Development of molecular tools for the study of intestinal and probiotic lactobacilli

Molecular tools were developed for the genetic characterization of intestinal and probiotic lactobacilli with an emphasis on Lactobacillus acidophilus and closely related species. A gene, gusA, encoding a new β-glucuronidase gene from Lactobacillus gasseri ADH was cloned by complementation of an Escherichia coli gus mutant. The L. gasseri GusA protein shared 39% identity with GusA from E. coli K-12. An analysis of other lactobacilli identified β-glucuronidase activity and gusA homologs in other L. gasseri isolates, but not in other Lactobacillus species tested. Overexpression of GusA was achieved in a β-glucuronidase-negative L. gasseri strain and preliminary characterization of the GusA protein from crude cell free extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. Comparisons of L. gasseri β-glucuronidase with E. coli β-glucuronidase showed that the Lactobacillus enzyme functions better at lower pH ranges than the E. coli enzyme, a beneficial characteristic for a reporter for acidifying bacteria. In order to assess the utility of gusA as a reporter gene, transcriptional fusions to the promoterless gusA gene were used to characterize three L. acidophilus promoters in six lactic acid bacteria (LAB). In addition, inducible expression of gusA was achieved in Lactococcus lactis using the lactococcal nisA promoter.; In order to facilitate strategies for gene disruption, gene deletion, and chromosomal stabilization of expression cassettes, an efficient method was developed for the generation of site-specific chromosomal integrations in L. acidophilus and L. gasseri. The strategy is an adaptation of the lactococcal pORI system (Leenhouts, K., G. Venema, and J. Kok. 1998. Methods Cell Sci. 20: 35–50) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstrated by the insertional inactivation of the L. acidophilus NCFM lacL gene encoding β-galactosidase and the L. gasseri ADH gusA and rmlA genes; Because the tools are based on broad-host-range plasmids and Lactobacillus-derived components, it is expected that they will greatly facilitate the study of other intestinal and probiotic lactobacilli.