Project description:Inflammatory diseases of the gut are associated with increased intestinal oxygen concentrations and high levels of inflammatory oxidants, including hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), which are antimicrobial compounds produced by the innate immune system. This contributes to dysbiotic changes in the gut microbiome, including increased populations of pro-inflammatory enterobacteria (Escherichia coli and related species) and decreased levels of health-associated anaerobic Firmicutes and Bacteroidetes. The pathways for H2O2 and HOCl resistance in E. coli have been well-studied, but little is known about how commensal and probiotic bacteria respond to inflammatory oxidants. In this work, we have characterized the transcriptomic response of the anti-inflammatory, gut colonizing Gram-positive probiotic Lactobacillus reuteri to both H2O2 and HOCl. L. reuteri mounts distinct responses to each of these stressors, and both gene expression and survival were strongly affected by the presence or absence of oxygen. Oxidative stress response in L. reuteri required several factors not found in enterobacteria, including the small heat shock protein Lo18, polyphosphate kinase 2, and RsiR, an L. reuteri-specific regulator of anti-inflammatory mechanisms. These results raise the intriguing possibility of developing treatments for inflammatory gut diseases that could sensitize pro-inflammatory enterobacteria to killing by the immune system while sparing anti-inflammatory, health-associated species.
Project description:Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent gastrointestinal system that adheres to the non-secretory epithelium of the forestomach and forms biofilms. Microarray analysis of the expression profile of L. reuteri 100-23 cells harvested from the stomach of ex-Lactobacillus-free mice, compared to those of L. reuteri 100-23 in laboratory culture, revealed an in vivo upregulation of a urease gene cluster by greater than 50-fold. Genes for urease production were absent in all publically available Lactobacillus genome sequences except L. reuteri 100-23 and have recently been identified as specific to rodent strains of L. reuteri (Frese et al. 2011). In the current study, the urease enzyme was shown to be functional. Supplementation with 2% urea allowed L. reuteri 100-23 to increase the pH of the culture medium. A mutant strain of L. reuteri 100-23 was developed by insertional inactivation of the ureC gene, which encodes the largest subunit of the urease enzyme. The mutant strain was unable to hydrolyze urea to increase the pH of culture medium, and did not survive acid stress at pH 2.5 for 6 h, even in the presence of urea. In contrast, the wild type strain was still viable after 6 h when 2% urea supplementation was included. When mice free of lactobacilli were inoculated with a mixture of equal numbers of wild type L. reuteri 100-23 and ureC mutant cells, the wild type constituted 99% of the resulting Lactobacillus population in the stomach, caecum and jejunum after one week (108 cells/gram of sample). This study has therefore shown the importance of a functional urease enzyme in the ecological fitness of L. reuteri 100-23.
Project description:This SuperSeries is composed of the following subset Series: GSE11860: The impact of glycerol on the metabolism of Lactobacillus reuteri - Exploratory experiment GSE11861: The impact of glycerol on the metabolism of Lactobacillus reuteri - Main experiment Refer to individual Series
Project description:Lactobacillus reuteri 100-23 is an autochthonous inhabitant of the rodent gastrointestinal system that adheres to the non-secretory epithelium of the forestomach and forms biofilms. Microarray analysis of the expression profile of L. reuteri 100-23 cells harvested from the stomach of ex-Lactobacillus-free mice, compared to those of L. reuteri 100-23 in laboratory culture, revealed an in vivo upregulation of a urease gene cluster by greater than 50-fold. Genes for urease production were absent in all publically available Lactobacillus genome sequences except L. reuteri 100-23 and have recently been identified as specific to rodent strains of L. reuteri (Frese et al. 2011). In the current study, the urease enzyme was shown to be functional. Supplementation with 2% urea allowed L. reuteri 100-23 to increase the pH of the culture medium. A mutant strain of L. reuteri 100-23 was developed by insertional inactivation of the ureC gene, which encodes the largest subunit of the urease enzyme. The mutant strain was unable to hydrolyze urea to increase the pH of culture medium, and did not survive acid stress at pH 2.5 for 6 h, even in the presence of urea. In contrast, the wild type strain was still viable after 6 h when 2% urea supplementation was included. When mice free of lactobacilli were inoculated with a mixture of equal numbers of wild type L. reuteri 100-23 and ureC mutant cells, the wild type constituted 99% of the resulting Lactobacillus population in the stomach, caecum and jejunum after one week (108 cells/gram of sample). This study has therefore shown the importance of a functional urease enzyme in the ecological fitness of L. reuteri 100-23. Analysis of the microarray data was obtained from two independent biological replicates.
