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Replication mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B resistance plasmid pT48
1Department of Biochemistry, University of Oxford, UK.
Abstract:
Copy-number mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non-inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three- to five-fold cis-dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual phenotype of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C-terminus of the wild-type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala-Asn-Glu-Ile-Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling-circle type replication.
Insights
Researchers identified Staphylococcus aureus plasmid mutants with altered copy numbers and multimerization. These changes are linked to specific mutations in the Rep protein, affecting rolling-circle replication termination.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Staphylococcus aureus harbors macrolide-lincosamide-streptogramin B (MLS) resistance plasmids, such as pT48.
- Plasmid copy number and multimerization are critical for bacterial genetics and antibiotic resistance dissemination.
Purpose of the Study:
- To investigate the genetic basis of copy-number and multimerization mutants of the pT48 MLS resistance plasmid in Staphylococcus aureus.
- To elucidate the role of the Rep protein in regulating plasmid replication and stability.
Main Methods:
- Isolation and characterization of copy-number mutants using tylosin resistance.
- Nucleotide sequencing to identify genetic alterations in mutant plasmids.
- Analysis of Rep protein alterations and their impact on plasmid multimerization.
Main Results:
- A cis-dominant, high-copy-number mutant (pcopD3) resulted from a single base change in the replication region.
- Other mutants exhibited increased plasmid multimerization and copy number, effective in trans.
- These mutants had deletions altering the C-terminus of the Rep protein, leading to a conserved pentapeptide sequence.
- The observed multimerization phenotype correlates with defective termination of rolling-circle replication.
Conclusions:
- Specific alterations in the Staphylococcus aureus pT48 Rep protein C-terminus can lead to increased plasmid copy number and multimerization.
- Defective termination of rolling-circle replication is a likely mechanism underlying the multimerization phenotype.
- Understanding these mechanisms is crucial for controlling antibiotic resistance gene spread.