L-tryptophan and copper interactions linked to reduced colibactin genotoxicity in pks+ Escherichia coli.

Colibactin, a nonribosomal peptide/polyketide produced by pks+ Enterobacteriaceae, is a virulence factor and putative carcinogen that damages DNA by interstrand crosslinking (ICL). While the clb genes for colibactin biosynthesis have been identified, studies are needed to elucidate the mechanisms regulating colibactin production and activity. Here we perform untargeted metabolomics of pks+ Escherichia coli cultures to identify L-tryptophan as a candidate repressor of colibactin activity. When pks+ E. coli is grown in a minimal medium supplemented with L-tryptophan in vitro ICL of plasmid DNA is reduced by >80%. L-tryptophan does not affect the transcription of clb genes but protects from copper toxicity and triggers the expression of genes to export copper to the periplasm where copper can directly inhibit the ClbP peptidase domain. Thus, L-tryptophan and copper interact and repress colibactin activity, potentially reducing its carcinogenic effects in the intestine. IMPORTANCE: Colibactin is a small molecule produced by pks+ Enterobacteriaceae that damages DNA, leading to oncogenic mutations in human genomes. Colibactin-producing Escherichia coli (pks+) cells promote tumorigenesis in mouse models of colorectal cancer (CRC) and are elevated in abundance in CRC patient biopsies, making it important to identify the regulatory systems governing colibactin production. Here, we apply a systems biology approach to explore metabolite repression of colibactin production in pks+ E. coli. We identify L-tryptophan as a repressor of colibactin genotoxicity that stimulates the expression of genes to export copper to the periplasm where it can inhibit ClbP, the colibactin-activating peptidase. These results work toward an antibiotic-sparing, prophylactic strategy to inhibit colibactin genotoxicity and its tumorigenic effects in the intestine.

Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics.

The Lachnospiraceae is a family of anaerobic bacteria in the class Clostridia with potential to advance the bio-economy and intestinal therapeutics. Some species of Lachnospiraceae metabolize abundant, low-cost feedstocks such as lignocellulose and carbon dioxide into value-added chemicals. Others are among the dominant species of the human colon and animal rumen, where they ferment dietary fiber to promote healthy gut and immune function. Here, we summarize recent studies of the physiology, cultivation, and genetics of Lachnospiraceae, highlighting their wide substrate utilization and metabolic products with industrial applications. We examine studies of these bacteria as Live Biotherapeutic Products (LBPs), focusing on in vivo disease models and clinical studies using them to treat infection, inflammation, metabolic syndrome, and cancer. We discuss key research areas including elucidation of intra-specific diversity and genetic modification of candidate strains that will facilitate the exploitation of Lachnospiraceae in industry and medicine.

Cell Type- and Stimulation-Dependent Transcriptional Programs Regulated by Atg16L1 and Its Crohn's Disease Risk Variant T300A.

Genome-wide association studies have identified common genetic variants impacting human diseases; however, there are indications that the functional consequences of genetic polymorphisms can be distinct depending on cell type-specific contexts, which produce divergent phenotypic outcomes. Thus, the functional impact of genetic variation and the underlying mechanisms of disease risk are modified by cell type-specific effects of genotype on pathological phenotypes. In this study, we extend these concepts to interrogate the interdependence of cell type- and stimulation-specific programs influenced by the core autophagy gene Atg16L1 and its T300A coding polymorphism identified by genome-wide association studies as linked with increased risk of Crohn's disease. We applied a stimulation-based perturbational profiling approach to define Atg16L1 T300A phenotypes in dendritic cells and T lymphocytes. Accordingly, we identified stimulus-specific transcriptional signatures revealing T300A-dependent functional phenotypes that mechanistically link inflammatory cytokines, IFN response genes, steroid biosynthesis, and lipid metabolism in dendritic cells and iron homeostasis and lysosomal biogenesis in T lymphocytes. Collectively, these studies highlight the combined effects of Atg16L1 genetic variation and stimulatory context on immune function.

Evolution of a Biomass-Fermenting Bacterium To Resist Lignin Phenolics.

Increasing the resistance of plant-fermenting bacteria to lignocellulosic inhibitors is useful to understand microbial adaptation and to develop candidate strains for consolidated bioprocessing. Here, we study and improve inhibitor resistance in Clostridium phytofermentans (also called Lachnoclostridium phytofermentans), a model anaerobe that ferments lignocellulosic biomass. We survey the resistance of this bacterium to a panel of biomass inhibitors and then evolve strains that grow in increasing concentrations of the lignin phenolic, ferulic acid, by automated, long-term growth selection in an anaerobic GM3 automat. Ultimately, strains resist multiple inhibitors and grow robustly at the solubility limit of ferulate while retaining the ability to ferment cellulose. We analyze genome-wide transcription patterns during ferulate stress and genomic variants that arose along the ferulate growth selection, revealing how cells adapt to inhibitors through changes in gene dosage and regulation, membrane fatty acid structure, and the surface layer. Collectively, this study demonstrates an automated framework for in vivo directed evolution of anaerobes and gives insight into the genetic mechanisms by which bacteria survive exposure to chemical inhibitors.IMPORTANCE Fermentation of plant biomass is a key part of carbon cycling in diverse ecosystems. Further, industrial biomass fermentation may provide a renewable alternative to fossil fuels. Plants are primarily composed of lignocellulose, a matrix of polysaccharides and polyphenolic lignin. Thus, when microorganisms degrade lignocellulose to access sugars, they also release phenolic and acidic inhibitors. Here, we study how the plant-fermenting bacterium Clostridium phytofermentans resists plant inhibitors using the lignin phenolic, ferulic acid. We examine how the cell responds to abrupt ferulate stress by measuring changes in gene expression. We evolve increasingly resistant strains by automated, long-term cultivation at progressively higher ferulate concentrations and sequence their genomes to identify mutations associated with acquired ferulate resistance. Our study develops an inhibitor-resistant bacterium that ferments cellulose and provides insights into genomic evolution to resist chemical inhibitors.

Quantitative proteomics using reductive dimethylation for stable isotope labeling.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.