GENETIC AND PHYSIOLOGICAL ANALYSIS OF A FORMYL-TETRAHYDROFOLATE SYNTHETASE MUTANT OF STREPTOCOCCUS-MUTANS

Previously we reported that transposon Tn917 mutagenesis of Streptococ cus mutans JH1005 yielded an isolate defective in its normal ability t o produce a mutacin (P. J. Crowley, J. D. Hillman, and A. S. Bleiweis, abstr. D55, p. 258 in Abstracts of the 95th General Meeting of the Am erican Society for Microbiology 1995, 1995). In this report we describ e the recovery of the mutated gene by shotgun cloning, Sequence analys is of insert DNA adjacent to Tn917 revealed homology to the gene encod ing formyl-tetrahydrofolate synthetase (Fhs) from both prokaryotic and eukaryotic sources. In many bacteria, Fhs catalyzes the formation of 10-formyl-tetrahydrofolate, which is used directly in purine biosynthe sis and formylation of Met-tRNA and indirectly in the biosynthesis of methionine, serine, glycine, and thymine, Analysis of the fhs mutant g rown anaerobically in a minimal medium demonstrated that the mutant ha d an absolute dependency only for adenine, although addition of methio nine was necessary for normal growth. Coincidently it was discovered t hat the mutant was sensitive to acidic pH; it grew more slowly than th e parent strain on complex medium at pH 5. Complementation of the muta nt with an integration vector harboring a copy of fhs restored its abi lity to grow in minimal medium and at acidic pH as well as to produce mutacin. This represents the first characterization of Fhs in Streptoc occus.