Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme
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Abstract:
A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Escherichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.
Insights
Researchers identified a Streptococcus pneumoniae gene mutation conferring sulfonamide resistance. The mutation altered the SulA polypeptide, an enzyme crucial for folate biosynthesis, suggesting a novel mechanism of resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Sulfonamide resistance is a significant challenge in treating bacterial infections.
- Understanding the genetic basis of sulfonamide resistance is crucial for developing effective antimicrobial strategies.
Purpose of the Study:
- To identify and characterize the genetic mutation responsible for sulfonamide resistance in Streptococcus pneumoniae.
- To elucidate the role of the SulA polypeptide in dihydropteroate synthase activity and sulfonamide resistance.
Main Methods:
- DNA sequencing to identify mutations in the resistant gene.
- Enzyme activity assays to confirm the function of the SulA polypeptide.
- Gene expression in minicells and protein purification to characterize the enzyme.
Main Results:
- A spontaneous mutation, sul-d, was identified in a Streptococcus pneumoniae gene.
- The mutation involved a 6-base pair insert and a repeat of an adjacent segment.
- The gene encoded a 34-kDa SulA polypeptide, which formed dimers or trimers constituting dihydropteroate synthase.
- Purification of the enzyme was achieved with high yield and homogeneity using an Escherichia coli expression vector.
- A deleted plasmid produced mutant dihydropteroate synthase but did not confer in vivo resistance.
Conclusions:
- The SulA polypeptide is a key component of dihydropteroate synthase, essential for folate biosynthesis.
- The sul-d mutation alters dihydropteroate synthase, but resistance may involve additional mechanisms.
- It is proposed that SulA might participate in another folate biosynthesis step, targeted by sulfonamides in vivo.