Functional genome analysis of Bifidobacterium breve UCC2003 reveals a major conserved host-colonization factor
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA138419 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Development of the human gut microbiota commences at birth with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date the genetic basis of bifidobacterial colonization and persistence remains poorly understood. Transcriptomic analysis of the 2,422,684 bp genome of Bifidobacterium breve UCC2003, a strain isolated from a nursling stool, during colonization of a mouse model revealed the differential expression of a type IVb or so-called Tad pilus-encoding cluster. Mutational analysis coupled with colonization experiments demonstrated that the UCC2003 tad gene cluster is essential for colonization. Immunogold transmission electron microscopy confirmed the presence of the Tad pili at the cell poles of B. breve UCC2003. Conservation of the Tad pilus-encoding locus among sequenced bifidobacterial genomes supports the notion of a ubiquitous and novel host colonization and persistence mechanism for bifidobacteria.
2011-07-01 | GSE27491 | GEO
Project description:Development of the human gut microbiota commences at birth with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date the genetic basis of bifidobacterial colonization and persistence remains poorly understood. Transcriptomic analysis of the 2,422,684 bp genome of Bifidobacterium breve UCC2003, a strain isolated from a nursling stool, during colonization of a mouse model revealed the differential expression of a type IVb or so-called Tad pilus-encoding cluster. Mutational analysis coupled with colonization experiments demonstrated that the UCC2003 tad gene cluster is essential for colonization. Immunogold transmission electron microscopy confirmed the presence of the Tad pili at the cell poles of B. breve UCC2003. Conservation of the Tad pilus-encoding locus among sequenced bifidobacterial genomes supports the notion of a ubiquitous and novel host colonization and persistence mechanism for bifidobacteria. Colonisation of BalbC mice: Seven-week-old male, BalbC mice were housed in individually vented cages (Animal care systems, Colorado) under a strict 12 h light cycle. Mice (n = 5 per group) were fed a standard polysaccharide-rich mouse chow diet and water ad libidum. Mice were inoculated by oral gavage (109 cfu of B. breve UCC2003 pPKCM7 or B. breve UCC 2003-tadA pPKCM7 in 100 ul of PBS). Fecal pellets were collected at intervals over 25 days to enumerate bacteria. Twenty five days after inoculation, mice were sacrificed and their intestinal tracts quickly dissected. Aliquots of small intestine, caecum and large intestine were harvested for determination of colony forming units (cfu) (serial dilution plating on RCA agar plates with appropriate antibiotics). Caeca were placed in RNA later for immediate RNA isolation (see below). Microarray procedures: DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, RNA isolation, RNA quality control were performed as described previously (van Hijum et al., 2005). Caeca, isolated from mice, were placed in RNAlater (Ambion) and immediately processed. Bacterial mRNA was extracted from caecal RNA preparations using the MicrobEnrich and MicrobExpress kits (Ambion) according to the manufacturer’s instructions. cDNA from bacterial mRNA was synthesized using the cDNA synthesis and labelling kit DSK-001 (Kreatech) according to the manufacturer’s instructions. 1 μl of each cDNA solution (10 ng) was amplified in triplicate using the GenomiPhi™ V2 DNA Amplification Kit (Amersham Biosciences) according to the manufacturer's protocol and labelled with Cy3 or Cy5 using Cy3-ULS and Cy5-ULS from the cDNA synthesis and labelling kit DSK-001 (Kreatech). Labelled and amplified 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 (publication number G4140-90050). Following hybridization, microarrays were washed as described in the manuals and scanned using Agilent's DNA microarray scanner G2565A. The scanning results were converted to data files with Agilent's Feature Extraction software (version 9.5). DNA microarray data was processed as previously described (Zomer et al. 2009). Differential expression tests were performed using a t test. A gene was considered differentially expressed between a test strain and control when an expression ratio of >5 or <0.2 relative to the result for the control was obtained with a corresponding P value that was <0.0001. DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, DNA isolation were performed as described previously (O'Riordan et al. 1998). Genomic DNA (gDNA) labeling for comparative genome hybridizations (CGH) was undertaken according to the BµG@S standard protocols (Hinds et al. 2002). In brief 5 µg of bifidobacterial genomic DNA was labeled with dCTP Cy3 (UCC2003) or dCTP Cy5 (test strain) using random primers (Promega) and a DNA polymerase I large Klenow fragment (Invitrogen). Labelled gDNA was hybridized using the Agilent CGH kit (part number 5188-5220) as described in the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis (v6.0) manual (publication number G4410-90010). Validation of the CGH was performed by comparing the hybridization efficiency of DNA of the B. breve type strain DSM 20213 to sequence identity of this strain with the probes on the microarray. A clear correlation was observed between sequence identity and hybridization efficiency, suggesting that an ln 2 fold change in signal intensity between target and control is sufficient to determine whether a gene is present or absent on a given genome. Therefore, a gene was considered absent when the ratio between a test strain and UCC2003 was < ln 2 with a corresponding P value that was <0.0001.
2011-07-01 | E-GEOD-27491 | biostudies-arrayexpress
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.
2016-12-23 | GSE92758 | GEO
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.
2010-08-11 | GSE17604 | GEO
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.
2010-08-11 | E-GEOD-17604 | biostudies-arrayexpress
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.
2012-09-06 | E-GEOD-34983 | biostudies-arrayexpress
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.
2014-05-29 | GSE56291 | GEO
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.
2012-09-06 | GSE34983 | GEO
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.
2014-12-17 | GSE59013 | GEO
Project description:Cellodextrin utilization by Bifidobacterium breve UCC2003
| PRJNA118557 | ENA