Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

摘要: New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains Streptococcus mutans(pTV1-OK) and subsequent recovery interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility Tn917 a strain S. mutans (JH1005) by showing (i) conditional replication pTV1-OK, repA(Ts) derivative broad-host-range plasmid pWVO1 harboring Tn9l7, JH1005 at permissive temperature (30 degrees C) versus that nonpermissive (45 C); (ii) transposition frequencies similar to those reported Bacillus subtilis (10(-5) 10(-4)) with curing 90 97% erythromycin-resistant survivors following shift 42 45 C; (iii) apparent randomness insertion as determined Southern hybridization analysis ability isolate nutritional mutants, mutants acid tolerance, bacteriocin production, ranging from 0.1 0.7%. Recovery transposon-interrupted was achieved two methods: marker rescue E. vector pTV21delta2TetM, tetracycline-resistant ampicillin-sensitive Tn9l7-pBR322 hybrid, "shotgun" cloning genomic libraries into pUC19. Sequence analyses revealed insertions five different genetic loci sequences displaying homologies Clostridium spp.fhs (66% identity), dfp (43% B. ylxM-ffh (58% icd (citC [69% identity]), argD (61% identity). Insertions caused requirements; one sensitivity, while fhs both sensitivity requirements. This paper describes construction pTV1-OK demonstrates it can be efficiently employed deliver some degree chromosome easily recovered characterization. represents first published report successful Tn9l7 genus Streptococcus.