Next generation sequencing based transcriptomic analysis of oxidative stress response in Bifidobacterium longum BBMN68
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ABSTRACT: Bifidobacterium longum strain BBMN68 is resistant to low concentrations of oxygen. In this study, a transcriptomic study was performed to detail the cellular response of B. longum strain BBMN68 to oxidative stress. Oxygen and its intermediate metabolites, reactive oxygen species (ROS), induced abundant changes in gene expression at the mRNA level. Increased expression was found for genes involved in ROS detoxification and the redox homeostasis system, protein and DNA synthesis and repair, the FeâS cluster assembly system, and biosynthesis of branched-chain amino acids and tetrahydrofolate. Among them, two classes of ribonucleotide reductase (RNR), which are important for deoxyribonucleotide biosynthesis, were rapidly and persistently induced: first, the class Ib RNR NrdHIEF and then the class III RNR NrdDG. The increased resistance to oxygen and hydrogen peroxide conferred by NADH oxidase was confirmed by its heterogeneous overexpression in B. longum strain NCC2705. In addition, cell-membrane and cell-wall compositions were modified, probably by an increase in cyclopropane fatty acids and a decrease in polysaccharides, respectively, resulting in improved cell hydrophobicity and autoaggregation; this subsequently reduced the permeation of dissolved oxygen into the cell. Taken together, the proposed cell model of B. longum responses to oxygen stress suggests that this bacterium employs a complex molecular defense mechanism against oxygen-induced stresses. Whole mRNA profiles of B. longum BBMN68 grown in the absence or presence of 3% oxygen were generated using AB SOLiD technology and differentially expressed genes were analyzed.
Project description:Bifidobacterium longum is one of the natural inhabitants in human gastrointestinal tract. In order to colonize and exert particular functions in the gut, it has to be tolerant to the physiological concentrations of bile salts. In this work, we used RNA-Seq transcriptomics based on the Next-Generation Sequencing to investigate the global response to bile in B. longum BBMN68, a potential probiotic strain isolated from a healthy centenarian. In the presence of 0.75 g liter-1 ox-bile, the transcription of 236 genes were regulated (M-bM-^IM-% 3-fold, p < 0.001). Function analysis and Gene Ontology suggested that the bile stress response of B. longum BBMN68 covers almost all biological processes, including bile stress resistance, general stress response, central metabolic process, transmembrane transport, gene expression, cell proliferation and interaction with the host. Remarkably, 3 two-component systems and 11 transcription factors were up- or down-regulated by bile, and target genes of the regulators were identified by bacterial one-hybrid system and bioinformatics methods, resulting in a putative regulatory network that controls bile stress response in B. longum for the first time. This study significantly develops our understanding on bile stress response and brings new insight to the regulatory mechanism in bifidobateria. Whole mRNA profiles of B. longum BBMN68 growing in the absence (CK) and presence (OG) of ox-bile were generated using AB SOLiD technology and differentially expressed genes were anylyzed.
Project description:Bifidobacterium longum strain BBMN68 is resistant to low concentrations of oxygen. In this study, a transcriptomic study was performed to detail the cellular response of B. longum strain BBMN68 to oxidative stress. Oxygen and its intermediate metabolites, reactive oxygen species (ROS), induced abundant changes in gene expression at the mRNA level. Increased expression was found for genes involved in ROS detoxification and the redox homeostasis system, protein and DNA synthesis and repair, the Fe–S cluster assembly system, and biosynthesis of branched-chain amino acids and tetrahydrofolate. Among them, two classes of ribonucleotide reductase (RNR), which are important for deoxyribonucleotide biosynthesis, were rapidly and persistently induced: first, the class Ib RNR NrdHIEF and then the class III RNR NrdDG. The increased resistance to oxygen and hydrogen peroxide conferred by NADH oxidase was confirmed by its heterogeneous overexpression in B. longum strain NCC2705. In addition, cell-membrane and cell-wall compositions were modified, probably by an increase in cyclopropane fatty acids and a decrease in polysaccharides, respectively, resulting in improved cell hydrophobicity and autoaggregation; this subsequently reduced the permeation of dissolved oxygen into the cell. Taken together, the proposed cell model of B. longum responses to oxygen stress suggests that this bacterium employs a complex molecular defense mechanism against oxygen-induced stresses.
Project description:Bifidobacterium longum subsp. infantis is a bacterial commensal that colonizes the breast-fed infant gut where it utilizes indigestible components delivered in human milk. Accordingly, human milk contains several non-protein nitrogenous molecules, including urea at high abundance. This project investigates the degree to which urea is utilized as a primary nitrogen source by Bifidobacterium longum subsp. infantis and incorporation of hydrolysis products into the expressed proteome.
Project description:Whole genome microarray comparisons (comparative genomic hybridizations) were used to associate genotypic biomarkers among 15 Bifidobacterium longum strains exhibiting various Human Milk Oligosaccharide utilization phenotypes and host associations.
