INSERTIONAL MUTAGENESIS AND RECOVERY OF INTERRUPTED GENES OF STREPTOCOCCUS-MUTANS BY USING TRANSPOSON TN917 - PRELIMINARY CHARACTERIZATION OF MUTANTS DISPLAYING ACID SENSITIVITY AND NUTRITIONAL-REQUIREMENTS

New vectors were constructed for efficient transposon Tn917-mediated m utagenesis of poorly transformable strains of Streptococcus mutans(pTV 1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21 Delta 2TetM). In this report, we demonstrate the utility of Tn91 7 mutagenesis of a poorly transformable strain of S. mutans (JH1005) b y showing (i) the conditional replication of pTV1-OK, a repA(Ts) deriv ative of the broad-host-range plasmid pWVO1 harboring Tn917, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpe rmissive temperature (45 degrees C); (ii) transposition frequencies si milar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant su rvivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupt ed genes was achieved by two methods: (i) marker rescue in E. coli wit h the recovery vector pTV21 Delta 2TetM, a tetracycline-resistant and ampicillin-sensitive Tn917-pBR322 hybrid, and (iii) ''shotgun'' clonin g of genetic loci in sequences displaying homologies to Clostridium sp p. fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylx M-ffh (58% identity), icd (citC [69% identity]), and argD (61% identit y). Insertions in icd and argD caused nutritional requirements; the on e in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caus ed both acid sensitivity and nutritional requirements. This paper desc ribes the construction of pTV1-OK and demonstrates that it can be effi ciently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome c an be easily recovered for subsequent characterization. This represent s the first published report of successful Tn917 mutagenesis in the ge nus Streptococcus.