Volker Döring

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

Chemical Evolution of a Bacterium’s Genome

We set out to develop a generic technology for evolving the chemical constitution of microbial populations by using the simplest possible algorithm. Extant living cells polymerize a restricted set of nucleic acid precursors, namely, four nucleoside triphosphates (UTP, CTP, ATP, GTP) and four deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP). Synthetic analogues, such as 5-halogenopyrimidines, 7-deazapurines, and 8-azapurines, are known to partially replace canonical bases in cellular RNA and DNA, yet were never demonstrated to sustain unlimited self-reproduction of an organism through complete genome or transcriptome substitution. A hamster cell line serially adapted to grow in the presence of bromodeoxyuridine, while dTMP synthesis was inhibited with aminopterin, has been reported to harbor DNA highly enriched in bromouracil over thymine. However, the significance of these findings could not be ascertained owing to the absence of a direct physical measurement of the base composition of the DNA and the absence of an assay of thymidylate biosynthesis, as well as the likely presence of metabolic components, such as nucleotides in the complex growth medium of the cells. Only certain DNA viruses are known to have undergone full transliteration of a canonical base through the biosynthesis of a noncanonical nucleoside triphosphate, for example, hydroxymethylcytosine in the T4 bacteriophage, presumably to counteract the restriction enzymes of their bacterial hosts. When Weiss and coworkers attempted to substitute thymine in the DNA of Escherichia coli with uracil, over 90% replacement was reached, but further growth was prevented. Genome-scale transliteration has apparently not evolved in any known living cell, possibly owing to a chemical barrier that natural biodiversity cannot overcome. Our experimental plan consisted of the combination of tight metabolic selection with the long-term automated cultivation of fast-growing asexual bacterial populations to change a canonical DNA base for a chemical ersatz. The cultivation setup was elaborated from the GM3 fluidic format (Figure 1), which features the cyclic transfer of the culture between twin growth chambers that alternately undergo sterilization. This cycle ensures that no internal surface of the device is spared from transient periodic cleansing with a sterilizing agent (5m sodium hydroxide), and therefore that no cultivated variant can escape dilution and selection for faster growth through the formation of biofilms. The active elimination of biofilms (wall growth) has proved critical for reprogramming and improving the metabolism of microbial populations. The GM3 cultivation device was connected to two nutrient reservoirs of different composition: a relaxing medium R that contains the canonical nutrient and a stressing medium S that contains the ersatz nutrient. Liquid pulses of defined volume are sent at regular intervals of time from these reservoirs to the culture, which is kept at a constant volume. Depending upon the state of the adapting cells, as measured by turbidity recording of the population density, the culture periodically receives a pulse of fixed volume of either medium R (if the population density falls below a fixed threshold) or medium S (if the density is higher than or equal to the threshold). Successive pulses thus renew the culture at a fixed dilution rate with a nutrient-medium flow whose composition varies with respect to the growth response of the population in such a way that the lowest tolerable concentration of canonical nutrient is automatically maintained over passing generations. We designate this mode of operation as the conditional pulse-feed regime. It qualifies as a simplified and generalized version of a method pioneered by Oliver. Mutations that confer a lower requirement for the canonical nutrient or a higher survival rate under starvation are expected to accumulate in the genome of the adapting population. No attempt was made to implement a finer regulation of differential nutrient supply than the coarse-grained control by medium-switch pulse feed described above. We thus relied on the robustness of biochemical machineries and their evolution to dampen oscillations. [*] Dr. P. Marli re Heurisko USA Inc., Delaware (USA)

In Vivo Assimilation of One-Carbon via a Synthetic Reductive Glycine Pathway in Escherichia coli

Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO2. Importantly, the biosynthesis of serine from formate and CO2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.

Evolution of a biomass-fermenting bacterium to resist lignin phenolics

ABSTRACT 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.