Abstract
A chloramphenicol acetyltransferase (catB7) gene containing two point mutations, 181A/G and 314A/G, has been recently reported to be a determinant for high-level chloramphenicol resistance phenotype in a Pseudomonas aeruginosa strain PAhcr1. The mutant CATB7 was further characterized in vitro and in vivo to elucidate the molecular basis of high-level resistance. CAT assay demonstrated that the mutant and wild-type recombinant CATB7 had similar specific activities. Dot blotting revealed that the accumulated amounts of CATB7 in P. aeruginosa strains PAO1 and PAhcr1 were proportionate to the respective anti-chloramphenicol level. Site-directed mutagenesis showed that G61S and Y105C contributed synergistically to the PAhcr1 resistance phenotype. It could be proposed that the mutant CATB7 was more structurally stable than catalytically efficient as a chloramphenicol resistance determinant in PAhcr1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetyl Coenzyme A / metabolism
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Amino Acid Substitution
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Chloramphenicol / metabolism*
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Chloramphenicol O-Acetyltransferase / chemistry
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Chloramphenicol O-Acetyltransferase / genetics*
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Chloramphenicol O-Acetyltransferase / isolation & purification
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Chloramphenicol O-Acetyltransferase / metabolism
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Chloramphenicol Resistance / genetics*
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Cloning, Molecular
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Enzyme Stability
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Genes, Bacterial
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Microbial Sensitivity Tests
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Mutagenesis, Site-Directed
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Mutation*
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Pseudomonas aeruginosa / drug effects
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Pseudomonas aeruginosa / enzymology*
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Pseudomonas aeruginosa / genetics*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
Substances
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Bacterial Proteins
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Recombinant Proteins
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Chloramphenicol
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Acetyl Coenzyme A
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Chloramphenicol O-Acetyltransferase