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. 1995 Jan;177(1):66–74. doi: 10.1128/jb.177.1.66-74.1995

A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae.

S A Lacks 1, B Greenberg 1, P Lopez 1
PMCID: PMC176557  PMID: 7798151

Abstract

Two genes, sulB and sulC, in a folate biosynthetic gene cluster of Streptococcus pneumoniae were identified after determination of the DNA sequence between two previously reported genes, sulA and sulD, in a cloned segment of chromosomal DNA containing a mutation to sulfonamide resistance. The gene products, SulB and SulC, correspond to polypeptides of 49 and 21 kDa, respectively. SulC has GTP cyclohydrolase activity and catalyzes the first step in the folate biosynthetic pathway. SulB apparently has dihydrofolate synthetase activity in that it complements a folC mutant of Escherichia coli and thus catalyzes the last step in the pathway. Prior work showed that SulA, a dihydropteroate synthase, and SulD, a bifunctional enzyme, catalyze three intervening steps. Mapping of the mRNA transcribed from the operon was consistent with its beginning at a promoter with a -35 site (gTGtCc) and an extended -10 site (T-TG-TAaAAT) and its termination at the end of a hairpin structure, which would give a transcript 3,745 nucleotides in length. SulC showed a considerable conservation of sequence by comparison with proven or putative GTP cyclohydrolases from four unrelated species, with 38 to 53% of the residues being identical. A similar comparison of SulB with dihydrofolate synthetases showed an identity of only 26 to 37%. Overall, comparisons of the five folate biosynthetic enzymes in different species suggest that S. pneumoniae is related more closely to other gram-positive bacteria, less closely to eucaryotes, and least closely to the gram-negative E. coli. The varied arrangements of folate biosynthetic genes in different species imply an early evolutionary period of fluidity in genomic rearrangement.

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Selected References

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  1. Babitzke P., Gollnick P., Yanofsky C. The mtrAB operon of Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis. J Bacteriol. 1992 Apr;174(7):2059–2064. doi: 10.1128/jb.174.7.2059-2064.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bacot C. M., Reeves R. H. Novel tRNA gene organization in the 16S-23S intergenic spacer of the Streptococcus pneumoniae rRNA gene cluster. J Bacteriol. 1991 Jul;173(13):4234–4236. doi: 10.1128/jb.173.13.4234-4236.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Cao G. J., Sarkar N. Identification of the gene for an Escherichia coli poly(A) polymerase. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10380–10384. doi: 10.1073/pnas.89.21.10380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dallas W. S., Dev I. K., Ray P. H. The dihydropteroate synthase gene, folP, is near the leucine tRNA gene, leuU, on the Escherichia coli chromosome. J Bacteriol. 1993 Dec;175(23):7743–7744. doi: 10.1128/jb.175.23.7743-7744.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dallas W. S., Gowen J. E., Ray P. H., Cox M. J., Dev I. K. Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100. J Bacteriol. 1992 Sep;174(18):5961–5970. doi: 10.1128/jb.174.18.5961-5970.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garrow T. A., Admon A., Shane B. Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9151–9155. doi: 10.1073/pnas.89.19.9151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilbert W. Genes-in-pieces revisited. Science. 1985 May 17;228(4701):823–824. doi: 10.1126/science.4001923. [DOI] [PubMed] [Google Scholar]
  11. Gollnick P., Ishino S., Kuroda M. I., Henner D. J., Yanofsky C. The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8726–8730. doi: 10.1073/pnas.87.22.8726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hatakeyama K., Inoue Y., Harada T., Kagamiyama H. Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway. J Biol Chem. 1991 Jan 15;266(2):765–769. [PubMed] [Google Scholar]
  13. 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]
  14. Katzenmeier G., Schmid C., Kellermann J., Lottspeich F., Bacher A. Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia coli. Biol Chem Hoppe Seyler. 1991 Nov;372(11):991–997. doi: 10.1515/bchm3.1991.372.2.991. [DOI] [PubMed] [Google Scholar]
  15. Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lacks S. Integration efficiency and genetic recombination in pneumococcal transformation. Genetics. 1966 Jan;53(1):207–235. doi: 10.1093/genetics/53.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu J. D., Parkinson J. S. Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control. J Bacteriol. 1989 Mar;171(3):1254–1261. doi: 10.1128/jb.171.3.1254-1261.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lopez P., Greenberg B., Lacks S. A. DNA sequence of folate biosynthesis gene sulD, encoding hydroxymethyldihydropterin pyrophosphokinase in Streptococcus pneumoniae, and characterization of the enzyme. J Bacteriol. 1990 Sep;172(9):4766–4774. doi: 10.1128/jb.172.9.4766-4774.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lopez P., Lacks S. A. A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities. J Bacteriol. 1993 Apr;175(8):2214–2220. doi: 10.1128/jb.175.8.2214-2220.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lopez P., Martinez S., Diaz A., Espinosa M., Lacks S. A. Characterization of the polA gene of Streptococcus pneumoniae and comparison of the DNA polymerase I it encodes to homologous enzymes from Escherichia coli and phage T7. J Biol Chem. 1989 Mar 5;264(7):4255–4263. [PubMed] [Google Scholar]
  25. Margolis P. S., Driks A., Losick R. Sporulation gene spoIIB from Bacillus subtilis. J Bacteriol. 1993 Jan;175(2):528–540. doi: 10.1128/jb.175.2.528-540.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. McLean J. R., Krishnakumar S., O'Donnell J. M. Multiple mRNAs from the Punch locus of Drosophila melanogaster encode isoforms of GTP cyclohydrolase I with distinct N-terminal domains. J Biol Chem. 1993 Dec 25;268(36):27191–27197. [PubMed] [Google Scholar]
  28. Mohan S., Aghion J., Guillen N., Dubnau D. Molecular cloning and characterization of comC, a late competence gene of Bacillus subtilis. J Bacteriol. 1989 Nov;171(11):6043–6051. doi: 10.1128/jb.171.11.6043-6051.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Oppenheim D. S., Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. doi: 10.1093/genetics/95.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Slock J., Stahly D. P., Han C. Y., Six E. W., Crawford I. P. An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene. J Bacteriol. 1990 Dec;172(12):7211–7226. doi: 10.1128/jb.172.12.7211-7226.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Talarico T. L., Ray P. H., Dev I. K., Merrill B. M., Dallas W. S. Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase. J Bacteriol. 1992 Sep;174(18):5971–5977. doi: 10.1128/jb.174.18.5971-5977.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Toy J., Bognar A. L. Cloning and expression of the gene encoding Lactobacillus casei folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure. J Biol Chem. 1990 Feb 15;265(5):2492–2499. [PubMed] [Google Scholar]
  41. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  42. Volpe F., Ballantine S. P., Delves C. J. The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. Eur J Biochem. 1993 Sep 1;216(2):449–458. doi: 10.1111/j.1432-1033.1993.tb18163.x. [DOI] [PubMed] [Google Scholar]
  43. Volpe F., Dyer M., Scaife J. G., Darby G., Stammers D. K., Delves C. J. The multifunctional folic acid synthesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase. Gene. 1992 Mar 15;112(2):213–218. doi: 10.1016/0378-1119(92)90378-3. [DOI] [PubMed] [Google Scholar]
  44. Winston D. J., Lau W. K., Gale R. P., Young L. S. Trimethoprim-sulfamethoxazole for the treatment of Pneumocystis carinii pneumonia. Ann Intern Med. 1980 Jun;92(6):762–769. doi: 10.7326/0003-4819-92-6-762. [DOI] [PubMed] [Google Scholar]

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