Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase

Abstract

The gene catIII, encoding a type III enterobacterial chloramphenicol acetyltransferase, was cloned from the transmissible plasmid R387 into pBR322 and bacteriophage M13 mp8. Nucleotide sequence analysis of 1160 bp of DNA identified an open reading frame encoding a protein of 213 amino acid residues and a calculated molecular mass of 24965 Da. The predicted N-terminal sequence is identical with that determined by Edman degradation of chloramphenicol acetyltransferase purified from Escherichia coli harbouring R387. Sequences equivalent to the consensus motifs for initiation and rho-factor-independent termination of transcription in E. coli occur 5' and 3' to the catIII open reading frame. In contrast with the catI gene, present on transposon Tn9 and many enterobacterial plasmids, expression of catIII is not subject to cyclic AMP-mediated catabolite repression in vivo and there is no sequence in the 5' non-coding DNA that resembles that deduced as the consensus for the binding of cyclic AMP receptor protein. Unique restriction-endonuclease cleavage sites were introduced adjacent to the catIII reading frame by using oligonucleotide-directed mutagenesis to facilitate insertion into E. coli expression vectors. Fully active chloramphenicol acetyltransferase represents 30-50% of the soluble protein component of cell-free extracts of E. coli containing the appropriate plasmids.

MOLECULAR DETECTION OF ANTIBIOTIC-RESISTANCE DETERMINANTS INESCHERICHIA COLIISOLATED FROM THE ENDANGERED AUSTRALIAN SEA LION (NEOPHOCA CINEREA)

“…For cassette array determination, overlapping sequence fragments were obtained using the primers HS458, HS459, and HS320 (Murray et al 1988). All sequencing was performed by Macrogen Inc. (Seoul, Korea) on a 3130x1 genetic analyzer (Applied Biosystems, Foster City, California, USA) using the standard run protocol for a 50-cm, 16-capillary array using a Big Dye terminator kit (Applied Biosystems).…”

The Structure of the Chloramphenicol Resistance Gene on a Transferable R Plasmid from the Fish Pathogen, Pasteurella piscicida

“…Several CP resistance genes have been sequenced ( 1,16,21,27). However, regardless of the bacteria from which cat was isolated, these enzymes revealed remarkable structural similarity especially around the active site.…”

“…4). Sequences of the pp-cat and its flanking region were compared with other cat genes previously reported (1,9,16,17,27). The pp-cat showed about 97, 56, 58, and 57% homology to the nucleotide sequence of CAT types I, II, III, and the CAT-VA gene, respectively.…”

“…Furthermore, CP resistance has arisen in a multiple drug-resistant plasmid linked to kanamycin, sulfonamide, and/or tetracycline resistance. In gram-negative bacteria, resistance to CP is due to the presence of the CP acetyltransferase (CAT) which is coded by the gene (cat) localized on plasmids or transposons and is expressed constitutively (1,15,16,22). Another resistant mechanism to CP, that is, chromosomal gene associated resistance which is ascribed to a permeability barrier to the drug, has also been reported (7,8,25 In human pathogenic bacteria, CP resistance mechanisms, including cat itself, have been studied in detail.…”

Identification of the C2‐1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C‐1H heteronuclear multiple quantum coherence method

“…Five resonances are readily identified in the HMQC 13C edited spectrum (Fig. Ib), out of a total of seven histidines in the translated nucleotide sequence of CATIII [9]. The twodimensional heteronuclear correlation r;pcctrum of the sample further separated the histidine C2-'H signals in the '%Z dimension, allowing six resonances to be distinguished (Fig.…”