The microbiota conditions a gut milieu that selects for wild-type Salmonella Typhimurium virulence.

Salmonella Typhimurium elicits gut inflammation by the costly expression of HilD-controlled virulence factors. This inflammation alleviates colonization resistance (CR) mediated by the microbiota and thereby promotes pathogen blooms. However, the inflamed gut-milieu can also select for hilD mutants, which cannot elicit or maintain inflammation, therefore causing a loss of the pathogen's virulence. This raises the question of which conditions support the maintenance of virulence in S. Typhimurium. Indeed, it remains unclear why the wild-type hilD allele is dominant among natural isolates. Here, we show that microbiota transfer from uninfected or recovered hosts leads to rapid clearance of hilD mutants that feature attenuated virulence, and thereby contributes to the preservation of the virulent S. Typhimurium genotype. Using mouse models featuring a range of microbiota compositions and antibiotic- or inflammation-inflicted microbiota disruptions, we found that irreversible disruption of the microbiota leads to the accumulation of hilD mutants. In contrast, in models with a transient microbiota disruption, selection for hilD mutants was prevented by the regrowing microbiota community dominated by Lachnospirales and Oscillospirales. Strikingly, even after an irreversible microbiota disruption, microbiota transfer from uninfected donors prevented the rise of hilD mutants. Our results establish that robust S. Typhimurium gut colonization hinges on optimizing its manipulation of the host: A transient and tempered microbiota perturbation is favorable for the pathogen to both flourish in the inflamed gut and also minimize loss of virulence. Moreover, besides conferring CR, the microbiota may have the additional consequence of maintaining costly enteropathogen virulence mechanisms.

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling.

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.

ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks.

13C-metabolic flux analysis (13C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current 13C-MFA approaches model label propagation starting from the extracellular 13C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current 13C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of Saccharomyces cerevisiae mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.

InhA, the enoyl-thioester reductase from Mycobacterium tuberculosis forms a covalent adduct during catalysis.

The enoyl-thioester reductase InhA catalyzes an essential step in fatty acid biosynthesis of Mycobacterium tuberculosis and is a key target of antituberculosis drugs to combat multidrug-resistant M. tuberculosis strains. This has prompted intense interest in the mechanism and intermediates of the InhA reaction. Here, using enzyme mutagenesis, NMR, stopped-flow spectroscopy, and LC-MS, we found that the NADH cofactor and the CoA thioester substrate form a covalent adduct during the InhA catalytic cycle. We used the isolated adduct as a molecular probe to directly access the second half-reaction of the catalytic cycle of InhA (i.e. the proton transfer), independently of the first half-reaction (i.e. the initial hydride transfer) and to assign functions to two conserved active-site residues, Tyr-158 and Thr-196. We found that Tyr-158 is required for the stereospecificity of protonation and that Thr-196 is partially involved in hydride transfer and protonation. The natural tendency of InhA to form a covalent C2-ene adduct calls for a careful reconsideration of the enzyme's reaction mechanism. It also provides the basis for the development of effective tools to study, manipulate, and inhibit the catalytic cycle of InhA and related enzymes of the short-chain dehydrogenase/reductase (SDR) superfamily. In summary, our work has uncovered the formation of a covalent adduct during the InhA catalytic cycle and identified critical residues required for catalysis, providing further insights into the InhA reaction mechanism important for the development of antituberculosis drugs.

An engineered Calvin-Benson-Bassham cycle for carbon dioxide fixation in Methylobacterium extorquens AM1.

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO2). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO2. The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO2 by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic 13C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms.

The One-carbon Carrier Methylofuran from Methylobacterium extorquens AM1 Contains a Large Number of α- and γ-Linked Glutamic Acid Residues.

