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Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut.

ABSTRACT: Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration.

SUBMITTER: Bottacini F

PROVIDER: S-EPMC3447821 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut.

Bottacini Francesca F Milani Christian C Turroni Francesca F Sánchez Borja B Foroni Elena E Duranti Sabrina S Serafini Fausta F Viappiani Alice A Strati Francesco F Ferrarini Alberto A Delledonne Massimo M Henrissat Bernard B Coutinho Pedro P Fitzgerald Gerald F GF Margolles Abelardo A van Sinderen Douwe D Ventura Marco M

PloS one 20120920 9

Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notio ...[more]

PMID: 23028506

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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.

Dataset Information

Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut.

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Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut.

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