Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases Transcriptional response to protease treatments (kallikrein, papain and chymotrypsin) of Bifidobacterium breve 210B
Project description:Members of the serpin (serine protease inhibitor) superfamily have been identified in higher, multicellular eukaryotes, as well as in bacteria, although surveillance of available genome sequences indicates that bacterial serpin-encoding (ser) homologs are not widely distributed. In members of the genus Bifidobacterium this gene appears to be present in at least five, and perhaps up to nine, out of 30 species tested. Moreover, phylogenetic analysis using available bacterial and eukaryotic serpin sequences revealed that bifidobacteria specify serpins that form a separate clade. We characterized the ser210B locus of Bifidobacterium breve 210B, which consists of a number of genes, whose deduced protein products display significant similarity to proteins encoded by corresponding loci found in several other bifidobacteria. Northern hybridization, primer extension, micro array analysis, RT-PCR and Quantitative Real Time (qRT) - PCR analysis revealed that a 3.5 kb polycistronic mRNA, encompassing the ser210B operon with a single transcriptional start site, is strongly induced following treatment of B. breve 210B cultures with particular proteases. In contrast, transcription of the ser homolog of other bifidobacteria, such as Bifidobacterium longum subsp. infantis, Bifidobacterium dentium and B. longum subsp. longum, appears to be triggered by a different set of proteases
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.
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.
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 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 seven strains belong to B. adolescentis species. Replicates were distributed on the chip at random, non-adjacent positions.
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.