A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia.

Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation.IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.

Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations.

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.

2-Deoxyribose-5-phosphate aldolase, a remarkably tolerant aldolase towards nucleophile substrates.

We explored a collection of 2-deoxyribose-5-phosphate aldolases (DERAs) from biodiversity for their nucleophile substrate promiscuity. The DERAs were screened using as nucleophiles propanone, propanal, cyclobutanone, cyclopentanone, dihydroxyacetone, and glycolaldehyde with l-glyceraldehyde-3-phosphate as an electrophile in aldol addition. A DERA from Arthrobacter chlorophenolicus (DERAArthro) efficiently allowed the synthesis of the corresponding aldol adducts in good yields, displaying complementarity in terms of configuration and substrate specificity with fructose-6-phosphate aldolase, the only previously known aldolase with a large nucleophile tolerance.

ABC Transporters Required for Hexose Uptake by Clostridium phytofermentans.

The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

De novo structure determination of 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid, a novel and abundant metabolite in Acinetobacter baylyi ADP1.

INTRODUCTION: Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. OBJECTIVES: This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). METHODS: We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D- and 2D-NMR (1H, 13C, 1H-1H-COSY, 1H-13C-HSQC, 1H-13C-HMBC and 1H-15N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. RESULTS: We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxybenzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the millimolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. CONCLUSION: Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.

Characterization of l-Carnitine Metabolism in Sinorhizobium meliloti.

l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from γ-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of l-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of l-carnitine biosynthesis and degradation in Sinorhizobium meliloti The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of l-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of l-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of l-carnitine as a ubiquitous nutrient.IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of l-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars.

Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrKmitis) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 106 s-1 M-1 and 2.7 105 s-1 M-1, respectively, but also significant ones toward D-tagatose (3.5 102 s-1 M-1) and the unnatural monosaccharides D-altrose (1.1 104 s-1 M-1) and D-talose (3.4 102 s-1 M-1). Specific activities measured for all hexoses showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.

Elucidation of the trigonelline degradation pathway reveals previously undescribed enzymes and metabolites.

Trigonelline (TG; N-methylnicotinate) is a ubiquitous osmolyte. Although it is known that it can be degraded, the enzymes and metabolites have not been described so far. In this work, we challenged the laboratory model soil-borne, gram-negative bacterium Acinetobacter baylyi ADP1 (ADP1) for its ability to grow on TG and we identified a cluster of catabolic, transporter, and regulatory genes. We dissected the pathway to the level of enzymes and metabolites, and proceeded to in vitro reconstruction of the complete pathway by six purified proteins. The four enzymatic steps that lead from TG to methylamine and succinate are described, and the structures of previously undescribed metabolites are provided. Unlike many aromatic compounds that undergo hydroxylation prior to ring cleavage, the first step of TG catabolism proceeds through direct cleavage of the C5-C6 bound, catalyzed by a flavin-dependent, two-component oxygenase, which yields (Z)-2-((N-methylformamido)methylene)-5-hydroxy-butyrolactone (MFMB). MFMB is then oxidized into (E)-2-((N-methylformamido) methylene) succinate (MFMS), which is split up by a hydrolase into carbon dioxide, methylamine, formic acid, and succinate semialdehyde (SSA). SSA eventually fuels up the TCA by means of an SSA dehydrogenase, assisted by a Conserved Hypothetical Protein. The cluster is conserved across marine, soil, and plant-associated bacteria. This emphasizes the role of TG as a ubiquitous nutrient for which an efficient microbial catabolic toolbox is available.

Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1-phosphate Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new C-C bond-forming enzyme for stereoselective synthesis of tertiary alcohols.

Complete Genome Sequence of the Facultative Methylotroph Methylobacterium extorquens TK 0001 Isolated from Soil in Poland.

Methylobacterium extorquens TK 0001 (DSM 1337, ATCC 43645) is an aerobic pink-pigmented facultative methylotrophic alphaproteobacterium isolated from soil in Poland. Here, we report the whole-genome sequence and annotation of this organism, which consists of a single 5.71-Mb chromosome.

Single-cell genomics of multiple uncultured stramenopiles reveals underestimated functional diversity across oceans.

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology.

Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.

Stereoselective synthesis of γ-hydroxy-α-amino acids through aldolase-transaminase recycling cascades.

Efficient bi-enzymatic cascades combining aldolases and α-transaminases were designed for the synthesis of γ-hydroxy-α-amino acids. These recycling cascades provide high stereoselectivity, atom economy, and an equilibrium shift of the transamination. l-syn or anti-4-hydroxyglutamic acid and d-anti-4,5-dihydroxynorvaline were thus prepared in 83-95% yield in one step from simple substrates.

