Genomic diversity of Bifidobacterium adolescentis group
Ontology highlight
ABSTRACT:
ORGANISM(S): Bifidobacterium adolescentis
SUBMITTER: Marco Ventura
PROVIDER: E-GEOD-38992 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium pseudocatenulatum. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of Bifidobacteriapseudocatenulatum DSM20438 (ABXX00000000.2). A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 M-CM-^W 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplierM-bM-^@M-^Ys instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols. We analysed ten strains belong to Bifidobacteriapseudocatenulatum and Bifidobacteriacatenulatum species. Replicates were distributed on the chip at random, non-adjacent positions.
2013-02-06 | E-GEOD-39047 | biostudies-arrayexpress
Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium pseudocatenulatum. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of Bifidobacteriapseudocatenulatum DSM20438 (ABXX00000000.2). A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 × 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplier’s instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols.
2013-02-06 | GSE39047 | GEO
Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium adolescentis. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of B. adolescentis ATCC15703 (NC_008618) . A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 × 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplier’s instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols.
2013-02-06 | GSE38992 | GEO
Project description:The aim of this study was to examine the effect of genetic disruption of the circadian clock on gene expression in the cortex across timepoints. Circadian clock protein regulate many critical aspects of cellular function, and Bmal1 knockout mice develop severe neuroinflammation, suggesting a role for circadian clock gene in brain homeostatic function. We compared brain-specific Bmal1 KO mice (Nestin-Cre;Bmal1(flox/flox) with Per1/2 double mutant mice, in order to assess the effects of deletion of the positive and negative limbs of the core clock. 11mo Cre-, NestinCre+/-;Bmal1(fx/fx), or Per1brdm,Per2brdm mice were entrained to 12h light:dark conditions with lights on at 6am for one month, then placed in constant darkness for 24 hours, after which mice were harvested at 6am (CT6) or 6pm (CT18), still in the dark. Mice were anesthetized in the dark, then perfused briefly with PBS+heparin. The brain was then quickly dissected on a cold surface, and the cerebral cortex flash frozen in liquid nitrogen. Cortex samples were mechanically dissociated with a Qiashredder device, then extracted with chloroform and diluted in 70% ethanol. RNA was extracted using Qiagen RNEasy kit according to manufacturers specifications. cDNAs were chemically labeled with Kreatech ULS RNA labeling kit (Kreatech Diagnostics) and Cy5-labeled cDNAs were hybridized to Agilent Mouse v2 4x44K microarrays (G4846A-026655).
2018-09-11 | E-MTAB-7151 | biostudies-arrayexpress
Project description:Amplified M.tb RNA derived from 15 subjects at multiple time intervals before and during chemotherapy (totalling 52 samples) was profiled alongside M.tb H37Rv RNA extracted from in vitro log phase bacilli (2 biological replicates hybridised in duplicate) as a standardised comparator. Amplified mycobacterial RNA (2 µg) were directly labelled with Cy3 fluorophore using the Universal Linkage System (ULS, Kreatech Diagnostics). Microarray hybridisations were conducted as previously described using an M.tb complex pan-genome microarray generated by the Bacterial Microarray Group at St. George’s (ArrayExpress accession number A-BUGS-41).
