A synthetic methylotrophic Escherichia coli as a chassis for bioproduction from methanol.

Methanol synthesized from captured greenhouse gases is an emerging renewable feedstock with great potential for bioproduction. Recent research has raised the prospect of methanol bioconversion to value-added products using synthetic methylotrophic Escherichia coli, as its metabolism can be rewired to enable growth solely on the reduced one-carbon compound. Here we describe the generation of an E. coli strain that grows on methanol at a doubling time of 4.3 h-comparable to many natural methylotrophs. To establish bioproduction from methanol using this synthetic chassis, we demonstrate biosynthesis from four metabolic nodes from which numerous bioproducts can be derived: lactic acid from pyruvate, polyhydroxybutyrate from acetyl coenzyme A, itaconic acid from the tricarboxylic acid cycle and p-aminobenzoic acid from the chorismate pathway. In a step towards carbon-negative chemicals and valorizing greenhouse gases, our work brings synthetic methylotrophy in E. coli within reach of industrial applications.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Assessing microbiome population dynamics using wild-type isogenic standardized hybrid (WISH)-tags.

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that require intrastrain resolution. Here we developed a genomic tagging system, termed wild-type isogenic standardized hybrid (WISH)-tags, that can be combined with quantitative polymerase chain reaction and next-generation sequencing for microbial strain enumeration. We experimentally validated the performance of 62 tags and showed that they can be differentiated with high precision. WISH-tags were introduced into model and non-model bacterial members of the mouse and plant microbiota. Intrastrain priority effects were tested using one species of isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. We observed colonization resistance against late-arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated Sphingomonas latecomers in a manner proportional to their presence at the late inoculation timepoint. This demonstrates that WISH-tags are a resource for deciphering population dynamics underlying microbiome assembly across biological systems.

mBARq: a versatile and user-friendly framework for the analysis of DNA barcodes from transposon insertion libraries, knockout mutants, and isogenic strain populations.

MOTIVATION: DNA barcoding has become a powerful tool for assessing the fitness of strains in a variety of studies, including random transposon mutagenesis screens, attenuation of site-directed mutants, and population dynamics of isogenic strain pools. However, the statistical analysis, visualization, and contextualization of the data resulting from such experiments can be complex and require bioinformatic skills. RESULTS: Here, we developed mBARq, a user-friendly tool designed to simplify these steps for diverse experimental setups. The tool is seamlessly integrated with an intuitive web app for interactive data exploration via the STRING and KEGG databases to accelerate scientific discovery. AVAILABILITY AND IMPLEMENTATION: The tool is implemented in Python. The source code is freely available (https://github.com/MicrobiologyETHZ/mbarq) and the web app can be accessed at: https://microbiomics.io/tools/mbarq-app.

Dashing Growth Curves: a web application for rapid and interactive analysis of microbial growth curves.

BACKGROUND: Recording and analyzing microbial growth is a routine task in the life sciences. Microplate readers that record dozens to hundreds of growth curves simultaneously are increasingly used for this task raising the demand for their rapid and reliable analysis. RESULTS: Here, we present Dashing Growth Curves, an interactive web application ( http://dashing-growth-curves.ethz.ch/ ) that enables researchers to quickly visualize and analyze growth curves without the requirement for coding knowledge and independent of operating system. Growth curves can be fitted with parametric and non-parametric models or manually. The application extracts maximum growth rates as well as other features such as lag time, length of exponential growth phase and maximum population size among others. Furthermore, Dashing Growth Curves automatically groups replicate samples and generates downloadable summary plots for of all growth parameters. CONCLUSIONS: Dashing Growth Curves is an open-source web application that reduces the time required to analyze microbial growth curves from hours to minutes.

Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens.

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.