Methylobacterium extorquens AM1 uses dedicated cofactors for one-carbon unit conversion. Based on the sequence identities of enzymes and activity determinations, a methanofuran analog was proposed to be involved in formaldehyde oxidation in Alphaproteobacteria. Here, we report the structure of the cofactor, which we termed methylofuran. Using an in vitro enzyme assay and LC-MS, methylofuran was identified in cell extracts and further purified. From the exact mass and MS-MS fragmentation pattern, the structure of the cofactor was determined to consist of a polyglutamic acid side chain linked to a core structure similar to the one present in archaeal methanofuran variants. NMR analyses showed that the core structure contains a furan ring. However, instead of the tyramine moiety that is present in methanofuran cofactors, a tyrosine residue is present in methylofuran, which was further confirmed by MS through the incorporation of a (13)C-labeled precursor. Methylofuran was present as a mixture of different species with varying numbers of glutamic acid residues in the side chain ranging from 12 to 24. Notably, the glutamic acid residues were not solely γ-linked, as is the case for all known methanofurans, but were identified by NMR as a mixture of α- and γ-linked amino acids. Considering the unusual peptide chain, the elucidation of the structure presented here sets the basis for further research on this cofactor, which is probably the largest cofactor known so far.

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules.

Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid-forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using ß-2-microglobulin (ß2m) as a model amyloid-forming protein along with two recently identified small molecule amyloid inhibitors (i.e., rifamycin SV and doxycycline), we demonstrate that covalent labeling and mass spectrometry (MS) can be used to map small-molecule binding sites for a rapidly aggregating protein. Specifically, three different covalent labeling reagents, namely diethylpyrocarbonate, 2,3-butanedione, and the reagent pair EDC/GEE, are used together to pinpoint the binding sites of rifamycin SV, doxycycline, and another molecule, suramin, which binds but does not inhibit Cu(II)-induced ß2m amyloid formation. The labeling results reveal binding sites that are consistent with the known effects of these molecules on ß2m amyloid formation and are in general agreement with molecular docking results. We expect that this combined covalent labeling approach will be applicable to other protein/small molecule systems that are difficult to study by traditional means.

Single-Cell Mass Spectrometry of Metabolites Extracted from Live Cells by Fluidic Force Microscopy.

Single-cell metabolite analysis provides valuable information on cellular function and response to external stimuli. While recent advances in mass spectrometry reached the sensitivity required to investigate metabolites in single cells, current methods commonly isolate and sacrifice cells, inflicting a perturbed state and preventing complementary analyses. Here, we propose a two-step approach that combines nondestructive and quantitative withdrawal of intracellular fluid with subpicoliter resolution using fluidic force microscopy, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed method enabled the detection and identification of 20 metabolites recovered from the cytoplasm of individual HeLa cells. The approach was further validated in 13C-glucose feeding experiments, which showed incorporation of labeled carbon atoms into different metabolites. Metabolite sampling, followed by mass spectrometry measurements, enabled the preservation of the physiological context and the viability of the analyzed cell, providing opportunities for complementary analyses of the cell before, during, and after metabolite analysis.

The use of ene adducts to study and engineer enoyl-thioester reductases.

An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.

Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella, most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress.

Bacillus methanolicus MGA3 is a model facultative methylotroph of interest for fundamental research and biotechnological applications. Previous research uncovered a number of pathways potentially involved in one-carbon substrate utilization. Here, we applied dynamic (13) C labeling to elucidate which of these pathways operate during growth on methanol and to uncover potentially new ones. B. methanolicus MGA3 uses the assimilatory and dissimilatory ribulose monophosphate (RuMP) cycles for conversion of the central but toxic intermediate formaldehyde. Additionally, the operation of two cofactor-dependent formaldehyde oxidation pathways with distinct roles was revealed. One is dependent on tri- and tetraglutamylated tetrahydrofolate (THF) and is involved in formaldehyde oxidation during growth on methanol. A second pathway was discovered that is dependent on bacillithiol, a thiol cofactor present also in other Bacilli where it is known to function in redox-homeostasis. We show that bacillithiol-dependent formaldehyde oxidation is activated upon an upshift in formaldehyde induced by a substrate switch from mannitol to methanol. The genes and the corresponding enzymes involved in the biosynthesis of bacillithiol were identified by heterologous production of bacillithiol in Escherichia coli. The presented results indicate metabolic plasticity of the methylotroph allowing acclimation to fluctuating intracellular formaldehyde concentrations.

Direct evidence for a covalent ene adduct intermediate in NAD(P)H-dependent enzymes.

