Comprehensive Analysis of Gene-Environmental Interactions with Temporal Gene Expression Profiles in Pseudomonas aeruginosa

To explore gene-environment interactions, based on temporal gene expression information, we analyzed gene and treatment information intensively and inferred interaction networks accordingly. The main idea is that gene expression reflects the response of genes to environmental factors, assuming that variations of gene expression occur under different conditions. Then we classified experimental conditions into several subgroups based on the similarity of temporal gene expression profiles. This procedure is useful because it allows us to combine diverse gene expression data as they become available, and, especially, allowing us to lay the regulatory relationships on a concrete biological basis. By estimating the activation points, we can visualize the gene behavior, and obtain a consensus gene activation order, and hence describe conditional regulatory relationships. The estimation of activation points and building of synthetic genetic networks may result in important new insights in the ongoing endeavor to understand the complex network of gene regulation

Genomic Instability in Regions Adjacent to a Highly Conserved \u3ci\u3epch\u3c/i\u3e Prophage in \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Generates Diversity in Expression Patterns of the LEE Pathogenicity Island

The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain variation in LEE expression has been observed, implying that expression patterns can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB, pchC, and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage

Genomic Instability in Regions Adjacent to a Highly Conserved \u3ci\u3epch\u3c/i\u3e Prophage in \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 Generates Diversity in Expression Patterns of the LEE Pathogenicity Island

The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain variation in LEE expression has been observed, implying that expression patterns can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB, pchC, and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage

Programming gene expression with combinatorial promoters

Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple −10 and −35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters

Monocyte-driven inflamm-aging reduces intestinal barrier function in females

Background: The intestinal barrier encompasses physical and immunological components that act to compartmentalize luminal contents, such as bacteria and endotoxins, from the host. It has been proposed that an age-related decline of intestinal barrier function may allow for the passage of luminal contents into the bloodstream, triggering a low-grade systemic inflammation termed inflamm-aging. Although there is mounting evidence to support this hypothesis in model species, it is unclear if this phenomenon occurs in humans. In addition, despite being well-established that biological sex impacts aging physiology, its influence on intestinal barrier function and inflamm-aging has not been explored. Results: In this study, we observed sex differences in markers of intestinal barrier integrity, where females had increased epithelial permeability throughout life as compared to males. With age, females had an age-associated increase in circulating bacterial products and metabolites such as LPS and kynurenine, suggesting reduced barrier function. Females also had age-associated increases in established markers of inflamm-aging, including peripheral blood monocytes as well as TNF and CRP. To determine if impaired barrier function was driving inflamm-aging, we performed a mediation analysis. The results show that the loss of intestinal barrier integrity was not the mediator of inflamm-aging in humans. Instead, persistent, low-grade inflammation with age preceded the increase in circulating bacterial products, which we confirmed using animal models. We found, as in humans, that sex modified age-associated increases in circulating monocytes in mice, and that inflammation mediates the loss of intestinal barrier function. Conclusion: Taken together, our results suggest that higher basal intestinal permeability in combination with age-associated inflammation, increases circulating LPS in females. Thus, targeting barrier permeability in females may slow the progression of inflamm-aging, but is unlikely to prevent it. <br/

Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis

Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis

Microbial communities reside in healthy tissues but are often disrupted during disease. Bacterial genomes and proteins are detected in brains from humans, nonhuman primates, rodents and other species in the absence of neurological disease. We investigated the composition and abundance of microbiota in frozen and fixed autopsied brain samples from patients with multiple sclerosis (MS) and age-and sex-matched nonMS patients as controls, using neuropathological, molecular and bioinformatics tools. 16s rRNA sequencing revealed Proteobacteria to be the dominant phylum with restricted diversity in cerebral white matter (WM) from MS compared to nonMS patients. Both clinical groups displayed 1,200-1,400 bacterial genomes/cm(3) and low bacterial rRNA: rDNA ratios in WM. RNAseq analyses showed a predominance of Proteobacteria in progressive MS patients' WM, associated with increased inflammatory gene expression, relative to a broader range of bacterial phyla in relapsing-remitting MS patients' WM. Although bacterial peptidoglycan (PGN) and RNA polymerase beta subunit immunoreactivities were observed in all patients, PGN immunodetection was correlated with demyelination and neuroinflammation in MS brains. Principal component analysis revealed that demyelination, PGN and inflammatory gene expression accounted for 86% of the observed variance. Thus, inflammatory demyelination is linked to an organ-specific dysbiosis in MS that could contribute to underlying disease mechanisms

Microbiota and host determinants of behavioural phenotype in maternally separated mice

Early-life stress is a determinant of vulnerability to a variety of disorders that include dysfunction of the brain and gut. Here we exploit a model of early-life stress, maternal separation (MS) in mice, to investigate the role of the intestinal microbiota in the development of impaired gut function and altered behaviour later in life. Using germ-free and specific pathogen-free mice, we demonstrate that MS alters the hypothalamic-pituitary-adrenal axis and colonic cholinergic neural regulation in a microbiota-independent fashion. However, microbiota is required for the induction of anxiety-like behaviour and behavioural despair. Colonization of adult germ-free MS and control mice with the same microbiota produces distinct microbial profiles, which are associated with altered behaviour in MS, but not in control mice. These results indicate that MS-induced changes in host physiology lead to intestinal dysbiosis, which is a critical determinant of the abnormal behaviour that characterizes this model of early-life stress

Decontamination of 16S rRNA gene amplicon sequence datasets based on bacterial load assessment by qPCR

Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators