Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Sep;172(9):4766–4774. doi: 10.1128/jb.172.9.4766-4774.1990

DNA sequence of folate biosynthesis gene sulD, encoding hydroxymethyldihydropterin pyrophosphokinase in Streptococcus pneumoniae, and characterization of the enzyme.

P Lopez 1, B Greenberg 1, S A Lacks 1
PMCID: PMC213129  PMID: 2168367

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.

Full text

PDF
4766

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bognar A. L., Osborne C., Shane B. Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J Biol Chem. 1987 Sep 5;262(25):12337–12343. [PubMed] [Google Scholar]
  3. Bognar A. L., Osborne C., Shane B., Singer S. C., Ferone R. Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. J Biol Chem. 1985 May 10;260(9):5625–5630. [PubMed] [Google Scholar]
  4. Currier T. C., Nester E. W. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal Biochem. 1976 Dec;76(2):431–441. doi: 10.1016/0003-2697(76)90338-9. [DOI] [PubMed] [Google Scholar]
  5. FUTTERMAN S. Enzymatic reduction of folic acid and dihydrofolic acid to tetrahydrofolic acid. J Biol Chem. 1957 Oct;228(2):1031–1038. [PubMed] [Google Scholar]
  6. Higgins R. C., Dahmus M. E. Rapid visualization of protein bands in preparative SDS-polyacrylamide gels. Anal Biochem. 1979 Mar;93(2):257–260. doi: 10.1016/s0003-2697(79)80148-7. [DOI] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Lacks S. A., Lopez P., Greenberg B., Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. doi: 10.1016/0022-2836(86)90026-4. [DOI] [PubMed] [Google Scholar]
  9. Lopez P., Espinosa M., Greenberg B., Lacks S. A. Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. J Bacteriol. 1987 Sep;169(9):4320–4326. doi: 10.1128/jb.169.9.4320-4326.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lopez P., Espinosa M., Lacks S. A. Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis. Mol Gen Genet. 1984;195(3):403–410. doi: 10.1007/BF00341440. [DOI] [PubMed] [Google Scholar]
  11. Mannarelli B. M., Balganesh T. S., Greenberg B., Springhorn S. S., Lacks S. A. Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4468–4472. doi: 10.1073/pnas.82.13.4468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Martinez S., Lopez P., Espinosa M., Lacks S. A. Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae. Gene. 1986;44(1):79–88. doi: 10.1016/0378-1119(86)90045-4. [DOI] [PubMed] [Google Scholar]
  13. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  14. Murray C. L., Rabinowitz J. C. Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes. J Biol Chem. 1982 Jan 25;257(2):1053–1062. [PubMed] [Google Scholar]
  15. Ortiz P. J. Dihydrofolate and dihydropteroate synthesis by partially purified enzynes from wild-type and sulfonamide-resistant pneumonococcus. Biochemistry. 1970 Jan 20;9(2):355–361. doi: 10.1021/bi00804a024. [DOI] [PubMed] [Google Scholar]
  16. Richey D. P., Brown G. M. The biosynthesis of folic acid. IX. Purification and properties of the enzymes required for the formation of dihydropteroic acid. J Biol Chem. 1969 Mar 25;244(6):1582–1592. [PubMed] [Google Scholar]
  17. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  18. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  19. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  20. Shiota T., Baugh C. M., Jackson R., Dillard R. The enzymatic synthesis of hydroxymethyldihydropteridine pyrophosphate and dihydrofolate. Biochemistry. 1969 Dec;8(12):5022–5028. doi: 10.1021/bi00840a052. [DOI] [PubMed] [Google Scholar]
  21. Stassi D. L., Lopez P., Espinosa M., Lacks S. A. Cloning of chromosomal genes in Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7028–7032. doi: 10.1073/pnas.78.11.7028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  23. de la Campa A. G., Springhorn S. S., Kale P., Lacks S. A. Proteins encoded by the DpnI restriction gene cassette. Hyperproduction and characterization of the DpnI endonuclease. J Biol Chem. 1988 Oct 15;263(29):14696–14702. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES