Application of Recombinase-Based In Vivo Expression Technology to Bifidobacterium longum subsp. longum for Identification of Genes Induced in the Gastrointestinal Tract of Mice

Bifidobacteria are one of the major components in human gut microbiota and well-known as beneficial microbes. However, clarification of commensal mechanisms of bifidobacteria in the intestines is still ongoing, especially in the presence of the gut microbiota. Here, we applied recombinase-based in vivo expression technology (R-IVET) using the bacteriophage P1 Cre/loxP system to Bifidobacterium longum subsp. longum 105-A (B. longum 105-A) to identify genes that are specifically expressed in the gastrointestinal tract of conventionally raised mice. Oral administration of the genomic DNA library of B. longum 105-A to conventionally raised mice resulted in the identification of 73 in vivo-induced genes. Four out of seven tested genes were verified in vivo-specific induction at least in the cecum by quantitative reverse transcription PCR. Although there is still room for improvement of the system, our findings can contribute to expanding our understanding of the commensal behavior of B. longum in the gut ecosystem.

Genetic analysis of Cheirostylis species based on microsatellite markers

Microsatellite markers specific to Cheirostylis yunnanensis Rolfe were developed using an enriched genomic DNA library technique. The library was constructed using (AG)20 and (CAG)20 oligonucleotide repeats. A total of 48 primer pairs were designed and tested with 48 C. yunnanensis Rolfe samples, resulting in 11 polymorphic loci. The number of alleles per locus ranged from 2 to 12, with an average of six alleles. The observed and expected heterozygosity ranged from 0.0426 to 0.8085 and 0.0421 to 0.9078, respectively. Of the 11 polymorphic loci, three showed a significant deviation from Hardy–Weinberg equilibrium and one exhibited linkage disequilibrium. Cross-species amplification was tested with five samples of Cheirostylis of unknown species resulting in eight loci that could be amplified, with the number of alleles ranging from one to two. The microsatellite markers developed in this study will be useful for the genetic analysis of C. yunnanensis in order to differentiate species as well as to establish a conservation plan for this species.