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:Transcriptional profiling of Bifidobacterium longum mutant versus wt strain in exponentional phase Keywords: Characterization of natural mutant One B. longum mutant (HPR2) was analysed versus the wt strain NCC2705 in: exponential phase 37°,pH 6.0, MRS, headspace flushing with CO2. Three biological replicates.
Project description:Nutrient starvation is an important survival challenge for bacteria during industrial production of functional foods. Lactobacilli are increasingly being used as probiotics in functional foods. As next-generation sequencing technology has greatly advanced, we performed integrative proteomic and genomic analysis to investigate the response of Lactobacillus casei Zhang to a glucose-restricted environment. L. casei Zhang strains were permitted to evolve in glucose-limited or normal medium from a common ancestor over a 3-year period, and they were sampled after 1000, 2000, 3000, 4000, 5000, 6000, 7000, and 8000 generations and subjected to proteomic and genomic analyses. Genomic resequencing data revealed different point mutations and other mutational events in each generation of L. casei Zhang under glucose limitation stress. The proteins expressed differentially under glucose limitation were found to be significantly related to fructose and mannose metabolism, carbohydrate metabolic processes, lyase activity, and amino acid-transporting ATPase activity. The integrative proteomic and genomic analysis revealed that the mutations protected L. casei Zhang against glucose starvation by regulating other cellular carbohydrate, fatty acid, and amino acid catabolism; phosphoenolpyruvate system pathway activation; glycogen synthesis; ATP consumption; pyruvate metabolism; and general stress response protein expression. The results help reveal the mechanisms of adapting to glucose starvation and provide new strategies for enhancing the industrial utility of L. casei Zhang.
Project description:Transcriptional profiling of Bifidobacterium longum mutants versus wt strain in exponentional phase, with or without heat-shock treatment, and in stationary phase Keywords: Characterization of natural mutants Two B. longum mutants (NCC2912 and NCC2913) were analysed versus the wt strain NCC2705 in three conditions : exponential phase 37°, exponential phase with 7 min 50° heat shock, stationary phase. Two biologic replicates and 2 technical replicates
Project description:Oxidative stress (OS) is caused by an imbalance between pro-oxidant and antioxidant reactions which leads to accumulation of reactive oxygen species (ROS) within cells. ROS can be harmful, due to their damaging effects on several cellular components, but are at the same time essential components of signaling cues. We investigate the effect of OS on the immediate early transcriptional response at a genomic level, by deep sequencing of nuclear and cytosolic RNA fractions, in in human fibroblasts treated for 30 or 120 minutes with sub-lethal doses of H2O2. In contrast to most of the protein-coding transcriptome, OS induces de novo transient transcription of thousands of previously uncharacterized genomic loci. We classify these stress induced long non coding RNAs (lncRNA) based on their genomic annotation and strand orientation relative to their nearest gene: distal, overlapping, terminal-associated or promoter-associated. The latter class of stress-induced promoter associated antisense lncRNAs (si-paancRNAs), is preferentially transcribed by PolII, which elongates from bidirectional promoters, enriched for specific transcription factor-binding sites (ZFP161, ZFX, MEF2A and divergent-FOS motives). Interestingly the associated sense-coding genes belong to specific functional categories (cellular response to stress and others). We finally demonstrate that a subset of si-paancRNAs associate better with the translation machinery, which is stalled, upon OS. Most studies to date have focused on the repertoire of lncRNAs present in physiological conditions. We here report the surprising result that thousands of lncRNAs are transcriptionally upregulated upon OS from specific loci possibly playing a role in physiological or pathological response to stress. RNA-Sequencing profiles of nuclear and cytosolic fractions of fibroblast cell lines after 0, 30 and 120 minutes of treatment with H2O2.
Project description:Lacticaseibacillus rhamnosus CM MSU 529 is the O2-tolerant facultative homofermentative lactic acid bacteria realizing respiratory metabolism when grown in a supplemented with hemin and vitamin K2 nutrient medium. This study describes the effect of aerobic and respiratory conditions on biomass and proteome of strain CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were down-regulated as detected by label-free LC-MS/MS. Up-regulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5'-phosphate oxidase), xylulose-5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npx), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), maturation and stability of ribosomal subunits (RbfA, VicX). Down-regulated proteins participate in ROS defense (AhpC), citrate and pyruvate consumption (CitE, PflB), oxaloacetate production (AvtA), arginine synthesis (ArgG), amino acid transport (GlnQ), and deoxy-nucleoside biosynthesis (RtpR). Overproduction of purine biosynthesis enzyme (PurE) distinguished cells grown under respiratory conditions from cells grown under aerobiosis. The data obtained shed light on mechanisms providing O2-tolerance and adaptation to aerobic/respiratory conditions in strain CM MSU 529.