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.

Identification of Streptococcus thermophilus Genes Specifically Expressed under Simulated Human Digestive Conditions Using R-IVET Technology

“…As mentioned before, no Spec S Kan R clone obtained throughout this work was devoid of an insert. However, we cannot formally exclude that at least certain inserts corresponded to false positives as proposed by Koguchi et al [ 27 ]. If we rule out the false positive hypothesis, then these data suggest the R-IVET technology as helpful for a better understanding of gene regulation, with potential use in genome annotation of a targeted strain such as S. thermophilus LMD-9.…”

“…Therefore, if a genomic DNA fragment inserted upstream of the recombinase gene displays promoter activity (activated R-IVET recombinant clone), the recombinase is expressed and the antibiotic resistance gene located in the chromosomal cassette is excised [ 22 , 23 ]. The R-IVET approach has already been developed in different bacteria, such as Bifidobacterium longum , Lactobacillus plantarum , Vibrio cholerae , and S. thermophilus [ 23 , 24 , 25 , 26 , 27 ]. Up to now, this technique was used to follow gene expression in complex digestive media, but mainly when using mice models [ 24 , 25 , 26 , 27 ]; alternatively, in a unique previous study by our team, it was used to assess the behavior of the S. thermophilus LMD-9 reference strain under simulated human gastric conditions [ 18 ].…”

Development of an improved colonization system for human-derived <i>Bifidobacterium longum</i> subsp. <i>longum</i> in conventional mice through the feeding of raffinose or 1-kestose

“…Bifidobacterium strains were cultured in an anaerobic chamber (80% N 2 , 10% CO 2 , and 10% H 2 ; Coy Laboratory Products, Inc., Grass Lake, MI, USA) at 37°C. Half-strength de Man, Rogosa, and Sharpe medium containing 1% (w/v) glucose, 0.34% (w/v) sodium ascorbate, and 0.02% (w/v) L-cysteine hydrochloride (1/2 MRSCS medium, pH 6.5) was used as the standard medium [ 22 ]. The glucose in the 1/2 MRSCS medium was replaced with one of 13 other carbohydrate sources (Supplementary Table 1) at 1% (w/v) to test their assimilation by the Bifidobacterium strain.…”

“…B. longum 105-A was transformed with two types of Escherichia coli-Bifidobacterium shuttle vectors harboring the Cm-resistance gene, pBFS38 [ 23 ] and pBFS63 [ 22 ], according to the protocol used in our previous study [ 9 ]. Transformants were selected and isolated using 1/2 MRSCS agar medium containing 2.5 µg/mL of Cm.…”

Molecular analysis of the replication functions of the bifidobacterial conjugative megaplasmid pMP7017

“…These reported health benefits have been the driving force behind a wave of research activities to study the biology and genetics of bifidobacteria and the way in which they interact with their host. Currently, knowledge on the health benefits of bifidobacteria is for a considerable part deduced from in silico analyses due to the recent surge of available bifidobacterial genome sequences, a 16.5‐fold increase in four years from 62 (O'Callaghan and van Sinderen, 2016) to 1021 (https://www.ncbi.nlm.nih.gov/taxonomy, accessed May 2020) and to a lesser degree based on findings from functional analyses (Cronin et al., 2007; O'Connell Motherway et al., 2009; Ruiz et al., 2013; Sakanaka et al., 2018; Koguchi et al., 2020). The paucity of functional data is partly due to the inherent resistance of Bifidobacterium to the artificial uptake of DNA due to natural barriers such as restriction‐modification systems (Bottacini et al., 2018), but most significantly due to the scarcity of molecular techniques and tools suited to these bacteria (Ventura et al., 2004; Cronin et al., 2011; O'Callaghan and van Sinderen, 2016).…”