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DNA sequence of folate biosynthesis gene sulD, encoding hydroxymethyldihydropterin pyrophosphokinase in Streptococcus
P Lopez1, B Greenberg, S A Lacks
1Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Abstract:
A cloned segment of the chromosome of Streptococcus pneumoniae, in which mutations to sulfonamide resistance occur, contains several genes encoding enzymes for folate biosynthesis. Determination of the DNA sequence of parts of this segment and identification of a putative promoter and terminator of transcription indicate an operon composed of four genes. The first, sulA, encodes the enzyme dihydropteroate synthase. The functions of the second and third possible genes, sulB and sulC, are not known. The last gene, sulD, encodes a 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase. The product of this enzyme is the substrate for dihydropteroate synthetase. The enzyme protein was partially purified and shown to consist of a single subunit of 31 kilodaltons, encoded by sulD. On the basis of gel filtration behavior, the native protein appears to be a trimer or tetramer. Subcloning of the sulD gene in an Escherichia coli expression vector increased expression of the pyrophosphokinase 1,000-fold over the level produced by a single copy of the chromosomal gene.
Insights
This study identifies an operon in Streptococcus pneumoniae involved in folate biosynthesis and sulfonamide resistance. It details genes sulA and sulD, encoding key enzymes for this pathway.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Sulfonamide resistance in Streptococcus pneumoniae is linked to mutations in folate biosynthesis genes.
- Understanding these genetic mechanisms is crucial for developing effective antimicrobial strategies.
Purpose of the Study:
- To characterize the genetic organization and function of genes involved in folate biosynthesis and sulfonamide resistance in Streptococcus pneumoniae.
- To identify and analyze the genes within a specific chromosomal segment associated with sulfonamide resistance.
Main Methods:
- DNA sequencing of a cloned chromosomal segment from Streptococcus pneumoniae.
- Identification of putative promoter and terminator sequences to define gene organization.
- Gene expression analysis through subcloning into an Escherichia coli expression vector.
Main Results:
- A four-gene operon was identified, including sulA (dihydropteroate synthase) and sulD (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase).
- The sulD gene encodes a 31 kDa protein subunit, which forms a trimer or tetramer in its native state.
- Expression of the sulD gene in E. coli resulted in a 1,000-fold increase in pyrophosphokinase activity.
Conclusions:
- The identified operon plays a significant role in folate biosynthesis in Streptococcus pneumoniae.
- The sulD gene product is essential for folate metabolism and is a target for understanding sulfonamide resistance mechanisms.