Reconstruction of the metabolic network of Pseudomonas aeruginosa to interrogate virulence factor synthesis

Virulence-linked pathways in opportunistic pathogens are putative therapeutic targets that may be associated with less potential for resistance than targets in growth-essential pathways. However, efficacy of virulence-linked targets may be affected by the contribution of virulence-related genes to metabolism. We evaluate the complex interrelationships between growth and virulence-linked pathways using a genome-scale metabolic network reconstruction of Pseudomonas aeruginosa strain PA14 and an updated, expanded reconstruction of P. aeruginosa strain PAO1. The PA14 reconstruction accounts for the activity of 112 virulence-linked genes and virulence factor synthesis pathways that produce 17 unique compounds. We integrate eight published genome-scale mutant screens to validate gene essentiality predictions in rich media, contextualize intra-screen discrepancies and evaluate virulence-linked gene distribution across essentiality datasets. Computational screening further elucidates interconnectivity between inhibition of virulence factor synthesis and growth. Successful validation of selected gene perturbations using PA14 transposon mutants demonstrates the utility of model-driven screening of therapeutic targets

Predicting Ecological Roles in the Rhizosphere Using Metabolome and Transportome Modeling

The ability to obtain complete genome sequences from bacteria in environmental samples, such as soil samples from the rhizosphere, has highlighted the microbial diversity and complexity of environmental communities. However, new algorithms to analyze genome sequence information in the context of community structure are needed to enhance our understanding of the specific ecological roles of these organisms in soil environments. We present a machine learning approach using sequenced Pseudomonad genomes coupled with outputs of metabolic and transportomic computational models for identifying the most predictive molecular mechanisms indicative of a Pseudomonad's ecological role in the rhizosphere: a biofilm, biocontrol agent, promoter of plant growth, or plant pathogen. Computational predictions of ecological niche were highly accurate overall with models trained on transportomic model output being the most accurate (Leave One Out Validation F-scores between 0.82 and 0.89). The strongest predictive molecular mechanism features for rhizosphere ecological niche overlap with many previously reported analyses of Pseudomonad interactions in the rhizosphere, suggesting that this approach successfully informs a system-scale level understanding of how Pseudomonads sense and interact with their environments. The observation that an organism's transportome is highly predictive of its ecological niche is a novel discovery and may have implications in our understanding microbial ecology. The framework developed here can be generalized to the analysis of any bacteria across a wide range of environments and ecological niches making this approach a powerful tool for providing insights into functional predictions from bacterial genomic data

'Systems toxicology' approach identifies coordinated metabolic responses to copper in a terrestrian non-model invertebrate, the earthworm Lumbricus rubellus

Background: New methods are needed for research into non-model organisms, to monitor the effects of toxic disruption at both the molecular and functional organism level. We exposed earthworms (Lumbricus rubellus Hoffmeister) to sub-lethal levels of copper (10–480 mg/kg soil) for 70 days as a real-world situation, and monitored both molecular (cDNA transcript microarrays and nuclear magnetic resonance-based metabolic profiling: metabolomics) and ecological/functional endpoints (reproduction rate and weight change, which have direct relevance to population-level impacts). Results: Both of the molecular endpoints, metabolomics and transcriptomics, were highly sensitive, with clear copper-induced differences even at levels below those that caused a reduction in reproductive parameters. The microarray and metabolomic data provided evidence that the copper exposure led to a disruption of energy metabolism: transcripts of enzymes from oxidative phosphorylation were significantly over-represented, and increases in transcripts of carbohydrate metabolising enzymes (maltase-glucoamylase, mannosidase) had corresponding decreases in small-molecule metabolites (glucose, mannose). Treating both enzymes and metabolites as functional cohorts led to clear inferences about changes in energetic metabolism (carbohydrate use and oxidative phosphorylation), which would not have been possible by taking a 'biomarker' approach to data analysis. Conclusion: Multiple post-genomic techniques can be combined to provide mechanistic information about the toxic effects of chemical contaminants, even for non-model organisms with few additional mechanistic toxicological data. With 70-day no-observed-effect and lowest-observed-effect concentrations (NOEC and LOEC) of 10 and 40 mg kg-1 for metabolomic and microarray profiles, copper is shown to interfere with energy metabolism in an important soil organism at an ecologically and functionally relevant level