Isolation and transposition properties of ISBlo11, an active insertion sequence belonging to the IS3 family, from Bifidobacterium longum 105-A

Sakanaka, Mikiyasu; Fukiya, Satoru; Kobayashi, Rei; Abe, Arisa; Hirayama, Yosuke; Kano, Yasunobu; Yokota, Atsushi

doi:10.1093/femsle/fnv032

Cited by 6 publications

(9 citation statements)

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“…Activity of ISBlo11 transposase translated from different start sites in E. coli. Translation of the ISBlo11 transposase was predicted to start at 51ATG (19), although the gene contains other candidate start codons, including 60ATG, 105ATG, and 159GTG. Therefore, we investigated the activity of the putative full-length protein (Tpase 51 ) and that of potentially truncated forms translated from other candidate start codons (Tpase 60 , Tpase 105 , Tpase 159 , Tpase 273 , and Tpase 354 ).…”

Section: Resultsmentioning

confidence: 99%

“…E. coli DH5␣ and JM109 were used as DNA cloning hosts. E. coli DH1/pCJ105 (19) and HB101 were used as a conjugation donor and recipient, respectively. E. coli was grown aerobically at 30°C or 37°C in LB or super optimal broth with catabolite repression (SOC) medium (39).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, pHSG298-IR-Sp-IR (19), which carries an Sp r gene sandwiched between the 5= terminus (87 bp) and 3= terminus (289 bp) of ISBlo11, was amplified by inverse PCR with primers Bam_Spec_Fw and Pr-Blo0085. After digestion with BamHI, the amplified product was self-ligated and cloned in E. coli.…”

Section: Dna Manipulations Pcr Primer Pairs and Template Dnas Are LImentioning

confidence: 99%

“…Transposition efficiency in E. coli. The transposition frequency of TnBlo11 in E. coli cells expressing different forms of transposase was analyzed by F-plasmid conjugation, as previously described (19), except that LB medium without glucose was used to culture E. coli DH1/pCJ105 cells transformed with pBFS24 to pBFS29 and pBFS33 and HB101 cells. Transposition frequency was calculated as the proportion of recipient cells harboring TnBlo11-transposed pCJ105 among total recipient cells.…”

Section: Dna Manipulations Pcr Primer Pairs and Template Dnas Are LImentioning

confidence: 99%

“…We previously identified ISBlo11, an active IS3 element, in B. longum 105-A, and analyzed its activity in Escherichia coli (19). Here, we describe an ISBlo11-based transposon mutagenesis system in B. longum 105-A, consisting of a transposase expression plasmid and an artificial transposon circle.…”

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confidence: 99%

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

Sakanaka

Nakakawaji

Nakajima

et al. 2018

Appl Environ Microbiol

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Dna Manipulations Pcr Primer Pairs and Template Dnas Are LImentioning

confidence: 99%

Section: Dna Manipulations Pcr Primer Pairs and Template Dnas Are LImentioning

confidence: 99%

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confidence: 99%

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