Dataset Information

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS3 Family Insertion Sequence, ISBlo11.

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 Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 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 ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/?g 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 B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative ?-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 B. longum subsp. longumIMPORTANCE 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 Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium 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.

SUBMITTER: Sakanaka M

PROVIDER: S-EPMC6102999 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Publications

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS3 Family Insertion Sequence, ISBlo11.

Sakanaka Mikiyasu M Nakakawaji Shingo S Nakajima Shin S Fukiya Satoru S Abe Arisa A Saburi Wataru W Mori Haruhide H Yokota Atsushi A

Applied and environmental microbiology 20180817 17

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 Bifido ...[more]

PMID: 29934330

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Project description:Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited ?-l-arabinofuranosidase activity toward p-nitrophenyl-?-l-arabinofuranoside but no activity toward p-nitrophenyl-?-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of ?1,2- and ?1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the ?1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan.IMPORTANCE We identified two novel ?-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on ?1,2/3-l-arabinofuranosyl linkages, while the latter acts on the ?1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.

Dataset Information

A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS3 Family Insertion Sequence, ISBlo11.

Publications

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

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