Housna Mouttaki

Physiology, Ecology, Phylogeny, and Genomics of Microorganisms Capable of Syntrophic Metabolism

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram‐positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

The genome of Syntrophus aciditrophicus: Life at the thermodynamic limit of microbial growth

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

Anaerobic degradation of non-substituted aromatic hydrocarbons

Aromatic hydrocarbons are among the most prevalent organic pollutants in the environment. Their removal from contaminated systems is of great concern because of the high toxicity effect on living organisms including humans. Aerobic degradation of aromatic hydrocarbons has been intensively studied and is well understood. However, many aromatics end up in habitats devoid of molecular oxygen. Nevertheless, anaerobic degradation using alternative electron acceptors is much less investigated. Here, we review the recent literature and very early progress in the elucidation of anaerobic degradation of non-substituted monocyclic (i.e. benzene) and polycyclic aromatic hydrocarbons (PAH such as naphthalene and phenanthrene). A focus will be on benzene and naphthalene as model compounds. This review concerns the microbes involved, the biochemistry of the initial activation and subsequent enzyme reactions involved in the pathway.

Cyclohexane Carboxylate and Benzoate Formation from Crotonate in Syntrophus aciditrophicus

ABSTRACT The anaerobic, syntrophic bacterium Syntrophus aciditrophicus grown in pure culture produced 1.4 ± 0.24 mol of acetate and 0.16 ± 0.02 mol of cyclohexane carboxylate per mole of crotonate metabolized. [U-13C]crotonate was metabolized to [1,2-13C]acetate and [1,2,3,4,5,7-13C]cyclohexane carboxylate. Cultures grown with unlabeled crotonate and [13C]sodium bicarbonate formed [6-13C]cyclohexane carboxylate. Trimethylsilyl (TMS) derivatives of cyclohexane carboxylate, cyclohex-1-ene carboxylate, benzoate, pimelate, glutarate, 3-hydroxybutyrate, and acetoacetate were detected as intermediates by comparison of retention times and mass spectral profiles to authentic standards. With [U-13C]crotonate, the m/z-15 ion of TMS-derivatized glutarate, 3-hydroxybutyrate, and acetoacetate each increased by +4 mass units, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +6 mass units. With [13C]sodium bicarbonate and unlabeled crotonate, the m/z-15 ion of TMS derivatives of glutarate, pimelate, cyclohex-1-ene carboxylate, benzoate, and cyclohexane carboxylate each increased by +1 mass unit, suggesting that carboxylation occurred after the synthesis of a four-carbon intermediate. With [1,2-13C]acetate and unlabeled crotonate, the m/z-15 ion of TMS-derivatized 3-hydroxybutyrate, acetoacetate, and glutarate each increased by +0, +2, and +4 mass units, respectively, and the m/z-15 ion of TMS-derivatized pimelate, cyclohex-1-ene carboxylate, benzoate, cyclohexane carboxylate, and 2-hydroxycyclohexane carboxylate each increased by +0, +2, +4, and +6 mass units. The data are consistent with a pathway for cyclohexane carboxylate formation involving the condensation of two-carbon units derived from crotonate degradation with CO2 addition, rather than the use of the intact four-carbon skeleton of crotonate.

Sequencing of Multiple Clostridial Genomes Related to Biomass Conversion and Biofuel Production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.

Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. Strain X514 via Isotopomer-Assisted Metabolite Analysis

ABSTRACT Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C5 and C6 sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via 13C-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining 13C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities.

Anaerobic Degradation of Benzene and Polycyclic Aromatic Hydrocarbons

Aromatic hydrocarbons such as benzene and polycyclic aromatic hydrocarbons (PAHs) are very slowly degraded without molecular oxygen. Here, we review the recent advances in the elucidation of the first known degradation pathways of these environmental hazards. Anaerobic degradation of benzene and PAHs has been successfully documented in the environment by metabolite analysis, compound-specific isotope analysis and microcosm studies. Subsequently, also enrichments and pure cultures were obtained that anaerobically degrade benzene, naphthalene or methylnaphthalene, and even phenanthrene, the largest PAH currently known to be degradable under anoxic conditions. Although such cultures grow very slowly, with doubling times of around 2 weeks, and produce only very little biomass in batch cultures, successful proteogenomic, transcriptomic and biochemical studies revealed novel degradation pathways with exciting biochemical reactions such as for example the carboxylation of naphthalene or the ATP-independent reduction of naphthoyl-coenzyme A. The elucidation of the first anaerobic degradation pathways of naphthalene and methylnaphthalene at the genetic and biochemical level now opens the door to studying the anaerobic metabolism and ecology of anaerobic PAH degraders. This will contribute to assessing the fate of one of the most important contaminant classes in anoxic sediments and aquifers.

Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non-substituted aromatic hydrocarbons

Polycyclic aromatic hydrocarbons such as naphthalene are recalcitrant environmental pollutants that are only slowly metabolized by bacteria under anoxic conditions. Based on metabolite analyses of culture supernatants, carboxylation or methylation of naphthalene have been proposed as initial enzymatic activation reactions in the pathway. However, the extremely slow growth of anaerobic naphthalene degraders with doubling times of weeks and the little biomass obtained from such cultures hindered the biochemical elucidation of the initial activation reaction, so far. Here, we provide biochemical evidence that anaerobic naphthalene degradation is initiated via carboxylation. Crude cell extracts of the sulfate-reducing enrichment culture N47 converted naphthalene and (13)C-labelled bicarbonate to 2-[carboxyl-(13)C]naphthoic acid at a rate of 0.12 nmol min(-1) mg protein(-1) . The enzyme, namely naphthalene carboxylase, catalysed a much faster exchange of (13) C-labelled bicarbonate with the carboxyl group of 2-[carboxyl-(12)C]naphthoic acid at a rate of 3.2 nmol min(-1) mg protein(-1), indicating that the rate limiting step of the carboxylation reaction is the activation of the naphthalene molecule rather than the carboxylation itself. Neither the carboxylation nor the exchange reaction activities necessitate the presence of ATP or divalent metal ions and they were not inhibited by avidin or EDTA. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.

Correlation of genomic and physiological traits of Thermoanaerobacter species with biofuel yields.

ABSTRACT Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B12 biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.

ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.