2016-04-12 | E-MTAB-3872 | biostudies-arrayexpress
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:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65M-BM-0C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
2014-09-01 | E-GEOD-52543 | biostudies-arrayexpress
Project description:To allow estimation of the complexity of the antennal transcriptome between sexes as well as in comparison with a non-antennal tissue, microarrays were designed based on an assembly of new 454 sequence data from the antenna and publicly available data including 454 data from the larval midgut. The microarrays were hybridized with samples generated from the repsecitve tissues from 3 (antenna) and 5 (midgut) animals per sample, with four independent samples per sex (antenna) and tissue (midgut). Sequence assembly of included Manduca antennal and gut ESTs (63) and all publicly available Genbank sequences was used with eArray (Agilent Technologies) for the design of 4 x 44K microarrays based on 60mer oligo probes. For sex-specific antennal microarray hybridizations, RNA of three individuals of one sex was pooled per preparation, and 5 larvae each were dissected for gut tissue isolation with four biological replicates per sex (antennae) and tissue (gut), respectively. Double purified total RNA was added to Agilent Technologies spike-in RNA and labeld using QuickAmp Amplification kit (Agilent Technologies) and the Kreatech ULS Fluorescent Labeling Kit with cyanine 3-CTP dye following the manufacturer´s instructions. Amplified cRNA samples were used for microarray hybridization, scanned with the Agilent Microarray Scanner and data was extracted from TIFF images with Agilent Feature Extraction software version 9.1. Raw data output files were analyzed using the GeneSpring GX11 and GeneSifter microarray analysis softwares.
2011-03-01 | E-GEOD-27470 | biostudies-arrayexpress
Project description:In this study, a whole-genome CombiMatrix Custom oligonucleotide tiling microarray with 90000 probes covering six sequenced Helicobacter pylori(H. pylori) genomes was designed and utilized for comparative genomic profiling of eight unsequenced strains isolated from patients with different gastroduodenal diseases in Heilongjiang province of China. Since significant genomic variation were found among these strains, an additional 76 H. pylori stains with different clinical outcomes isolated from various provinces of China were further tested by PCR to demonstrate this distinction. We observed several highly variable regions among strains of gastritis, gastric ulceration and gastric cancer. They are involved in genes associated with bacterial type I, type II and type III R-M system as well as in a virB gene neighboring the well studied cag pathogenic island. Previous studies have reported the diverse genetic characterization of this pathogenic island, but it is conserved in the strains tested by microarray in this study. Moreover, a number of genes involved in the type IV secretion system related to DNA horizontal transfer between H. pylori strains were identified based on the comparative analysis of the strain specific genes. These findings may provide new insights for discovering biomarkers for prediction of gastric diseases. Here we describe the design and use of a high-density oligonucleotide microarray covering six sequenced H. pylori genomes as well as several sequenced plasmids. The performance of this microarray is evaluated, and its utility is illustrated for the hybridization of genomic DNA in order to compare eight uncharacterized H. pylori strains which have not been sequenced with the six known, sequenced strains. We utilize this microarray to identify variable genomic region among H. pylori strains isolated from patients with different gastroduodenal diseases in a Chinese patient population. H. pylori isolates from 2 patients with chronic superficial gastritis, 2 patients with atrophic gastritis, 2 patients with gastric ulcer, and 2 patients with gastric cancer were studied. All eight strains were isolated from Heilongjiang province of China. A number of variable regions with high genetic diversity was identified. 26 selected genes were validated by large scale PCR in both microarray tested strains while an additional 76 strains were isolated from eight provinces.
2013-11-10 | E-GEOD-32107 | biostudies-arrayexpress
Project description:Custom oligonucleotide array CGH. We designed two fine-tiling oligonucleotide microarrays to cover the specific amplicons observed at chromosome 2p22.1 and 10q11.21 This was undertaken using the Agilent custom array design tool e-Array (Agilent, Santa Clara, CA, USA; https://earray.chem.agilent.com/), and comprised 700 probes covering 43.56Ð43.70 Mb on chromosome 10 and 5000 probes covering 39Ð40 Mb on chromosome 10 with a median probe interval of 200 bp on 2x105K microarray. Due to limited amount of material, DNA was whole genome amplified (WGA) using the GenomePlex Complete Whole Genome Amplification Kit (Sigma, Gillingham, UK) starting with 10 ng of sample and control DNA, and following the manufacturers protocol. WGA DNA was labelled using the Agilent Genomic DNA ULS labelling kit, hybridised as per manufacturers instructions, and scanned on the Agilent 2505B Microarray Scanner System.
2014-03-01 | E-MTAB-2340 | biostudies-arrayexpress