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

Abstract

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli(pTV21⌬2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn917, in JH1005 at the permissive temperature (30؇C) versus that at the nonpermissive temperature (45؇C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10 ؊5 to 10 ؊4) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45؇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-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21⌬2TetM, a tetracycline-resistant and ampicillin-sensitive Tn917-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp. fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn917 mutagenesis in the genus Streptococcus.

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

“…In order to establish that the insertion of Tn917 was responsible for the observed acid-sensitive phenotype, a genetic backcross was performed. For this purpose, chromosomal DNA isolated from AS17 was used to transform S. mutans strain NG8 as described previously (Gutierrez et al, 1996). Em' transformants were isolated and assessed for their sensitivities to acid when compared to the parent strains NG8 and JHlOO5 and mutant AS17.…”

“…This mutant was also sensitive to high osmolarity and high temperature. We have been involved in similar research but have used transposon Tn917 to isolate acid-sensitive mutants of S. mutans (Gutierrez et al, 1996). This small and relatively stable mutagenic element was delivered in pTV1-OK, a broad-host-range, conditional temperature-sensitive (T,) replication vector for transformation-independent mutagenesis.…”

“…One such mutant (AS17) was recovered with the transposon insertion in the intergenic region between the S. mutans y 1 x M -B homologues. We have named this locus sat (for secretion and acid tolerance; Gutierrez et al, 1996). In Bacillus subtilis (Honda et al, 1993) this operon encodes YlxM, a putative 13.2 kDa protein of unknown function which displays no significant homology to other known proteins in databases.…”

“…Plasmid pJG301 (Gutierrez et al, 1996) served as the template. The amplified product was gel-purified and ligated to SrnaIdigested S. mutans-E. coli shuttle vector pDL278 (Dunny et al, 1991) to generate plasmid pPC300, which was used to transform E. coli strain WAM113 (ffh) and S. mutans strains AS17 and AS17N.…”

“…This small and relatively stable mutagenic element was delivered in pTV1-OK, a broad-host-range, conditional temperature-sensitive (T,) replication vector for transformation-independent mutagenesis. Our work resulted in the isolation of a variety of acid-sensitive mutants, suggesting that acidurance in S. mutans is a multifactorial phenomenon (Gutierrez et al, 1996 ;Crowley et al, 1997). One such mutant (AS17) was recovered with the transposon insertion in the intergenic region between the S. mutans y 1 x M -B homologues.…”