Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(3):650–655. doi: 10.1128/jb.179.3.650-655.1997

Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.

D G Cvitkovitch 1, J A Gutierrez 1, A S Bleiweis 1
PMCID: PMC178743  PMID: 9006016

Abstract

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Brown, J. D. Hillman, P. Youngman, and A. S. Bleiweis, J. Bacteriol. 178:4166-4175, 1996), a Tn917 transposon-generated mutant of Streptococcus mutans JH1005 unable to synthesize glutamate anaerobically was isolated and the insertion point of the transposon was determined to be in the icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd gene of S. mutans has now been isolated from an S. mutans genomic plasmid library by complementation of an icd mutation in Escherichia coli host strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encoded an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa. The nucleotide sequence contained regions of high (60 to 72%) homology with icd genes from three other bacterial species. Immediately 5' of the icd gene, we discovered an ORF of 1,119 nucleotides in length, designated citZ, encoding a homolog of known citrate synthase genes from other bacteria. This ORF encoded a predicted protein of 372 amino acids with a molecular mass of 43 kDa. Furthermore, plasmid pJG400 was also able to complement a citrate synthase (gltA) mutation of E. coli W620. The enzyme activities of both ICDH, found to be NAD+ dependent, and citrate synthase were measured in cell extracts of wild-type S. mutans and E. coli mutants harboring plasmid pJG400. The region 5' from the citZ gene also revealed a partial ORF encoding 264 carboxy-terminal amino acids of a putative aconitase gene. The genetic and biochemical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to alpha-ketoglutarate, which is necessary for the synthesis of glutamic acid. Indeed, S. mutans JH1005 was shown to assimilate ammonia as a sole source of nitrogen in minimal medium devoid of organic nitrogen sources.

Full Text

The Full Text of this article is available as a PDF (302.2 KB).

