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Genetic, molecular, and functional analysis of Streptococcus faecalis R plasmid pJH1
Abstract:
Streptococcus faecalis JH1 contains two conjugative plasmids, pJH1, an R plasmid that codes for resistance to kanamycin, streptomycin, erythromycin, and tetracycline, and pJH2, a hemolysin-bacteriocin plasmid. Strain JH1 was used as an antibiotic resistance donor in conjugation experiments with two plasmid-free S. faecalis recipient strains, JH2-2 and OG1-RF1. Plasmid pJH1 was purified from one transconjugant, DL77, and subjected to restriction endonuclease analyses. Five restriction enzymes, EcoRI, XbaI, BamHI, SalI, and XhoI, yielding 10, 9, 3, 2, and 2 fragments, respectively, were used to determine the size (80.7 kilobases) of pJH1 and to construct a restriction endonuclease map of the plasmid. Twenty-eight percent of the antibiotic-resistant transconjugants examined expressed only part of the resistance pattern (Kmr Smr Emr Tcr) associated with pJH1, that is, they were resistant to kanamycin, streptomycin, and erythromycin; to erythromycin and tetracycline; or to erythromycin or to tetracycline only. Most of these strains also produced hemolysin and bacteriocin, and several contained a hybrid plasmid consisting of pJH2 and specific segments of pJH1 DNA. Several of these hybrid plasmids, as well as a deletion derivative of pJH1 that coded for resistance to tetracycline but not to kanamycin, streptomycin, or erythromycin, were purified and used to confirm the arrangement of restriction endonuclease fragments on the pJH1 map and to locate the resistance determinants on this map.
Insights
This study maps the antibiotic resistance plasmid pJH1 in Streptococcus faecalis. Researchers identified hybrid plasmids and deletion derivatives, revealing the location of resistance genes.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus faecalis JH1 harbors two conjugative plasmids: pJH1 (R plasmid) and pJH2 (hemolysin-bacteriocin plasmid).
- Antibiotic resistance in bacteria is a significant public health concern, often mediated by plasmids.
Purpose of the Study:
- To construct a restriction endonuclease map of the pJH1 plasmid.
- To locate antibiotic resistance determinants on the pJH1 map.
- To investigate the formation and characteristics of hybrid plasmids.
Main Methods:
- Conjugation experiments using Streptococcus faecalis strains.
- Purification of plasmid DNA (pJH1).
- Restriction endonuclease analysis with enzymes like EcoRI, XbaI, BamHI, SalI, and XhoI.
- Characterization of transconjugants and hybrid plasmids.
Main Results:
- A restriction map of the 80.7 kilobase pJH1 plasmid was constructed.
- Twenty-eight percent of transconjugants showed partial antibiotic resistance patterns.
- Hybrid plasmids, combining pJH1 and pJH2, were identified, some conferring hemolysin and bacteriocin production.
- Deletion derivatives of pJH1 were analyzed to confirm gene arrangement and locate resistance determinants.
Conclusions:
- The study successfully mapped the pJH1 plasmid and identified the locations of its antibiotic resistance genes.
- The formation of hybrid plasmids suggests mechanisms for the dissemination of antibiotic resistance and other traits.
- Understanding plasmid structure and gene organization is crucial for combating antibiotic resistance.