OBJECTIVETo construct an acid-sensitive mutant of Streptococcus mutans (S. mutans) by transposon mutagenesis and to find a new gene related to the acid tolerance of S. mutans.

METHODSThe transposon Tn917 was delivered into S. mutans UA159 by the temperature-sensitive plasmid pTV1-OK bearing Tn917 and transposition of Tn917 was induced after incubation at non-permissive temperature (42 degrees C). Transposants harboring Tn917 in the chromosome were screened for the selection of mutant that had diminished growth at low pH. Southern analysis was performed with EcoRI (no cut within Tn917) digests of S. mutans UA159 and the selected aid-sensitive mutant, with DIG-labeled probe of 4.3 kb KpnI fragment of pTV1-OK containing Tn917. Genetic backcross experiment was performed by transforming the genome of the mutant to another S. mutans strain MT8148 to determine the linkage of Tn917 insertion to the change of phenotype (acid-sensitivity). Comparison of the abilities to grow at low pH, the glycolytic pH drop and killing pH values were done between the acid-sensitive mutant and the parent strain. The asymmetric PCR method was used to obtain the fragment flanking Tn917 and the PCR products were cloned to pMD18-T vector for sequencing.

RESULTSOne mutant that showed no growth at pH 5.0 was isolated from 2 316 transposants and was named as b23. Southern analysis and genetic backcross experiment confirmed the linkage between single Tn917 insertion into the chromosome and the phenotypic change (acid sensitive). b23 was less acid tolerant than UA159 in that it showed poorer growth at low pH and higher glycolytic pH minimum and higher killing pH. BLAST results indicated that Tn917 inserted into the genome of S. mutans UA159 at the site of 996 123 bp.

CONCLUSIONAn acid-sensitive mutant of S. mutans was successfully constructed and a new gene that is responsible for the acid tolerance in S. mutans UA159 was revealed.