Molecular cloning and genetic analysis of a chloramphenicol acetyltransferase determinant from Clostridium difficile

Abstract

A gene bank from a clinical isolate of Clostridium difficile expressing high chloramphenicol acetyltransferase activity was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the plasmid vector pUC13. Among 1,020 clones tested, 11 were resistant to chloramphenicol; 1 of these, with an insert size of 1.9 kilobases (pPPM9), was studied further. The clone pPPM9 was mapped using a variety of restriction enzymes, and a 0.27-kilobase EcoRV-TaqI restriction fragment was shown to be within the chloramphenicol resistance (Cmr) gene by using transposon (Tn1000) mutagenesis. The 0.27-kilobase fragment and the 1.9-kilobase insert were radiolabeled and used as DNA probes in hybridization studies. Southern blot analysis with the gene probes against chromosomal DNA from Cmr strains of C. difficile obtained from five distinct geographical locations revealed that at least two copies of the same chloramphenicol acetyltransferase gene were present for each strain. Hybridization of the gene probes against Cmr strains of Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella edwardsii, Escherichia coli, and to four other clostridial species revealed no homology even under conditions of low stringency.

Hybridization analysis of three chloramphenicol resistance determinants from Clostridium perfringens and Clostridium difficile

“…The chloramphenicol resistance gene from C. difficile, catD, was previously cloned in E. coli (25). Hybridization experiments have shown that the catD and catP genes are homologous.…”

“…4). However, the catP probe did hybridize to the 1.9-kb Sau3A fragment of recombinant plasmid pPPM9 (25), which carried the C. difficile-derived chloramphenicol resistance determinant, designated catD (Fig. 4).…”

“…Restriction endonucleases and other enzymes were from Boehringer Mannheim Biochemicals, Bethesda Research Laboratories, Inc., Pharmacia, or New England BioLabs, Inc. Restriction endonuclease analysis and agarose gel electrophoresis of plasmid DNA were as described previously (2,7,17,25). DNA fragments were purified from low-melting-temperature agarose gels (SeaPlaque) by phenol extraction (17).…”

“…E. coli (1,4,25), C. perfringens (2), and S. aureus (16) DNAs were prepared as described previously. DNA from B. subtilis was made by using the C. perfringens method (2).…”

“…Recently, the chloramphenicol resistance determinant from a clinical isolate of Clostridium difficile has been cloned and analyzed. This gene, which is carried on pPPM9, is expressed in only one orientation in E. coli (25).…”