Project description:The transcription of the cldEFGC gene cluster of Bifidobacterium breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating these genes in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this bacterium. HPAEC-PAD analysis of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose and cellopentaose, with cellotriose representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 was shown to be the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity towards various cellodextrins.
Project description:The transcription of the cldEFGC gene cluster of Bifidobacterium breve UCC2003 was shown to be induced upon growth on cellodextrins, implicating these genes in the metabolism of these sugars. Phenotypic analysis of a B. breve UCC2003::cldE insertion mutant confirmed that the cld gene cluster is exclusively required for cellodextrin utilization by this bacterium. HPAEC-PAD analysis of medium samples obtained during growth of B. breve UCC2003 on a mixture of cellodextrins revealed its ability to utilize cellobiose, cellotriose, cellotetraose and cellopentaose, with cellotriose representing the preferred substrate. The cldC gene of the cld operon of B. breve UCC2003 was shown to be the first described bifidobacterial β-glucosidase exhibiting hydrolytic activity towards various cellodextrins. In order to investigate differences in gene expression patterns of B. breve UCC2003 when grown on cellobiose or cellodextrins as compared to growth on glucose, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003 cultures grown on cellobiose, cellodextrins, or glucose (see Materials and Methods). The cultures were harvested at the time points that ensured that B. breve UCC2003 was metabolizing cellobiose or cellodextrins as opposed to the residual glucose present in the cellodextrin preparation. Analysis of the DNA microarray data was obtained from two independent biological replicates.
Project description:Phenotypic screening of a random mutant library combined with microarray analysis of the transcriptional response of B. breve UCC2003 to iron limitation, allowed the identification of a number of genes implicated in the survival of Bifidbacterium breve UCC2003 under iron-limiting conditions. Of the identified genes, two putative iron-uptake systems, were further characterised: (i) a presumed ferrous iron uptake system, designated here as bfeUO, and (ii) a predicted ferric iron/siderophore uptake system, designated sifABCDE. In silico analysis also illustrated that these two clusters are highly conserved across members of the genus Bifidobacterium and are invariably co-located. Murine colonization studies demonstrated that B. breve UCC2003-bfeU and B. breve UCC2003-sifA insertion mutants are able to colonize a healthy murine gut as efficiently as the wild type B. breve strain, indicating that these genes are not crucial for gut survival or colonization in a healthy host.
Project description:A conserved two-component signal transduction system controls the response to phosphate starvation in Bifidobacterium breve UCC2003
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.
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.
Project description:This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by phoRP in Bifidobacterium breve UCC2003, which controls the response to phosphate (Pi) starvation. The response regulator PhoP was shown to bind to the promoter region of pstSCAB, specifying a predicted Pi transporter system, as well as that of phoU, which specifies a putative Pi-responsive regulatory protein. This interaction is assumed to cause transcriptional activation under conditions of Pi limitation. The phoRP genes appear to be subject to positive auto-regulation, and with pstSCAB and phoU, represent the complete regulon controlled by the phoRP-encoded 2CRS in B. breve UCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together with phoRP is conserved among many high GC-content Gram+ bacteria. The importance of the phoRP 2CRS in the response of B. breve to Pi starvation conditions was confirmed by generating a B. breve::phoP insertion mutant which exhibited decreased growth under phosphate-limiting conditions as compared to its parent strain UCC2003. In order to investigate differences in global gene expression upon growth of B. breve UCC2003 under low phosphate conditions compared to normal growing cells, DNA microarray experiments were performed in CDM. Total RNA was isolated from B. breve UCC2003 cultures under normal conditions and cultures grown under low phosphate conditions. Starvation experiments with wildtype and overexpression strains were performed in triplicate, while knockout array experiments where performed as single experiments and targets were confirmed with QRT-PCR.
Project description:This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by phoRP in Bifidobacterium breve UCC2003, which controls the response to phosphate (Pi) starvation. The response regulator PhoP was shown to bind to the promoter region of pstSCAB, specifying a predicted Pi transporter system, as well as that of phoU, which specifies a putative Pi-responsive regulatory protein. This interaction is assumed to cause transcriptional activation under conditions of Pi limitation. The phoRP genes appear to be subject to positive auto-regulation, and with pstSCAB and phoU, represent the complete regulon controlled by the phoRP-encoded 2CRS in B. breve UCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together with phoRP is conserved among many high GC-content Gram+ bacteria. The importance of the phoRP 2CRS in the response of B. breve to Pi starvation conditions was confirmed by generating a B. breve::phoP insertion mutant which exhibited decreased growth under phosphate-limiting conditions as compared to its parent strain UCC2003.
Project description:Bifidobacteria constitute commensal bacteria that commonly inhabit the mammalian gastro intestinal tract. The gut commensal Bifidobacterium breve UCC2003 was previously shown to utilise a variety of plant/diet-derived carbohydrates, including cellodextrin, starch and galactan. In the current study, we investigated the ability of this strain to utilize (parts of) a host-derived source of carbohydrate, namely the mucin glycoprotein. Here, we demonstrate that B. breve UCC2003 exhibits growth properties in a mucin-based medium, but only when in the presence of Bifidobacterium bifidum PRL2010, which is known to metabolize mucin. Based on HPAEC analysis, transcriptome data and insertion mutagenesis, it appears that B. breve UCC2003 sustains this improved survival in co-culture by cross-feeding on a combination of fucose, sialic acid and galactose-containing oligosaccharides.
Project description:A bacterial nursling stool isolate, Bifidobacterium breve UCC2003, encodes two putative sulfatases. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support growth of B. breve UCC2003, while three other tested sulfated monosaccharides, N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate and N-acetylgalactosamine-6-sulfate, did not. Using a combination of transcriptomic and functional genomic approaches, a gene cluster, designated ats2, was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is shown to be regulated by a ROK-family transcriptional repressor. Expression profiling by array