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MalR regulon of B. breve UCC2003; malto-oligosachrides and beyond

ABSTRACT: Bifidobacteria resident in the gastrointestinal tract are subject to many stresses such as bile stress, osmotic stress and starvation. Adaption to these stresses requires a high amount of energy and rapid changes in gene transcription. Four Bifidobacterium breve UCC2003-encoded Lac I type transcriptional regulators had been proposed to be involved in the utilisation of maltose, maltodextrins and related polymers such as starch, amylopectin, amylose, glycogen and pullulan. However, it now appears that these regulators are also involved in the utilisation of other carbon sources such as ribose and cellobiose. Interestingly, in vitro these regulators often cross regulate the same carbohydrate utilization genes, along with regulating each other. This hierarchical regulatory system controls the transcription of genes involved in carbohydrate uptake, storage, breakdown and central metabolism. These four regulators respond to differing effectors in vitro, these effectors include sugars such as turanose or galactose, thus indicating that each regulator is responsible for a different aspect of carbon metabolism. This complex network of gene regulation provide novel insights into the decision making process of the cell and the metabolic adaption of bifidobacteria to its environment.

ORGANISM(S): Bifidobacterium breve UCC2003

PROVIDER: GSE108950 | GEO | 2018/03/09

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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MalR regulon of B. breve UCC2003; malto-oligosachrides and beyond

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