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Genetic analysis of the conjugal transfer determinants encoded by the streptococcal broad-host-range plasmid pIP501
1Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond 23298-0678.
Abstract:
pIP501 is a 30.2-kilobase (kb) broad-host-range conjugative streptococcal plasmid which encodes chloramphenicol and erythromycin resistance. A smaller conjugation-proficient derivative of pIP501 has been constructed and designated pVA1702. pVA1702 is 25.2 kb, encodes only kanamycin resistance, and confers conjugative ability in an Enterococcus faecalis mating system. The temperature-sensitive transposon delivery vectors pTV1ts and pTV32 carrying Tn917 and Tn917lac, respectively, were used to create insertion mutants of pVA1702 in E. faecalis. These insertions enabled us to localize the determinants conferring conjugation to two separate regions of pVA1702: the 7.5-kb region A and the 8.8-kb region B. A nested set of deletions of the 7.5-kb region was subcloned in Escherichia coli, and the expression of these deletions was examined in an E. coli minicell system. The genes for three conjugal proteins have been located and designated cnjA, cnjB, and cnjC (for conjugation). Two other peptides that are transcribed in a divergent direction from the cnj genes were encoded in the A region, but it is not known whether they are involved in conjugation. These studies indicate that the conjugation genes of pIP501 are arranged in multiple transcriptional units.
Insights
Researchers mapped the conjugation genes on the pVA1702 plasmid, a derivative of pIP501. They identified three key genes (cnjA, cnjB, cnjC) essential for plasmid transfer in bacteria.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- pIP501 is a 30.2 kb broad-host-range streptococcal plasmid conferring chloramphenicol and erythromycin resistance.
- A derivative, pVA1702 (25.2 kb), confers kanamycin resistance and conjugative ability in Enterococcus faecalis.
Purpose of the Study:
- To identify and localize the genetic determinants responsible for the conjugative ability of the pVA1702 plasmid.
- To characterize the transcriptional organization of conjugation genes.
Main Methods:
- Construction of insertion mutants of pVA1702 in E. faecalis using temperature-sensitive transposon delivery vectors (pTV1ts, pTV32).
- Localization of conjugation determinants to specific regions (Region A and Region B) through mutant analysis.
- Subcloning and expression analysis of deletions within Region A in Escherichia coli minicells.
Main Results:
- Conjugation determinants were localized to two distinct regions: 7.5-kb Region A and 8.8-kb Region B.
- Three genes, designated cnjA, cnjB, and cnjC, encoding conjugal proteins, were identified within Region A.
- Two additional peptides, transcribed divergently from cnj genes, were encoded in Region A, with unknown function.
Conclusions:
- The conjugation genes of pIP501 are organized into multiple transcriptional units.
- The identified cnj genes are crucial for the conjugative transfer mediated by the pVA1702 plasmid.