Tn917-lac mutagenesis of Streptococcus mutans to identify environmentally regulated genes

Previously, we demonstrated successful Tn917 mutagenesis of the oral pathog en Streptococcus mutans using PTV 1-OK (Km(r), repATs), a temperature condi tional replicative delivery vector carrying a lactococcal pWVO1Ts backbone. In this report we describe the construction and utilization of pTV32-OK, a plasmid harboring Tn917-lac (em(r), beta-gal(+)) that was employed to isol ate transcriptional fusions of the Escherichia coil lacZ reporter gene with streptococcal promoters in S. mutans strain NG8, Tn917-lac transposition o ccurred at a frequency of ca. 10(-6) with 20% of the resultant em(r) clones displaying varying levels of lacZ expression. Tn917-lac mutants that expre ssed beta-galactosidase activity under growth conditions of glucose limitat ion, acidic pH, 35 mM NaCl, and elevated (42 degrees C) temperature were is olated. Further characterization of one of the mutants with increased P-gal activity under glucose limitation, strain AS42, revealed maximal activity in batch culture in stationary phase after glucose depletion. The P-gal act ivity of AS42 also was found to be repressed 3-fold in medium containing 2% glucose relative to measured activity from cells suspended in the same med ium containing no glucose. Further phenotypic analysis revealed that AS42 h ad a 30% lower growth yield than the parent strain NG8 when grown in pH 5 m edium. Sequence analysis of the region harboring the transposon revealed th at the lacZ fusion occurred near the 3'-end of a gene encoding a homolog of an ATP binding protein from a family of Gram-positive ABC transporters. Th ese findings demonstrate that Tn917-lac mutagenesis can be used to identify environmentally regulated genes in S. mutans and possibly in other medical ly relevant streptococcal species. (C) 2000 Federation of European Microbio logical Societies. Published by Elsevier Science B.V. All rights reserved.