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:Bifidobacteria represents one of the dominant group of microorganisms colonizing the intestine of infants. However, the genetic determinants supporting the establishment and the interaction with the human hosts are still largely unknown. Most commensal bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that modulate interaction with host cell receptors or with soluble macromolecules. Whole genome transcription profiling of B. bifidum PRL2010, a strain isolated from infant stool, under in vitro as well as in vivo conditions revealed the expression of few common extracellular proteins among which type 1 pili encoding genes.
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. 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:In this study, we investigated the transcriptome of Bifidobacterium bifidum PRL2010 during in vitro growth by micro array technology. When B. bifidum PRL2010 was grown in liquid broth, 425 of the 1644 PRL2010 genes represented on the array were expressed in at least one of the three investigated growth phases, i.e., lag-, exponential and stationary phase. These transcriptional analyses identified a core in vitro transcriptome encompassing 150 genes, which resulted expressed in all phases. A proportion of the latter genes were further investigated as potential reference genes by Quantitative Real Time PCR (qRT-PCR) assays. Their expression stability was evaluated under different growth conditions, encompassing cultivation on different carbon sources, exposure to environmental stresses (thermal, acidic and osmotic) and growth phases. Our analyses validated six reference genes suitable for normalizing mRNA expression levels in qRT-PCR experiments applied to bifidobacteria.
Project description:Bifidobacteria represents one of the dominant group of microorganisms colonizing the intestine of infants. However, the genetic determinants supporting the establishment and the interaction with the human hosts are still largely unknown. Most commensal bacteria interacting with eukaryotic hosts express adhesive molecules on their surfaces that modulate interaction with host cell receptors or with soluble macromolecules. Whole genome transcription profiling of B. bifidum PRL2010, a strain isolated from infant stool, under in vitro as well as in vivo conditions revealed the expression of few common extracellular proteins among which type 1 pili encoding genes. To investigate the molecular mechanisms sustaining the interaction of PRL2010 strain with the human gut, we first explored the global genome transcription profiling of this strain in a in vitro human model such as in the presence of HT29 cell lines. The transcriptome was analyzed using a custom B. bifidum PRL2010 array representing the 90% of this organismM-bM-^@M-^Ys protein coding genes. To better evaluate the conserved responses by B. bifidum, the in vivo transcriptomes were quantified against a diverse set of transcriptome patterns identified for in vitro laboratory cultures of the strain, i.e., B. bifidum responses after growth on an cellM-bM-^@M-^Ys monolayers growth medium (DMEM); B.bifidum responses after growth on synthetic medium (MRS). Briefly, we analized five conditions, two of which are also used as references. Every experiment was performed in duplicate and in vivo condition was performed in quadruplicate.