The pyridine nucleotides NADH and NADPH (NAD(P)H) are ubiquitous redox coenzymes that are present in all living cells. Although about 16% of all characterized enzymes use pyridine nucleotides as hydride donors or acceptors during catalysis, a detailed understanding of how the hydride is transferred between NAD(P)H and the corresponding substrate is lacking for many enzymes. Here we present evidence for a new mechanism that operates during enzymatic hydride transfers using crotonyl-CoA carboxylase/reductase (Ccr) as a case study. We observed a covalent ene intermediate between NADPH and the substrate, crotonyl-CoA, using NMR, high-resolution MS and stopped-flow spectroscopy. Preparation of the ene intermediate further allowed direct access to the catalytic cycle of other NADPH-dependent enzymes-including those from type II fatty acid biosynthesis-in an unprecedented way, suggesting that formation of NAD(P)H ene intermediates is a more general principle in catalysis.

DynaMet: a fully automated pipeline for dynamic LC-MS data.

Dynamic isotope labeling data provides crucial information about the operation of metabolic pathways and are commonly generated via liquid chromatography-mass spectrometry (LC-MS). Metabolome-wide analysis is challenging as it requires grouping of metabolite features over different samples. We developed DynaMet for fully automated investigations of isotope labeling experiments from LC-high-resolution MS raw data. DynaMet enables untargeted extraction of metabolite labeling profiles and provides integrated tools for expressive data visualization. To validate DynaMet we first used time course labeling data of the model strain Bacillus methanolicus from (13)C methanol resulting in complex spectra in multicarbon compounds. Analysis of two biological replicates revealed high robustness and reproducibility of the pipeline. In total, DynaMet extracted 386 features showing dynamic labeling within 10 min. Of these features, 357 could be fitted by implemented kinetic models. Feature identification against KEGG database resulted in 215 matches covering multiple pathways of core metabolism and major biosynthetic routes. Moreover, we performed time course labeling experiment with Escherichia coli on uniformly labeled (13)C glucose resulting in a comparable number of detected features with labeling profiles of high quality. The distinct labeling patterns of common central metabolites generated from both model bacteria can readily be explained by one versus multicarbon compound metabolism. DynaMet is freely available as an extension package for Python based eMZed2, an open source framework built for rapid development of LC-MS data analysis workflows.

Engineering Escherichia coli for methanol conversion.

Methylotrophic bacteria utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy. For this purpose, these bacteria evolved a number of specialized enzymes and pathways. Here, we used a synthetic biology approach to select and introduce a set of "methylotrophy genes" into Escherichia coli based on in silico considerations and flux balance analysis to enable methanol dissimilation and assimilation. We determined that the most promising approach allowing the utilization of methanol was the implementation of NAD-dependent methanol dehydrogenase and the establishment of the ribulose monophosphate cycle by expressing the genes for hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi). To test for the best-performing enzymes in the heterologous host, a number of enzyme candidates from different donor organisms were selected and systematically analyzed for their in vitro and in vivo activities in E. coli. Among these, Mdh2, Hps and Phi originating from Bacillus methanolicus were found to be the most effective. Labeling experiments using (13)C methanol with E. coli producing these enzymes showed up to 40% incorporation of methanol into central metabolites. The presence of the endogenous glutathione-dependent formaldehyde oxidation pathway of E. coli did not adversely affect the methanol conversion rate. Taken together, the results of this study represent a major advancement towards establishing synthetic methylotrophs by gene transfer.

High-level production of ethylmalonyl-CoA pathway-derived dicarboxylic acids by Methylobacterium extorquens under cobalt-deficient conditions and by polyhydroxybutyrate negative strains.

