Cross-feeding by Bifidobacterium breve UCC2003 during Bifidobacterium bifidum PRL2010 growth on a mucin-based medium.
Ontology highlight
ABSTRACT:
ORGANISM(S): Bifidobacterium breve UCC2003
SUBMITTER: Mary O'Connell Motherway
PROVIDER: E-GEOD-59013 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Cross-feeding by Bifidobacterium breve UCC2003 during co-cultivation with Bifidobacterium bifidum PRL2010 in a mucin-based medium.
Egan Muireann M Motherway Mary O'Connell MO Kilcoyne Michelle M Kane Marian M Joshi Lokesh L Ventura Marco M van Sinderen Douwe D
BMC microbiology 20141125
<h4>Background</h4>Bifidobacteria constitute a specific group of commensal bacteria that commonly inhabit the mammalian gastrointestinal tract. Bifidobacterium breve UCC2003 was previously shown to utilize a variety of plant/diet/host-derived carbohydrates, including cellodextrin, starch and galactan, as well as the mucin and HMO-derived monosaccharide, sialic acid. In the current study, we investigated the ability of this strain to utilize parts of a host-derived source of carbohydrate, namely ...[more]
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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. 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