Role of the citrate pathway in glutamate biosynthesis by Streptococcus mutans.
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.
Cvitkovitch
D G
DG
Department of Oral Biology, College of Dentistry, University of Florida, Gainesville, USA. dcvitkov@dental.ufl.edu
Gutierrez
J A
JA
Bleiweis
A S
AS
D000154:Aconitate Hydratase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000096:biosynthesis*; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D003470:Culture Media; D011994:Recombinant Proteins; D019343:Citric Acid; D018698:Glutamic Acid; D007521:Isocitrate Dehydrogenase; D002950:Citrate (si)-Synthase; D000154:Aconitate Hydratase