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Molecular cloning, purification, and properties of a plasmid-encoded chloramphenicol acetyltransferase from
1Institut für Bakteriologie und Immunologie, Justus Liebig Universität Giessen, Federal Republic of Germany.
Abstract:
A small chloramphenicol resistance (Cmr) plasmid of approximately 3.75 kb, designated pSCS5, was isolated from Staphylococcus haemolyticus. This plasmid encoded an inducible chloramphenicol acetyltransferase (CAT; EC 2.3.1.28). The cat gene of pSCS5 was cloned into the Escherichia coli plasmid vector pBluescript SKII+. It differed in its nucleotide sequence and deduced amino acid sequence from the cat genes described previously in staphylococci and other gram-positive bacteria. The CAT enzyme was purified from cell-free lysates by ammonium sulfate precipitation, ion-exchange chromatography, and fast protein liquid chromatography. The native enzyme had an Mr of 70,000 and was composed of three identical subunits, each with an Mr of approximately 23,000. Its isoelectric point was at pH 6.15. CAT from pSCS5 exhibited Km values of 2.81 and 51.8 microM for chloramphenicol and acetyl coenzyme A, respectively. The optimum pH for activity was 7.8. CAT encoded by pSCS5 proved to be relatively heat stable, but sensitive to mercury ions. The observed differences in the nucleotide sequence and the biochemical characteristics of the enzyme allowed the identification of the pSCS5-encoded CAT from S. haemolyticus as a CAT variant different from those described previously in gram-positive bacteria.
Insights
A novel chloramphenicol acetyltransferase (CAT) variant was identified in Staphylococcus haemolyticus. This enzyme, encoded by the pSCS5 plasmid, exhibits unique biochemical properties distinct from previously characterized CAT enzymes.
Area of Science:
- Microbiology
- Molecular Biology
- Enzymology
Background:
- Antibiotic resistance is a growing global health concern.
- Chloramphenicol resistance is often mediated by chloramphenicol acetyltransferase (CAT) enzymes.
- Understanding novel resistance mechanisms is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To isolate and characterize a novel chloramphenicol resistance determinant from Staphylococcus haemolyticus.
- To investigate the genetic and biochemical properties of the pSCS5-encoded chloramphenicol acetyltransferase (CAT).
- To compare the novel CAT variant with previously described enzymes.
Main Methods:
- Isolation and characterization of the pSCS5 plasmid from Staphylococcus haemolyticus.
- Cloning of the cat gene into an Escherichia coli expression vector.
- Purification of the CAT enzyme using ammonium sulfate precipitation, ion-exchange chromatography, and FPLC.
- Biochemical characterization including subunit analysis, isoelectric point determination, kinetic analysis (Km values), and determination of optimal pH and stability.
Main Results:
- A 3.75 kb plasmid, pSCS5, encoding an inducible chloramphenicol acetyltransferase (CAT) was isolated.
- The nucleotide and deduced amino acid sequences of the pSCS5 cat gene differed from previously reported staphylococcal and Gram-positive bacterial cat genes.
- The purified native CAT enzyme (Mr 70,000) consisted of three identical subunits (Mr ~23,000), with an isoelectric point of pH 6.15.
- The enzyme exhibited Km values of 2.81 µM for chloramphenicol and 51.8 µM for acetyl-CoA, with optimal activity at pH 7.8.
- The CAT enzyme demonstrated relative heat stability but sensitivity to mercury ions.
Conclusions:
- The pSCS5-encoded CAT enzyme represents a novel variant distinct from those previously identified in Gram-positive bacteria.
- The unique biochemical characteristics of this CAT variant highlight the diversity of antibiotic resistance mechanisms.
- Further investigation into this novel CAT variant could provide insights into evolving antibiotic resistance patterns.