Project description:Lactobacillus reuteri is a heterofermentative lactic acid bacterium best known for its ability to co-ferment glucose and glycerol. Its genome sequence has recently been deduced enabling the implementation of genome-wide analysis. In this study we developed a dedicated cDNA microarray platform and a genome-scale metabolic network model of L. reuteri and use them to revisit the co-fermentation of glucose and glycerol. The model was used to simulate experimental conditions and to visualize and integrate experimental data in particular the global transcriptional response of L. reuteri to the presence of glycerol. We show how the presence of glycerol affects cell physiology and triggers specific regulatory mechanisms allowing simultaneously a better yield and more efficient biomass formation. Furthermore we were able to predict and demonstrate for this well-studied condition the involvement of previously unsuspected metabolic pathways for instance related to amino acids and vitamins. These could be used as leads in future studies aiming at the increased production of industrially relevant compounds such as vitamin B12 or 1 3- propanediol. Keywords: cell type comparison
Project description:Lactobacillus reuteri is a heterofermentative lactic acid bacterium best known for its ability to co-ferment glucose and glycerol. Its genome sequence has recently been deduced enabling the implementation of genome-wide analysis. In this study we developed a dedicated cDNA microarray platform and a genome-scale metabolic network model of L. reuteri and use them to revisit the co-fermentation of glucose and glycerol. The model was used to simulate experimental conditions and to visualize and integrate experimental data in particular the global transcriptional response of L. reuteri to the presence of glycerol. We show how the presence of glycerol affects cell physiology and triggers specific regulatory mechanisms allowing simultaneously a better yield and more efficient biomass formation. Furthermore we were able to predict and demonstrate for this well-studied condition the involvement of previously unsuspected metabolic pathways for instance related to amino acids and vitamins. These could be used as leads in future studies aiming at the increased production of industrially relevant compounds such as vitamin B12 or 1 3- propanediol. Keywords: cell type comparison
Project description:Lactobacillus reuteri is a heterofermentative lactic acid bacterium best known for its ability to co-ferment glucose and glycerol. Its genome sequence has recently been deduced enabling the implementation of genome-wide analysis. In this study we developed a dedicated cDNA microarray platform and a genome-scale metabolic network model of L. reuteri and use them to revisit the co-fermentation of glucose and glycerol. The model was used to simulate experimental conditions and to visualize and integrate experimental data in particular the global transcriptional response of L. reuteri to the presence of glycerol. We show how the presence of glycerol affects cell physiology and triggers specific regulatory mechanisms allowing simultaneously a better yield and more efficient biomass formation. Furthermore we were able to predict and demonstrate for this well-studied condition the involvement of previously unsuspected metabolic pathways for instance related to amino acids and vitamins. These could be used as leads in future studies aiming at the increased production of industrially relevant compounds such as vitamin B12 or 1 3- propanediol. Keywords: cell type comparison Dye swap
Project description:Lactobacillus reuteri is a heterofermentative lactic acid bacterium best known for its ability to co-ferment glucose and glycerol. Its genome sequence has recently been deduced enabling the implementation of genome-wide analysis. In this study we developed a dedicated cDNA microarray platform and a genome-scale metabolic network model of L. reuteri and use them to revisit the co-fermentation of glucose and glycerol. The model was used to simulate experimental conditions and to visualize and integrate experimental data in particular the global transcriptional response of L. reuteri to the presence of glycerol. We show how the presence of glycerol affects cell physiology and triggers specific regulatory mechanisms allowing simultaneously a better yield and more efficient biomass formation. Furthermore we were able to predict and demonstrate for this well-studied condition the involvement of previously unsuspected metabolic pathways for instance related to amino acids and vitamins. These could be used as leads in future studies aiming at the increased production of industrially relevant compounds such as vitamin B12 or 1 3- propanediol. Keywords: cell type comparison Loop design
Project description:Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We found that microbial metabolites from healthy mice or humans were growth-repressive, and this response was attenuated in mice and patients with CRC. Microbial profiling revealed that Lactobacillus reuteri and its metabolite, reuterin were downregulated in mouse and human CRC. Reuterin altered redox balance, and reduced survival, and proliferation in colon cancer cells. Reuterin induced selective protein oxidation, and inhibited ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricted mouse colon tumor growth, increased tumor reactive oxygen species, and decreased protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.