Selected References

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

  1. Abbe K., Takahashi S., Yamada T. Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions. J Bacteriol. 1982 Oct;152(1):175–182. doi: 10.1128/jb.152.1.175-182.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyd D. A., Cvitkovitch D. G., Hamilton I. R. Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans. J Bacteriol. 1995 May;177(10):2622–2627. doi: 10.1128/jb.177.10.2622-2627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlsson J., Johansson T. Sugar and the production of bacteria in the human mouth. Caries Res. 1973;7(4):273–282. doi: 10.1159/000259851. [DOI] [PubMed] [Google Scholar]
  4. Carlsson J., Kujala U., Edlund M. B. Pyruvate dehydrogenase activity in Streptococcus mutans. Infect Immun. 1985 Sep;49(3):674–678. doi: 10.1128/iai.49.3.674-678.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlsson J. Nutritional requirements of Streptococcus mutans. Caries Res. 1970;4(4):305–320. doi: 10.1159/000259653. [DOI] [PubMed] [Google Scholar]
  6. David M., Lubinsky-Mink S., Ben-Zvi A., Suissa M., Ulitzur S., Kuhn J. Citrate synthase from Mycobacterium smegmatis. Cloning, sequence determination and expression in Escherichia coli. Biochem J. 1991 Aug 15;278(Pt 1):225–234. doi: 10.1042/bj2780225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Jong M. H., Van der Hoeven J. S. The growth of oral bacteria on saliva. J Dent Res. 1987 Feb;66(2):498–505. doi: 10.1177/00220345870660021901. [DOI] [PubMed] [Google Scholar]
  8. Dingman D. W., Sonenshein A. L. Purification of aconitase from Bacillus subtilis and correlation of its N-terminal amino acid sequence with the sequence of the citB gene. J Bacteriol. 1987 Jul;169(7):3062–3067. doi: 10.1128/jb.169.7.3062-3067.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fortnagel P., Freese E. Analysis of sporulation mutants. II. Mutants blocked in the citric acid cycle. J Bacteriol. 1968 Apr;95(4):1431–1438. doi: 10.1128/jb.95.4.1431-1438.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fouet A., Jin S. F., Raffel G., Sonenshein A. L. Multiple regulatory sites in the Bacillus subtilis citB promoter region. J Bacteriol. 1990 Sep;172(9):5408–5415. doi: 10.1128/jb.172.9.5408-5415.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Griffith C. J., Carlsson J. Mechanism of ammonia assimilation in streptococci. J Gen Microbiol. 1974 Jun;82(2):253–260. doi: 10.1099/00221287-82-2-253. [DOI] [PubMed] [Google Scholar]
  13. Gutierrez J. A., Crowley P. J., Brown D. P., Hillman J. D., Youngman P., Bleiweis A. S. Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements. J Bacteriol. 1996 Jul;178(14):4166–4175. doi: 10.1128/jb.178.14.4166-4175.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamada S., Slade H. D. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol Rev. 1980 Jun;44(2):331–384. doi: 10.1128/mr.44.2.331-384.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  16. Jin S., Sonenshein A. L. Identification of two distinct Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4669–4679. doi: 10.1128/jb.176.15.4669-4679.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LaPorte D. C. The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology. J Cell Biochem. 1993 Jan;51(1):14–18. doi: 10.1002/jcb.240510104. [DOI] [PubMed] [Google Scholar]
  18. Muir J. M., Russell R. J., Hough D. W., Danson M. J. Citrate synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus. Protein Eng. 1995 Jun;8(6):583–592. doi: 10.1093/protein/8.6.583. [DOI] [PubMed] [Google Scholar]
  19. Muro-Pastor M. I., Reyes J. C., Florencio F. J. The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria. J Bacteriol. 1996 Jul;178(14):4070–4076. doi: 10.1128/jb.178.14.4070-4076.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nichols B. J., Perry A. C., Hall L., Denton R. M. Molecular cloning and deduced amino acid sequences of the alpha- and beta- subunits of mammalian NAD(+)-isocitrate dehydrogenase. Biochem J. 1995 Sep 15;310(Pt 3):917–922. doi: 10.1042/bj3100917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pearce J., Leach C. K., Carr N. G. The incomplete tricarboxylic acid cycle in the blue-green alga Anabaena variabilis. J Gen Microbiol. 1969 Mar;55(3):371–378. doi: 10.1099/00221287-55-3-371. [DOI] [PubMed] [Google Scholar]
  22. REISSIG J. L., WOLLMAN E. L. TRANSDUCTION DES MARQUEURS GALACTOSE PAR LES BACT'ERIOPHAGES TEMP'ER'ES 82 ET 434 D'ESCHERICHIA COLI. Ann Inst Pasteur (Paris) 1963 Oct;105:774–779. [PubMed] [Google Scholar]
  23. Smith A. J., London J., Stanier R. Y. Biochemical basis of obligate autotrophy in blue-green algae and thiobacilli. J Bacteriol. 1967 Oct;94(4):972–983. doi: 10.1128/jb.94.4.972-983.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  25. St Martin E. J., Wittenberger C. L. Regulation and function of ammonia-assimilating enzymes in Streptococcus mutans. Infect Immun. 1980 Apr;28(1):220–224. doi: 10.1128/iai.28.1.220-224.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Stern J. R., Bambers G. Glutamate biosynthesis in anaerobic bacteria. I. The citrate pathways of glutamate synthesis in Clostridium kluyveri. Biochemistry. 1966 Apr;5(4):1113–1118. doi: 10.1021/bi00868a001. [DOI] [PubMed] [Google Scholar]
  27. Terleckyj B., Shockman G. D. Amino acid requirements of Streptococcus mutans and other oral streptococci. Infect Immun. 1975 Apr;11(4):656–664. doi: 10.1128/iai.11.4.656-664.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thorsness P. E., Koshland D. E., Jr Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem. 1987 Aug 5;262(22):10422–10425. [PubMed] [Google Scholar]
  29. Yamada T., Carlsson J. Phosphoenolpyruvate carboxylase and ammonium metabolism in oral streptococci. Arch Oral Biol. 1973 Jul;18(7):799–812. doi: 10.1016/0003-9969(73)90051-4. [DOI] [PubMed] [Google Scholar]
  30. van Houte J. Bacterial specificity in the etiology of dental caries. Int Dent J. 1980 Dec;30(4):305–326. [PubMed] [Google Scholar]
  31. van Houte J. Role of micro-organisms in caries etiology. J Dent Res. 1994 Mar;73(3):672–681. doi: 10.1177/00220345940730031301. [DOI] [PubMed] [Google Scholar]

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

RESOURCES