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Bifidobacterium kashiwanohense expresses fucosidases necessary for fucosyllactose utilisation as an adaptation to the infant gut environment

ABSTRACT: Bifidobacteria dominate the composition of the neonatal gut microbiota in the first number of weeks following birth. A number of species in particular are found with a significantly higher frequency in the microbiome of breastfed infants, owing to their ability to rely on Human Milk Oligosacchraides (HMOs) as their sole carbohydrate substrate; namely B. bifidum, B. longum spp. infantis and B. breve. Bifidobacterium kashiwanohense is a species that has been isolated previously only from the faeces of infants, but extremely infrequently at that. Relatively little is currently known about the species itself, let alone the metabolic pathways that allow it to successfully establish a population in the infant gut. We have isolated a novel strain of B. kashiwanohense from the faeces of a breastfed infant on the basis of its ability to utilise the HMO component fucosyllactose as its sole carbohydrate source. In this study, we read and annotate the full genome sequence of this novel strain, and use the data obtained to direct our further experimental analysis of fucosyllactose metabolism in B. kashiwanohense. Using transcriptomic and growth analysis results, we identify the genes responsible for B. kashiwanohense to utilise fucosyllactose, and employ a combination of cloning, in vitro hydrolysis assays, and further, recombinant transcriptomic and growth assays to elucidate the pathway for fucosyllactose metabolism in B. kashiwanohense, as well as revealing insight into fucosyllactose and fucose metabolism in Bifidobacteria as whole.

ORGANISM(S): Bifidobacterium breve UCC2003 Bifidobacterium catenulatum subsp. kashiwanohense

PROVIDER: GSE107439 | GEO | 2019/11/12

REPOSITORIES: GEO

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Project description:Members of the genus Bifidobacterium are Gram-positive bacteria which are commonly found in the gastrointestinal tract (GIT) of mammals, including humans. Growth of bifidobacteria has been shown to be selectively stimulated by various carbohydrates found in the human diet. To extend our understanding of the regulation of bifidobacterial carbohydrate utilization systems, we investigated the regulation of two carbohydrate utilisation clusters dedicated to the metabolism of raffinose type sugars and melezitose. Transcriptomic and functional genomic approaches clearly identified that the raffinose utilisation system is positively regulated by the activator RafR, while the melezitose utilisation system is negatively regulated by lacI type transcriptional regulators. A B. breve UCC2003-rafR insertion mutant was incapable of utilising raffinose containing sugars or melibiose as a sole carbohydrate source, while the UCC2003-lacI1 and UCC2003-lacI2 insertion mutants retained their ability to utilise melezitose as a sole carbohydrate source. In silico analysis and DNA/protein interaction studies revealed a novel conserved 22 bp palindromic sequence as the RafR binding operator sequence in the rafB promoter region. Within the melezitose utilisation cluster a 20bp palindromic sequence for the melA promoter region and a 24bp palindromic sequence for the Bbr_1863 promoter region DNA-microarrays containing oligonucleotide primers representing each of the 1864 annotated genes on the genome of B. breve UCC2003 (O'Connell Motherway et al., 2011) were designed by and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labeling were performed as described previously (Pokusaeva et al., 2009). Labeled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent's Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described (Garcia De La Nava et al., 2003). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test (Long et al., 2001). A gene was considered differentially expressed when p < 0.001 and an expression ratio of >3 or <0.33 relative to the control.

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Bifidobacterium kashiwanohense expresses fucosidases necessary for fucosyllactose utilisation as an adaptation to the infant gut environment

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