Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases Transcriptional response to protease treatments (kallikrein, papain and chymotrypsin) of Bifidobacterium breve 210B
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:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases
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: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 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.
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: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: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.