OF THE CITRATE PATHWAY IN GLUTAMATE BIOSYNTHESIS BY STREPTOCOCCUS-MUTANS

In work previously reported (J. A. Gutierrez, P. J. Crowley, D. P. Bro wn, 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 isolat ed and the insertion point of the transposon was determined to be in t he icd gene encoding isocitrate dehydrogenase (ICDH). The intact icd g ene of S. mutans has now been isolated from an S. mutans genomic plasm id library by complementation of an icd mutation in Escherichia coli h ost strain EB106. Genetic analysis of the complementing plasmid pJG400 revealed an open reading frame (ORF) of 1,182 nucleotides which encod ed an enzyme of 393 amino acids with a predicted molecular mass of 43 kDa, The nucleotide sequence contained regions of high (60 to 72%) hom ology 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 wa s also able to complement a citrate synthase (gltA) mutation of E. col i W620. The enzyme activities of both ICDH, found to be NAD(+) depende nt, and citrate synthase were measured in cell extracts of wild-type S . mutans and E. coli mutants harboring plasmid pJG400. The region 5' f rom the citZ gene also revealed a partial ORF encoding 264 carboxy-ter minal amino acids of a putative aconitase gene. The genetic and bioche mical evidence indicates that S. mutans possesses the enzymes required to convert acetyl coenzyme A and oxalacetate to ol-ketoglutarate, whi ch 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.