acetyltransferases or efflux proteins involved in chloramphenicol and/or florfenicol resistance or to other proteins whose functions are known.
Journal of Applied Microbiology, 1992
M. CARDOSO AND S. SCHWARZ. 1992. The 4.6 kb chloramphenicol resistance (CmR) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by CmR plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of CmR-determinants between Staph. aureus of human and bovine biotype.
Microbiology-sgm, 1992
The two 4.6 kb chloramphenicol resistance (CmR) plasmids pSCs6 and pScS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed M, values of 70000 on FPLC and were composed of three identical subunits, each of Mr approximately 23000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCs6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The K, values for chloramphenicol and acetyl-CoA of the CAT from pSCs6 were 2.5 pM and 58.8 PM, respectively, while those of the CAT from pSCS7 were 2.7 p~ and 55.5 PM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.
Two genes for chloramphenicol resistance common to Staphylococci and streptococci
European Journal of Epidemiology, 1988
Southern blot hybridization and pneumococcal transformation were used to study the epidemiology at a molecular level of the genes for chloramphenicol resistance (cat) in streptococci and staphylococci. The cat gene of staphylococcal plasmid pC194 showed homology to the cat genes of the chromosomal elements of 5 different strains of Streptococcus pneurnoniae and of Streptococcus agaIactiae B109. DNA sequence homology was also detected between the cat gene of staphylococcal plasmid pC221 and the cat gene of broad host range conjugative plasmid piP501, originally isolated from S. agalactiae. Two different cat genes appear to be present in clinical isolates of both streptococci and staphylooocci.
To characterise the chloramphenicol gene it was necessary to use gel electrophoresis on restriction fragments of the vector, containing the camr, gene that was used to produce recombinant DNA. The Camr gene gave resistance to the antibiotic chloramphenicol. The empty vector contains a resistance gene, in this instance to the antibiotic ampicillin. This was characterised in the same manor. These genes were amplified using the gene cloning method using resistance to the antibiotics as selective markers. Only 0.4% of the total competent E.coli cells were transformed to develop antibiotic resistance by use of the plasmid vector. The viable cell count of the same E-coli cells was shown to be 183 when diluted 100 fold. See table 1. The size of the restriction fragments generated by the restriction enzyme, HindIII of the vector that contained the camr gene proved to be 995 bp (base pairs) and 3020 bp giving a total length of 3975 bp. The fragment generated by the restriction digestion of the empty vector was shown to be 3020 bp. This method proved inadequate to characterise E.coli chromosomal DNA as the fragments generated proved to be too numerous to resolve when visualised by UV transillumination