Related Experiment Videos
Characterization of the chloramphenicol acetyltransferase variants encoded by the plasmids pSCS6 and pSCS7 from
1Institut für Bakteriologie und Immunologie, Justus Liebig Universität Giessen, FRG.
Abstract:
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 pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed Mr values of 70,000 on FPLC and were composed of three identical subunits, each of Mr approximately 23,000. 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 Km values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5 microM and 58.8 microM, respectively, while those of the CAT from pSCS7 were 2.7 microM and 55.5 microM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.
Insights
Two chloramphenicol resistance plasmids from bovine mastitis Staphylococcus aureus encode distinct chloramphenicol acetyltransferase (CAT) enzymes. These CAT variants exhibit differences in charge, isoelectric point, and sensitivity to mercuric ions, despite similar structures and optimal activity.
Area of Science:
- Molecular Biology
- Enzymology
- Antimicrobial Resistance
Background:
- Subclinical bovine mastitis is a significant concern in dairy farming.
- Antibiotic resistance, particularly to chloramphenicol, poses a threat to animal and human health.
- Plasmids carrying resistance genes, such as chloramphenicol resistance (CmR) plasmids, are key drivers of resistance spread.
Purpose of the Study:
- To characterize the chloramphenicol acetyltransferase (CAT) enzymes encoded by the CmR plasmids pSCS6 and pSCS7.
- To compare the biochemical and biophysical properties of the CAT variants from pSCS6 and pSCS7.
- To understand the molecular basis of chloramphenicol resistance in Staphylococcus aureus isolates from bovine mastitis.
Main Methods:
- Purification of CAT variants using ammonium sulphate precipitation, ion-exchange chromatography, and Fast Protein Liquid Chromatography (FPLC).
- Determination of molecular weight (Mr) and subunit composition using FPLC.
- Analysis of enzyme kinetics, including Michaelis constants (Km) for chloramphenicol and acetyl-CoA.
- Assessment of enzyme activity at different pH values and thermostability.
- Evaluation of sensitivity to mercuric ions.
Main Results:
- Both pSCS6 and pSCS7 encode inducible CAT enzymes with native Mr values of 70,000, composed of three identical subunits of approximately 23,000 Mr.
- The CAT variants differ in net charge and isoelectric points (pI 5.7 for pSCS6-CAT, pI 5.2 for pSCS7-CAT).
- Both enzymes exhibit optimal activity at pH 8.0, with similar Km values for chloramphenicol (2.5-2.7 µM) and acetyl-CoA (55.5-58.8 µM).
- The CAT variants are relatively thermostable, with the pSCS6-encoded enzyme showing lower sensitivity to mercuric ions compared to the pSCS7 variant.
Conclusions:
- The study elucidates distinct biochemical properties of two chloramphenicol acetyltransferase variants from Staphylococcus aureus.
- Differences in charge and ion sensitivity suggest potential variations in the active site or overall protein structure.
- These findings contribute to understanding the diversity of chloramphenicol resistance mechanisms in bovine mastitis pathogens.