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Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens
P N Rather1, R Mierzwa, R S Hare
1Schering-Plough Research Institute, Bloomfield, New Jersey 07003.
Abstract:
The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Bräu, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.
Insights
A novel aminoglycoside acetyltransferase gene, aac(3)-Vb, was identified in Serratia marcescens, conferring resistance to aminoglycoside antibiotics. This gene is distinct from the known aac(3)-Va and is rare in clinical settings.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The AAC(3)-V resistance mechanism confers high-level resistance to specific aminoglycosides.
- Serratia marcescens strain 82041944 possesses an AAC(3)-V mechanism but lacks homology to the previously identified aac(3)-Va gene.
Purpose of the Study:
- To clone and characterize the gene responsible for the AAC(3)-V resistance mechanism in Serratia marcescens 82041944.
- To compare the newly identified gene with the known aac(3)-Va gene.
Main Methods:
- High-pressure liquid chromatography (HPLC) to analyze sisomicin acetylation products.
- Cloning of the acetyltransferase gene.
- Nucleotide and amino acid sequence comparisons.
- DNA hybridization using an internal aac(3)-Vb probe.
Main Results:
- An AAC(3) enzyme was detected in S. marcescens 82041944 extracts.
- The gene encoding this enzyme was cloned and designated aac(3)-Vb.
- aac(3)-Vb shares 72% nucleotide identity and 75% amino acid identity with aac(3)-Va.
- The aac(3)-Vb gene is rare in clinical isolates with an AAC(3)-V mechanism.
Conclusions:
- A new aminoglycoside acetyltransferase gene, aac(3)-Vb, has been identified and characterized.
- The aac(3)-Vb gene is evolutionarily related but distinct from aac(3)-Va.
- The aac(3)-Vb gene appears to be uncommon in clinical aminoglycoside resistance.