A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS 3 Family Insertion Sequence, IS Blo11

Abstract

Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for subsp. 105-A (JCM 31944) based on IS, a native IS family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the IS transposase. An artificial transposon plasmid, pBFS12, in which IS terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >10 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of subsp. Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for subsp. , a representative health-promoting species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.

Revisiting the Metabolic Capabilities of Bifidobacterium longum susbp. longum and Bifidobacterium longum subsp. infantis from a Glycoside Hydrolase Perspective

“…Subsequently, a tree was constructed taking into account the sequences of the 16S rRNA gene, using only the sequences available in the SILVA database of those strains for which a subspecies has been assigned ( Figure 1B). Furthermore, a clustering analysis considering full or partial sequences of recA, tuf, and ldh genes, previously used for subspecies identification in B. longum [27][28][29][30], was also carried out with the strains included in Figure 1A ( Supplementary File 3). Neither of the trees have included strains for which the subspecies is not specified.…”

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

“…longum 105-A harboring pBFS63 was cultured anaerobically in 1/2MRSCS-Cm broth until the mid-log phase (OD 660 = 0.5-0.7). The cells were pretreated with RNAprotect bacteria reagent (Qiagen) and total RNA was extracted as described in our previous study using enzymatic cell wall digestion and mechanical cell disruption with zirconia beads [38]. Total RNA was also extracted from pretreated mouse cecal contents (see Section 2.8.1) using the same protocol.…”

“…Total RNA was also extracted from pretreated mouse cecal contents (see Section 2.8.1) using the same protocol. cDNA synthesis and qRT-PCR analysis were conducted as described previously [38]. A relative standard curve method was used to calculate the relative expression of the target gene against the reference gene.…”

“…Among them, BL105A_1294 was not identified in the R-IVET analysis, but this gene was used as an expected positive control gene for in vivo-specific expression because the β-fructofuranosidase encoded by this gene is necessary to degrade 1-kestose in the mouse diet [39]. BL105A_1946 (rnpA encoding the RNase P protein component) was used as a reference gene [17,38]. Gene-specific primers for qRT-PCR analysis are shown in Table 2 nos.…”

Methionine utilization by bifidobacteria: possible existence of a reverse transsulfuration pathway

“…Bifidobacterial strains, including Bifidobacterium longum subsp. longum 105-A ( B. longum 105-A, JCM 31944) [ 9 , 10 ], were routinely cultured in 1/2 MRSCS [ 11 ]. Sulfur-containing amino acid assimilation was examined in bifidobacterial minimal medium (BMM; ingredients provided in Supplementary Table 1 ) [ 5 ] supplemented with 2 mM of cysteine (BMM+Cys) or methionine (BMM+Met), (242 mg/L for cysteine and 298 mg/L for methionine).…”