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

Abstract

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.

Keywords: Bifidobacterium longum subsp. longum; Cre recombinase; R-IVET; bifidobacteria; gut microbiota; in vivo gene expression; qRT-PCR.

Figure 1

Overview of the recombinase-based in vivo expression technology (R-IVET) system constructed in this study. The loxP-Sp strain harbored a loxP-Sp^R-loxP cassette that was inserted between BL105A_1451 and BL105A_1452 on the chromosome of B. longum 105-A. Random DNA fragments of B. longum 105-A were independently inserted upstream of the promoterless Cre gene in pBFK86. The resulting plasmids were introduced into the loxP-Sp strain, generating the genomic DNA library consisting of ~120,000 clones. The library was cultured in Sp-containing medium to exclude Sp^S strains in which the Cre gene was expressed by the DNA fragment with in vitro promoter activity. The library was then administered orally to mice and collected from feces. Finally, the Sp^S strains in which the Cre gene had been expressed during passage through the gastrointestinal tract were identified to determine in vivo-induced gene promoters. Sp^R, spectinomycin resistance; Sp^S, spectinomycin sensitive; Cm^R, chloramphenicol resistance.

Figure 2

Proportion of Sp^R strains when each Cre expression plasmid was independently introduced into the loxP-Sp strain. Detailed methods are described in Section 2.5 of the Materials and Methods. The proportion of Sp^R strains in the tested strains is shown as a percentage. The values in parenthesis indicate the number of Sp^R strains among the tested strains. Sp^R, spectinomycin resistance; Cm^R, chloramphenicol resistance. Green box with an arrow, terminator; yellow box, ribosome-binding site (RBS); pink box, promoter.

Figure 3

Summary of the results leading to identification of candidate in vivo-induced genes in the four trials of the R-IVET analysis. First and second trials were performed with four mice, respectively. Third and fourth trials were carried out with two mice, respectively. See Section 2.2 and Section 2.7 of the Materials and Methods for detailed procedures.

Figure 4

In vitro and in vivo relative expression levels of the genes identified by R-IVET. BL105A_1294 (encoding β-fructofuranosidase (glycoside hydrolase family 32)) was used as a positive control (PC) in qRT-PCR analysis, while the other genes were used to verify in vivo-induced expression in the cecum. BL105A_1946 (rnpA) was used as a reference gene. Data obtained from in vitro (n = 4) and in vivo (n = 6) conditions are expressed as the mean ± standard deviation together with each data plot. After testing the equality of variance by the F-test, Student’s or Welch two-tailed t-tests were used to evaluate statistical significance. p-values of the t-tests are also indicated in each panel and p < 0.05 was considered as statistically significant.