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Physical characterization of Bacteroides fragilis R plasmid pBF4
Abstract:
Bacteroides fragilis V479-1 has previously been shown to harbor a self-transmissible 27 X 10(6)-dalton plasmid (pBF4) which confers lincosamide-macrolide resistance. The present study has focused on the physical properties of pBF4. The plasmid was found to be present in 1 to 2 copies per chromosomal equivalent. pBF4 was genetically stable, although spontaneously occurring plasmidless segregants could be detected at low frequency (approximately 1%). This frequency was unaffected by growth of cells in ethidium bromide. About one-third of all spontaneously occurring macrolide-lincosamide-sensitive clones of strain V479-1 were found to contain pBF4 molecules that carried deletions. Ten independently obtained deletion derivatives of pBF4 from lincosamide-macrolide-sensitive strains were compared with the parental pBF4 by restriction endonuclease cleavage analysis. A restriction site map of pBF4 was constructed, and the location of the deletions was approximated. Self-annealed pBF4 molecules, examined by electron microscopy, revealed the presence of two pairs of inverted repeat (IR) sequences on the plasmid. IR-1 was about 400 base pairs in length, and its two component members were separated by an intervening sequence of about 15 kilobases. IR-2 was about 75 base pairs in length, and its component members were separated by 4.2 kilobases. Each of the deletions of pBF4 studied had a terminus at or near the same IR-2 sequence.
Insights
Bacteroides fragilis V479-1 plasmid pBF4 confers antibiotic resistance. Deletions in this plasmid, often near inverted repeat sequences, lead to loss of resistance, impacting bacterial genetics.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacteroides fragilis V479-1 harbors a self-transmissible plasmid, pBF4.
- pBF4 confers resistance to lincosamide and macrolide antibiotics.
Purpose of the Study:
- To investigate the physical properties of the pBF4 plasmid.
- To characterize the genetic stability and deletion patterns of pBF4.
Main Methods:
- Plasmid copy number determination.
- Analysis of spontaneous plasmid loss and deletion derivatives.
- Restriction endonuclease cleavage analysis and mapping.
- Electron microscopy of self-annealed plasmid DNA.
Main Results:
- pBF4 exists at 1-2 copies per chromosomal equivalent and is genetically stable.
- Approximately one-third of sensitive segregants contained pBF4 with deletions.
- Deletions were mapped and often terminated near an inverted repeat sequence (IR-2).
- Two pairs of inverted repeat sequences (IR-1 and IR-2) were identified on pBF4.
Conclusions:
- The pBF4 plasmid's physical and genetic properties were elucidated.
- Deletions within pBF4, associated with inverted repeats, are a mechanism for loss of antibiotic resistance in Bacteroides fragilis.