Gluconate metabolism in Lactobacillus and its role in persistence in the human intestine

The demand for foods that provide a health benefit beyond basic nutrition is on the rise. Prebiotics are non-viable food components that promote growth of a number of beneficial bacteria that naturally inhabit the human intestine. Some of these beneficial bacteria, such as Lactobacillus and Bifidobacterium species, are capable of eliciting health benefits such as reduced incidence of lactose intolerance and diarrhea, improved immune response and reduced cancer risk. The present studies address the effect of gluconate, a component of many foods and food supplements, on human intestinal microflora. Genes involved in gluconate metabolism in Lactobacillus reuteri were characterized. The effect of dietary gluconate supplementation on human intestinal populations of Lactobacillus, Bifidobacterium, Propionibacterium , and Escherchia coli was assessed over a 7-week period. Weekly stool samples were collected from 12 subjects and selected bacterial populations were enumerated. Colonies were subsequently grown on medium containing glucose or gluconate as the primary carbon source. At the level tested, dietary calcium gluconate did not significantly affect the overall quantity of Lactobacillus, Bifidobacterium, E. coli or Propionibacterium in most of the subjects tested. However, the proportion of gluconate-fermenting Lactobacillus and Propionibacterium strains increased upon gluconate consumption in some subjects, suggesting that a shift in species composition may occur. Speciation using the 16S-23S rRNA intergenic spacer region showed 2-6 different gluconate-fermenting Lactobacillus species in each subject and 11 different species overall. A 500 base pair (bp) putative gluconate kinase and a 750 bp putative gluconate permease fragments were found in the Lactobacillus reuteri 100-23 genome by amplifying DNA with degenerate primers. A gluconate-negative mutant of L. reuteri 100-23 was constructed via homologous recombination utilizing the putative gluconate permease fragment. The mutant (100-23D) and wild-type strains were characterized for gluconokinase, 6-phosphogluconate dehydrogenase and gluconate uptake activities. The mutant had significantly lower gluconokinase and gluconate uptake activities compared to wild-type. This suggests that gluconate genes in L. reuteri 100-23 are induced by gluconate and potentially arranged in an operon. Transcriptional analysis of wild-type and mutant strains showed a single transcript, indicating the gluconate permease gene is constitutively transcribed, and not inducible, in the presence of glucose or gluconate.