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Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in
Abstract:
The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study shows how a Streptococcus pneumoniae gene conferring sulfonamide resistance can be amplified in plasmids, leading to higher resistance levels. It also demonstrates cross-species gene expression in Bacillus subtilis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus pneumoniae possesses a dihydropteroate synthase gene with a sul-d allele.
- This allele confers resistance to sulfanilamide, with varying levels depending on chromosomal or plasmid location.
Purpose of the Study:
- To investigate the sul-d allele's function and expression in different genetic contexts.
- To explore mechanisms of gene transfer and expression between bacterial species.
Main Methods:
- Cloning of the sul-d allele into the pLS80 plasmid.
- Restriction analysis to map the mutation.
- Plasmid manipulation and selection for antibiotic resistance.
- Transformation experiments in Bacillus subtilis.
Main Results:
- The sul-d allele confers higher sulfanilamide resistance (2.0 mg/ml) on multicopy plasmids compared to single chromosomal copies (0.2 mg/ml).
- Chromosomal facilitation and plasmid deletion/restoration mechanisms were observed.
- The pneumococcal gene demonstrated cross-species expression in Bacillus subtilis, conferring sulfanilamide resistance.
Conclusions:
- Plasmid amplification significantly enhances sulfonamide resistance conferred by the sul-d allele.
- Mechanisms like chromosomal facilitation and equilibration play roles in gene transfer and maintenance.
- The sul-d gene is functionally expressed across different bacterial species.