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Molecular analysis of an antibiotic resistance plasmid, pAV5, and its derivative plasmids in Acinetobacter
Abstract:
The non-conjugative plasmid pAV5 specifies resistance to kanamycin/neomycin (KmR) and tetracycline (TcR). Physical evidence is presented to show that pAV5 gives rise to two plasmids, pAV51 (KmR) and pAV52 (TcR), which are formed by deletion of apparently non-overlapping segments of pAV5. Expression of TcR has been obtained in Escherichia coli and is associated with a 1.9 kb HindIII fragment found in pAV5 and in pAV52. Expression of KmR has been obtained in E. coli and is associated with a 1.3 kb PstI fragment found in pAV5 and pAV51. Evidence is presented that the KmR gene is flanked by inverted repeat sequences and is therefore tentatively identified as a transposon, designated Tn4411. The KmR gene specifies an aminoglycoside 3'-phosphotransferase-type I (APH(3')-I) enzyme.
Insights
The plasmid pAV5 separates into two smaller plasmids, conferring kanamycin/neomycin and tetracycline resistance. The kanamycin resistance gene is identified as a transposon, Tn4411, encoding an APH(3’)-I enzyme.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The non-conjugative plasmid pAV5 confers resistance to kanamycin/neomycin (KmR) and tetracycline (TcR).
- Understanding plasmid evolution and gene organization is crucial in microbial genetics.
Purpose of the Study:
- To physically characterize the plasmid pAV5 and its resistance determinants.
- To identify the genetic elements responsible for kanamycin/neomycin and tetracycline resistance.
Main Methods:
- Plasmid DNA isolation and analysis.
- Restriction enzyme digestion and fragment analysis.
- Gene expression studies in Escherichia coli.
Main Results:
- Plasmid pAV5 was shown to resolve into two distinct plasmids: pAV51 (KmR) and pAV52 (TcR).
- Tetracycline resistance (TcR) expression in E. coli is linked to a 1.9 kb HindIII fragment.
- Kanamycin resistance (KmR) expression in E. coli is linked to a 1.3 kb PstI fragment.
- The KmR gene is flanked by inverted repeats, suggesting it is a transposon (Tn4411).
- The KmR gene encodes an aminoglycoside 3'-phosphotransferase-type I (APH(3')-I) enzyme.
Conclusions:
- Plasmid pAV5 undergoes deletion to form smaller, functional resistance plasmids.
- The kanamycin resistance gene, Tn4411, is a mobile genetic element.
- The identified enzyme APH(3')-I is responsible for kanamycin/neomycin resistance.