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Bifidobacterium breve UCC2003 employs multiple transcriptional regulators in a series of glycan-induced positive feedback loops to regulate HMO metabolism

ABSTRACT: Regulation of carbohydrate metabolism in Bifidobacterium breve has been studied in detail for a variety of both plant and human-derived glycans, particularly in the model strain UCC2003. We have recently comprehensively elucidated the precise metabolic pathways by which the human milk oligosaccharide (HMO) components LNT, LNnT and LNB are utilized by B. breve UCC2003. However, no work has been carried out to date in identifying and understanding the regulatory mechanisms that control transcription of the genetic loci involved in these metabolic pathways. In the current study, we identified and characterized several transcriptional regulators involved in controlling the expression of HMO loci, and the regulatory sequences of each locus required for these regulatory mechanisms. A series of positive feedback loops inducing the expression of each locus, with the function of each regulator itself dependent on the presence of the specific monosaccharide metabolite released by the breakdown of the glycan targeted by the individual locus controlled by each respective regulator. Furthermore, we dissect the regulatory regions of each affected gene, and identify the key features within them.

ORGANISM(S): Bifidobacterium breve UCC2003

PROVIDER: GSE105108 | GEO | 2018/03/14

REPOSITORIES: GEO

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Project description:Bifidobacteria constitute a specific group of commensal bacteria which inhabit the gastrointestinal tract of humans and other mammals. Bifidobacterium breve UCC2003 has previously been shown to utilise several plant-derived carbohydrates that include cellodextrins, starch and galactan. In the current study, we investigate the ability of this strain to utilise the mucin- and human milk oligosaccharide (HMO)-derived carbohydrate, sialic acid. Using a combination of transcriptomic and functional genomic approaches, we identified a gene cluster dedicated to the uptake and metabolism of sialic acid. Furthermore, we demonstrate that B. breve UCC2003 can cross feed on sialic acid derived from the metabolism of 3’ sialyllactose, a HMO, by Bifidobacterium bifidum PRL2010. 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.

Project description:Bbr_0838 from Bifidobacterium breve UCC2003 encodes a 683 residue membrane protein, that contains a permease domain displaying similarity to transporters belonging to the major facilitator superfamily, as well as a CBS (cystathionine beta synthase) domain. The high level of similarity to bile-efflux pumps from other bifidobacteria, suggests a significant role for Bbr_0838 in bile tolerance of B. breve UCC2003. Bbr_0838 transcription was shown to be monocistronic and strongly induced upon exposure to bile. Further analysis delineated the transcriptional start site and the minimal region required for promoter activity and bile regulation. Insertional inactivation of Bbr_0838 in B. breve UCC2003 resulted in a strain that exhibited reduced survival upon cholate exposure as compared to the parent strain, a phenotype that was reversed when a functional Bbr_0838 gene was introduced into UCC2003::838800. Transcriptome analysis of UCC2003::838800 grown in the presence or absence of bile demonstrated that transcription of Bbr_0832, which is predicted to encode a macrolide-efflux transporter gene, was significantly increased in the presence of bile, representing a likely compensatory mechanism for bile removal in the absence of Bbr_0838. This study represents the first in depth analysis of a bile-inducible locus in bifidobacteria, identifying a key gene relevant for bifidobacterial bile tolerance. In order to investigate differences in global gene expression upon growth or exposure of B. breve UCC2003-delta0838 to cholic acid compared to normal growing cells, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003-delta0838 cultures under normal conditions and cultures grown on or exposed to cholic acid. All experiments were performed in duplo and targets where confirmed with QRT-PCR. In addition transcriptome analyse was performed of B. breve UCC2003 compared to that of B. breve UCC2003-delta0838 both exposed to 0.1 % cholic acid. This was performed as a single experiment and targets were confirmed by QRT-PCR

Project description:This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK, which is believed to control the expression of the ser2003 locus in Bifidobacterium breve UCC2003. The ser2003 locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser2003 and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis, coupled to transcriptional fusion and EMSA assays. The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating B. breve-s-serR and B. breve-::serU insertion mutants, which exhibited altered ser2003 transcriptional induction patterns as compared to their parent strain UCC2003. 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 breve UCC2003 employs multiple transcriptional regulators in a series of glycan-induced positive feedback loops to regulate HMO metabolism

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