Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter

Sakanaka, Mikiyasu; Tamai, Saki; Hirayama, Yosuke; Onodera, Ai; Koguchi, Hiroka; Kano, Yasunobu; Yokota, Atsushi; Fukiya, Satoru

doi:10.1016/j.jbiosc.2014.05.002

Cited by 17 publications

(30 citation statements)

References 37 publications

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“…The amplified fragment was also sequenced to ensure the correct recombination event. Complementation plasmid pMSK128 was constructed by ligating PCR-amplified xfp (xylulose 5-phosphate/fructose 6-phosphate phosphoketolase) promoter region (P xfp ) and the type 4 ldh coding region with PstI- and SalI-digested pBFS38 77 using the In-Fusion cloning kit, by which type 4 ldh was placed under the control of P xfp . Primer pairs of Pr-598/Pr-599 and Pr-600/Pr-601 were used for amplifying P xfp from pBFS48 77 and the type 4 ldh gene from the B. longum subsp.…”

Section: Methodsmentioning

confidence: 99%

Breastmilk-promoted bifidobacteria produce aromatic amino acids in the infant gut

Laursen

Sakanaka

Burg

et al. 2020

Preprint

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26Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune 27 development. However, few breastmilk-dependent microbial metabolites mediating host-microbiota 28 interactions are currently known. We here demonstrate that breastmilk-promoted Bifidobacterium 29 species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective 30 aromatic lactic acids (indolelactate, phenyllactate and 4-hydroxyphenyllactate) via a previously 31 unrecognised aromatic lactate dehydrogenase. By longitudinal profiling of the gut microbiota 32 composition and metabolome of stool samples of infants obtained from birth until 6 months of age, 33 we show that stool concentrations of aromatic lactic acids is determined by the abundance of human 34 milk oligosaccharide degrading Bifidobacterium species containing the aromatic lactate 35 dehydrogenase. Finally, we demonstrate that stool concentrations of Bifidobacterium-derived 36 indolelactate are associated with the capacity of infant stool samples to activate the aryl 37 hydrocarbon receptor, a receptor important for maintenance of intestinal homeostasis and immune 38 system development. These findings open up new directions towards understanding the role of 39 breastmilk-promoted Bifidobacterium in mediating host-microbiota interactions in early life. 40 INTRODUCTION 41Human breastmilk is a perfectly adapted nutritional supply for the infant 1 . Breastfeeding provides 42 children with important short-term protection against infections, and may also provide long-term 43 metabolic benefits 1,2 . These benefits may partly be mediated through the gut microbiota, since 44 breastfeeding is the strongest determinant of gut microbiota composition and function during 45 infancy 3-5 . Human breastmilk contains human milk oligosaccharides (HMOs), which are complex, 46 highly abundant sugars serving as substrates for specific microbes including certain species of 47 Bifidobacterium 6 . This co-evolution between bifidobacteria and the host, mediated by HMOs, to a 48 large extent directs the colonization of the gut in early life, which has critical impact on the immune 49 system 7 . Depletion of specific microbes, including Bifidobacterium, in early life has been associated 50 with increased risk of allergy and asthma development in childhood 8,9 , and is suggested to 51 compromise immune function and lead to increased susceptibility to infectious disease 10,11 . Despite 52 Bifidobacterium dominating the gut of breastfed infants and being widely acknowledged as 53 beneficial, mechanistic insights on the contribution of these bacteria and their metabolites to 54 immune development during infancy remain limited. Recent studies show that microbial aromatic 55 amino acid metabolites including tryptophan-derived indoles 12 , via activation of the aryl 56 hydrocarbon receptor (AhR), can fortify the intestinal barrier 13,14 , protect against pathogenic 57 infections 15,16 and influence host metabolism 13,17,18 , which makes this...

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Section: Methodsmentioning

confidence: 99%

Breastmilk-promoted bifidobacteria produce aromatic amino acids in the infant gut

Laursen

Sakanaka

Burg

et al. 2020

Preprint

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Section: Discussionmentioning

confidence: 93%

“…We also investigated whether the promoter driving transposase expression affects transposition efficiency. In the presence of glucose to repress xylose-inducible p fruEKFG_Blo activity and Tpase 51 expression in 105-A/pBFS100 (20), pBFS12 transposition decreased 10-fold to 125 Ϯ 63 CFU/g DNA (Table 2). It was supported by the quantitative real-time PCR (qRT-PCR) analysis revealing that Tpase 51 expression in 105-A/pBFS100 was significantly lower in the presence of glucose than in the presence of xylose (Table S4).…”

Section: Resultsmentioning

confidence: 99%

“…In our system, the expression of transposase is driven by the bifidobacterial pro- moters p fruEKFG_Blo and p xfp_Bbr that were previously characterized by measuring the promoter activities using a reporter assay system (20). The activities of p fruEKFG_Blo on 4% (wt/vol) xylose and of p xfp_Bbr on 1% (wt/vol) glucose were at a similar level and were over 25-fold higher than that of p fruEKFG_Blo on 1% (wt/vol) glucose (20). Accordingly, strong promoter activity in 105-A/pBFS100 and 105-A/pBFS101 grown on 4% (wt/vol) xylose and 1% (wt/vol) glucose, respectively, resulted in over 10-fold higher transposition efficiency than weak promoter activity in 105-A/pBFS100 grown on 1% (wt/vol) glucose ( Table 2), suggesting a positive correlation between promoter strength and transposition efficiency.…”

Section: Discussionmentioning

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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“…Klijn et al (2006) constructed a reporter plasmid based on a B. longum cryptic plasmid and the Escherichia coli gusA gene. Wang et al (2012) analysed the expression level in B. bifidum promoters using b-glucosidase activity and recently Sakanaka et al (2014) analyzed Bifidobacterium promoters using as the reporter the a-galactosidase gene. The use of protein fluorescence as reporter has a several advantages, the GFP does not require any exogenous substrate, complex medium, or expensive equipment.…”

Section: Discussionmentioning

confidence: 99%

Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein

et al. 2015

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Functional analysis of bifidobacterial promoters in Bifidobacterium longum and Escherichia coli using the α-galactosidase gene as a reporter

Cited by 17 publications

References 37 publications

Breastmilk-promoted bifidobacteria produce aromatic amino acids in the infant gut

Breastmilk-promoted bifidobacteria produce aromatic amino acids in the infant gut

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

Analysis of gene expression of bifidobacteria using as the reporter an anaerobic fluorescent protein

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