Transcriptome analysis of probiotic Lactobacillus casei Zhang during fermentation in soymilk
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ABSTRACT: Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China. Gene expression dynamics of L. casei Zhang during growth in soymilk was investigated in attempt to reveal the mechanisms involved in growth stimulation for growing probiotics in. Comparison of different transcripts next to each other revealed 162 and 63 significantly induced genes in late logarithmic phase and stationary phase, of which the expression was at least 3 fold up-regulated and down-regulated, respectively. Approximately, 38.4% of the up-regulated genes were associated with amino acid transport and metabolism notably for histidine and lysine biosynthesis, followed by genes/gene clusters involved in carbohydrate transport and metabolism, lipid transport and metabolism, and inorganic ion transport and metabolism. The analysis results suggest that the stimulatory effect of soymilk-based ecosystem on the L. casei Zhang growth is more complex than amino acids or oligopeptides supply. To study gene expression dynamics of L. casei Zhang during growth in soymilk, a whole genome microarray was used to screen for differentially expressed genes when grown to lag phase, late logarithmic phase, and stationary phase.
Project description:Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China that has been commercially used as a starter in the manufacture of dairy products. To study the gene expression profiles of L. casei Zhang during growth in milk, a whole-genome microarray was used. Compared to L. casei Zhang grown to late logarithmic phase in milk, 61 genes were significantly up-regulated (>5 fold) in stationary phase, whereas 26 genes were down-regulated. Collectively, these data showed that the majority of the identified genes was involved in carbohydrate metabolism and energy production, followed by genes involved in nucleotide metabolism, inorganic ion transport, amino acid transport and metabolism, chaperone, etc. This study demonstrates the fundamental effects of cultural conditions on the transcriptome of L. casei Zhang. Moreover, it improves the understanding of the growth and survival mechanism of the bacterium during the late stage of milk fermentation. L. casei Zhang was grown in milk for 14h (late logarithmic phase) or 18h (stationary phase). 2 biological replicates each.
Project description:Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China that has been commercially used as a starter in the manufacture of dairy products. To study the gene expression profiles of L. casei Zhang during growth in milk, a whole-genome microarray was used. Compared to L. casei Zhang grown to late logarithmic phase in milk, 61 genes were significantly up-regulated (>5 fold) in stationary phase, whereas 26 genes were down-regulated. Collectively, these data showed that the majority of the identified genes was involved in carbohydrate metabolism and energy production, followed by genes involved in nucleotide metabolism, inorganic ion transport, amino acid transport and metabolism, chaperone, etc. This study demonstrates the fundamental effects of cultural conditions on the transcriptome of L. casei Zhang. Moreover, it improves the understanding of the growth and survival mechanism of the bacterium during the late stage of milk fermentation.
Project description:Lactobacillus casei Zhang is a probiotic bacterium isolated from koumiss in Inner Mongolia of China. Gene expression dynamics of L. casei Zhang during growth in soymilk was investigated in attempt to reveal the mechanisms involved in growth stimulation for growing probiotics in. Comparison of different transcripts next to each other revealed 162 and 63 significantly induced genes in late logarithmic phase and stationary phase, of which the expression was at least 3 fold up-regulated and down-regulated, respectively. Approximately, 38.4% of the up-regulated genes were associated with amino acid transport and metabolism notably for histidine and lysine biosynthesis, followed by genes/gene clusters involved in carbohydrate transport and metabolism, lipid transport and metabolism, and inorganic ion transport and metabolism. The analysis results suggest that the stimulatory effect of soymilk-based ecosystem on the L. casei Zhang growth is more complex than amino acids or oligopeptides supply.
Project description:Growth phase dependent transcriptional and translational changes of Hb. salinarum were examined. In the genome-wide transcriptome analysis, gene expression in exponential versus stationary growth phase was monitored on DNA microarrays. In the global study of translational regulation, the relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase using DNA microarrays.
Project description:Growth phase dependent translational regulation of Hf. volcanii was analyzed on a genome-wide scale. The relative amounts of free versus polysome-bound mRNA (separated by gradient centrifugation) were quantified in exponential as well as stationary growth phase by the use of shotgun DNA microarrays.
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:Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of glutamate decarboxylase (GAD) system, the most efficient acid resistance mechanism in E. coli. The full contribution of GadE to the acid resistance and virulence of pathogenic E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles with that of wild type in exponential and stationary phases of growth using microarrays containing 6088 ORFs from three E. coli genomes. gadE inactivation significantly altered the expression of 60 genes independent of growth phase and 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly down-regulated the expression of gadA, gadB, gadC and many acid fitness island genes in a growth phase-dependent manner. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Altogether, our data indicate that GadE is critical for acid resistance of E. coli O157:H7 and plays an important role in virulence by down-regulating expression of LEE. The results are based on O157:H7 Sakai wild type and gadE mutant exponential and stationary phase cultures grown in MOPS minimal medium. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), strain (wild type or mutant) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+strain+phase*strain. We incorporated the dye-swaps among the biological replicates.
Project description:Escherichia coli O157:H7 has caused serious outbreaks of foodborne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns after exposure to apple juice. Transcriptomes of mid-exponential and stationary phase cells were evaluated after 10 minutes in model apple juice (pH3.5) using microarrays probing 4,886 ORFs. Significant changes in gene expression were determined using R/MAANOVA and the Fs test. A total of 331 ORFs were significantly induced upon exposure of cells to model apple juice and included genes involved in the acid and osmotic stress responses as well as the oxidative stress response and envelope stress. Genes involved in the acid and osmotic stress responses, including asr, osmC, osmB, and osmY were significantly induced in response to model apple juice. Genes involved in the envelope stress response, known to be controlled by CpxR (cpxP, degP, and htpX), were significantly induced 2 to 15 fold upon exposure to apple juice, independent of growth phase. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 ORFs induced in model apple juice, 104 are O157-specific ORFs, including those encoding type three secretion effectors espJ, espB, espM2, espL3 and espZ. By elucidating the response of O157:H7 to acidic foods, we hope to gain insights into how this pathogen is able to survive in food matrices and how exposure to foods affects subsequent transmission and virulence. Keywords: stress The results are based on O157:H7 Sakai exponential and stationary phase cultures grown in MOPS minimal medium and then exposed to model apple juice (pH 3.5, 37C) for 10 minutes. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), treatment (MOPS or MAJ) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+treatment+phase*treatment. We incorporated the dye-swaps among the biological replicates.
Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR. Hybridizations measured transcriptional differences between an rpoN null and wild-type (WT) strain of E. coli O157:H7 Sakai at logarithmic and transition phase. Image files (TIFF) of hybridized microarray slides were generated using an Axon 4000B scanner (Molecular Devices), and analyzed using GenePix Pro software (Molecular Devices, ver. 6.0). The resulting microarray intensity data was log2-transformed, and normalized using the LOWESS algorithm in MAANOVA ver. 0.98-8 (R ver. 2.2.1).
Project description:DNA methylation is an important epigenetic modification. DNA methylation phenomenon exists widely in bacteria, plants and animals and is involved in a variety of biological processes.The wild Lactobacillus casei Zhang and its mutant Lactobacillus casei Zhang Δpglx were used as the research subjects.Proteomics was used to explore the effects of DNA methylation on various aspects of Lactobacillus casei.