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.

Global repositioning of transcription start sites in a plant-fermenting bacterium.

Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5' mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.

A scalable assembly-free variable selection algorithm for biomarker discovery from metagenomes.

BACKGROUND: Metagenomics holds great promises for deepening our knowledge of key bacterial driven processes, but metagenome assembly remains problematic, typically resulting in representation biases and discarding significant amounts of non-redundant sequence information. In order to alleviate constraints assembly can impose on downstream analyses, and/or to increase the fraction of raw reads assembled via targeted assemblies relying on pre-assembly binning steps, we developed a set of binning modules and evaluated their combination in a new "assembly-free" binning protocol. RESULTS: We describe a scalable multi-tiered binning algorithm that combines frequency and compositional features to cluster unassembled reads, and demonstrate i) significant runtime performance gains of the developed modules against state of the art software, obtained through parallelization and the efficient use of large lock-free concurrent hash maps, ii) its relevance for clustering unassembled reads from high complexity (e.g., harboring 700 distinct genomes) samples, iii) its relevance to experimental setups involving multiple samples, through a use case consisting in the "de novo" identification of sequences from a target genome (e.g., a pathogenic strain) segregating at low levels in a cohort of 50 complex microbiomes (harboring 100 distinct genomes each), in the background of closely related strains and the absence of reference genomes, iv) its ability to correctly identify clusters of sequences from the E. coli O104:H4 genome as the most strongly correlated to the infection status in 53 microbiomes sampled from the 2011 STEC outbreak in Germany, and to accurately cluster contigs of this pathogenic strain from a cross-assembly of these 53 microbiomes. CONCLUSIONS: We present a set of sequence clustering ("binning") modules and their application to biomarker (e.g., genomes of pathogenic organisms) discovery from large synthetic and real metagenomics datasets. Initially designed for the "assembly-free" analysis of individual metagenomic samples, we demonstrate their extension to setups involving multiple samples via the usage of the "alignment-free" d2S statistic to relate clusters across samples, and illustrate how the clustering modules can otherwise be leveraged for de novo "pre-assembly" tasks by segregating sequences into biologically meaningful partitions.

Fungal lysis by a soil bacterium fermenting cellulose.

Recycling of plant biomass by a community of bacteria and fungi is fundamental to carbon flow in terrestrial ecosystems. Here we report how the plant fermenting, soil bacterium Clostridium phytofermentans enhances growth on cellulose by simultaneously lysing and consuming model fungi from soil. We investigate the mechanism of fungal lysis to show that among the dozens of different glycoside hydrolases C. phytofermentans secretes on cellulose, the most highly expressed enzymes degrade fungi rather than plant substrates. These enzymes, the GH18 Cphy1799 and Cphy1800, synergize to hydrolyse chitin, a main component of the fungal cell wall. Purified enzymes inhibit fungal growth and mutants lacking either GH18 grow normally on cellulose and other plant substrates, but have a reduced ability to hydrolyse chitinous substrates and fungal hyphae. Thus, C. phytofermentans boosts growth on cellulose by lysing fungi with its most highly expressed hydrolases, highlighting the importance of fungal interactions to the ecology of cellulolytic bacteria.

Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach.

Expansive knowledge of bacterial metabolism has been gained from genome sequencing output, but the high proportion of genes lacking a proper functional annotation in a given genome still impedes the accurate prediction of the metabolism of a cell. To access to a more global view of the functioning of the soil bacterium Acinetobacter baylyi ADP1, we adopted a multi 'omics' approach. Application of RNA-seq transcriptomics and LC/MS-based metabolomics, along with the systematic phenotyping of the complete collection of single-gene deletion mutants of A. baylyi ADP1 made possible to interrogate on the metabolic perturbations encountered by the bacterium upon a biotic change. Shifting the sole carbon source from succinate to quinate elicited in the cell not only a specific transcriptional response, necessary to catabolize the new carbon source, but also a major reorganization of the transcription pattern. Here, the expression of more than 12 % of the total number of genes was affected, most of them being of unknown function. These perturbations were ultimately reflected in the metabolome, in which the concentration of about 50 % of the LC/MS-detected metabolites was impacted. And the differential regulation of many genes of unknown function is probably related to the synthesis of the numerous unidentified compounds that were present exclusively in quinate-grown cells. Together, these data suggest that A. baylyi ADP1 metabolism involves unsuspected enzymatic reactions that await discovery.