Bio-based production of dicarboxylic acids is an emerging research field with remarkable progress during the last decades. The recently established synthesis of the ethylmalonyl-CoA pathway (EMCP)-derived dicarboxylic acids, mesaconic acid and (2S)-methylsuccinic acid, from the alternative carbon source methanol (Sonntag et al., Appl Microbiol Biotechnol 98:4533-4544, 2014) gave a proof of concept for the sustainable production of hitherto biotechnologically inaccessible monomers. In this study, substantial optimizations of the process by different approaches are presented. Abolishment of mesaconic and (2S)-methylsuccinic acid reuptake from culture supernatant and a productivity increase were achieved by 30-fold decreased sodium ion availability in culture medium. Undesired flux from EMCP into polyhydroxybutyrate (PHB) cycle was hindered by the knockout of polyhydroxyalkanoate synthase phaC which was concomitant with 5-fold increased product concentrations. However, frequently occurring suppressors of strain ΔphaC lost their beneficial properties probably due to redirected channeling of acetyl-CoA. Pool sizes of the product precursors were increased by exploiting the presence of two cobalt-dependent mutases in the EMCP: Fine-tuned growth-limiting cobalt concentrations led to 16-fold accumulation of mesaconyl- and (2S)-methylsuccinyl-CoA which in turn resulted in 6-fold increased concentrations of mesaconic and (2S)-methylsuccinic acids, with a combined titer of 0.65 g/l, representing a yield of 0.17 g/g methanol. This work represents an important step toward an industrially relevant production of ethylmalonyl-CoA pathway-derived dicarboxylic acids and the generation of a stable PHB synthesis negative Methylobacterium extorquens strain.

Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis.

Carboxylating enoyl-thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α,ß-unsaturated CoA-thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl-CoA during assimilation of the central metabolite acetyl-CoA. In secondary metabolism they provide distinct α-carboxyl-acyl-thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5-enoyl-CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof-of-principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification.

eMZed: an open source framework in Python for rapid and interactive development of LC/MS data analysis workflows.

SUMMARY: The Python-based, open-source eMZed framework was developed for mass spectrometry (MS) users to create tailored workflows for liquid chromatography (LC)/MS data analysis. The goal was to establish a unique framework with comprehensive basic functionalities that are easy to apply and allow for the extension and modification of the framework in a straightforward manner. eMZed supports the iterative development and prototyping of individual evaluation strategies by providing a computing environment and tools for inspecting and modifying underlying LC/MS data. The framework specifically addresses non-expert programmers, as it requires only basic knowledge of Python and relies largely on existing successful open-source software, e.g. OpenMS. AVAILABILITY: The framework eMZed and its documentation are freely available at http://emzed.biol.ethz.ch/. eMZed is published under the GPL 3.0 license, and an online discussion group is available at https://groups.google.com/group/emzed-users. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate.

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.

C-di-AMP levels modulate Staphylococcus aureus cell wall thickness, response to oxidative stress, and antibiotic resistance and tolerance.

IMPORTANCE: Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting.

Complete genome sequence and metabolic potential of the quinaldine-degrading bacterium Arthrobacter sp. Rue61a.

BACKGROUND: Bacteria of the genus Arthrobacter are ubiquitous in soil environments and can be considered as true survivalists. Arthrobacter sp. strain Rue61a is an isolate from sewage sludge able to utilize quinaldine (2-methylquinoline) as sole carbon and energy source. The genome provides insight into the molecular basis of the versatility and robustness of this environmental Arthrobacter strain. RESULTS: The genome of Arthrobacter sp. Rue61a consists of a single circular chromosome of 4,736,495 bp with an average G + C content of 62.32%, the circular 231,551-bp plasmid pARUE232, and the linear 112,992-bp plasmid pARUE113 that was already published. Plasmid pARUE232 is proposed to contribute to the resistance of Arthrobacter sp. Rue61a to arsenate and Pb2+, whereas the linear plasmid confers the ability to convert quinaldine to anthranilate. Remarkably, degradation of anthranilate exclusively proceeds via a CoA-thioester pathway. Apart from quinaldine utilization, strain Rue61a has a limited set of aromatic degradation pathways, enabling the utilization of 4-hydroxy-substituted aromatic carboxylic acids, which are characteristic products of lignin depolymerization, via ortho cleavage of protocatechuate. However, 4-hydroxyphenylacetate degradation likely proceeds via meta cleavage of homoprotocatechuate. The genome of strain Rue61a contains numerous genes associated with osmoprotection, and a high number of genes coding for transporters. It encodes a broad spectrum of enzymes for the uptake and utilization of various sugars and organic nitrogen compounds. A. aurescens TC-1 is the closest sequenced relative of strain Rue61a. CONCLUSIONS: The genome of Arthrobacter sp. Rue61a reflects the saprophytic lifestyle and nutritional versatility of the organism and a strong adaptive potential to environmental stress. The circular plasmid pARUE232 and the linear plasmid pARUE113 contribute to heavy metal resistance and to the ability to degrade quinaldine, respectively.