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.