Involvement of NADH:Acceptor Oxidoreductase and Butyryl Coenzyme A Dehydrogenase in Reversed Electron Transport during Syntrophic Butyrate Oxidation by Syntrophomonas wolfei

Methanogenic oxidation of butyrate to acetate requires a tight cooperation between the syntrophically fermenting Syntrophomonas wolfei and the methanogen Methanospirillum hungatei, and a reversed electron transport system in S. wolfei was postulated to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH for H₂ generation. The metabolic activity of butyrate-oxidizing S. wolfei cells was measured via production of formazan and acetate from butyrate, with 2,3,5-triphenyltetrazolium chloride as electron acceptor. This activity was inhibited by trifluoperazine (TPZ), an antitubercular agent known to inhibit NADH:menaquinone oxidoreductase. In cell extracts of S. wolfei, the oxidation of NADH could be measured with quinones, viologens, and tetrazolium dyes as electron acceptors, and also this activity was inhibited by TPZ. The TPZ-sensitive NADH:acceptor oxidoreductase activity appeared to be membrane associated but could be dissociated from the membrane as a soluble protein and was semipurified by anion-exchange chromatography. Recovered proteins were identified by peptide mass fingerprinting, which indicated the presence of an NADH:acceptor oxidoreductase as part of a three-component [FeFe] hydrogenase complex and a selenocysteine-containing formate dehydrogenase. Furthermore, purification of butyryl-CoA dehydrogenase (Bcd) activity and peptide mass fingerprinting revealed two Bcd proteins different from the Bcd subunit of the Bcd/electron-transfer flavoprotein complex (Bcd/EtfAB) predicted from the genome sequence of S. wolfei. The results suggest that syntrophic oxidation of butyrate in S. wolfei involves a membrane-associated TPZ-sensitive NADH:acceptor oxidoreductase as part of a hydrogenase complex similar to the recently discovered “bifurcating” hydrogenase in Thermotoga maritima and butyryl-CoA dehydrogenases that are different from Bcd of the Bcd/EtfAB complex.Butyrate is fermented to methane and CO₂ by syntrophic communities in which a methanogenic partner organism maintains a low hydrogen partial pressure to allow the oxidation of butyrate to acetate (19, 20, 29). Only under such conditions can butyrate-oxidizing bacteria such as Syntrophomonas wolfei gain energy from the latter reaction in a range of approximately −20 kJ per mol of butyrate, which is just sufficient to support microbial growth (29). It was postulated that S. wolfei has to invest some of the ATP that is formed in the acetate kinase reaction during the β-oxidation of butyrate into an ATP-driven reversed electron transport in order to shift electrons from butyryl coenzyme A (butyryl-CoA) oxidation to the redox potential of NADH (34).Experimental evidence for the involvement of a proton gradient and of ATPase activity in this process was obtained with intact cell suspensions (36), and it was hypothesized that menaquinone-7 could play an essential role in this reaction (36). This would imply that membrane-bound enzymes similar to complex I of the aerobic respiratory chain, i.e., NADH dehydrogenase (NDH), operate in reverse to reduce NAD⁺ with butyrate electrons.Another option for a reversed electron transport during butyrate oxidation and hydrogen formation in S. wolfei could be a reversal of the so-called Buckel-Thauer reaction. In this reaction that was described for ethanol-acetate fermentation by Clostridium kluyveri, electrons from NADH are disproportionated to reduce both crotonyl-CoA and ferredoxin simultaneously. The reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase/electron-transfer flavoprotein (Bcd/EtfAB) complex (13, 18). Very recently, another “bifurcating” electron pathway has been described for an NADH- and ferredoxin-coaccepting di-iron hydrogenase complex in Thermotoga maritima (30). Here, electrons from NADH and from ferredoxin are combined to produce hydrogen, and the genome sequence of S. wolfei has been shown to contain candidate genes for such a three-component hydrogenase complex (30). Nonetheless, the energetic situation of syntrophic butyrate oxidation is basically different from that of ethanol or glucose degradation: electrons arise at comparably positive redox potentials, i.e., at −125 mV/−10 mV (12, 28) and −250 mV, and there is no oxidation step involved that could be coupled directly with ferredoxin reduction.In the present study, we report that butyrate oxidation by S. wolfei cell suspensions can be inhibited by trifluoperazine (TPZ), an antitubercular agent that has been shown to inhibit type II NADH:menaquinone oxidoreductase NDH-2 in Mycobacterium tuberculosis (40), and that a TPZ-sensitive NADH:acceptor oxidoreductase activity can be measured in cell extracts of S. wolfei cells. This enzyme system and a butyryl-CoA dehydrogenase were enriched by anion-exchange chromatography, and the obtained proteins were identified by peptide mass fingerprinting.     

Phospholipid Fatty Acid Composition of the Syntrophic Anaerobic Bacterium Syntrophomonas wolfei

The membrane phospholipid fatty acids (PLFAs) from several cocultures and a pure culture of Syntrophomonas wolfei were determined by capillary column gas chromatography. Cocultures of S. wolfei with a Desulfovibrio sp. contained PLFAs from both organisms, whereas PLFAs from a coculture with Methanospirillum hungatei contained very little biomass to analyze. The pure culture of S. wolfei grown on crotonate provided the best material for analysis of the PLFAs. The predominant PLFAs of S. wolfei were the monounsaturated 16:1ω7c and 16:1ω9c and the saturated 16:0 and 14:0. A low concentration of the diunsaturated 18:2ω6 was detected. The PLFA analysis provides additional information for consideration in the determination of the profile of PLFAs obtained from anaerobic environments. In addition, this information may aid in the understanding of the physiology and phylogeny of S. wolfei and other syntrophic bacteria.     

Separation of Syntrophomonas wolfei from Methanospirillum hungatii in Syntrophic Cocultures by Using Percoll Gradients

Percoll gradient centrifugation effectively separated Syntrophomonas wolfei cells from Methanospirillum hungatii cells, resulting in a 70- to 80-fold enrichment of S. wolfei cells relative to M. hungatii cells. The separated S. wolfei cells were viable. Gram quantities of cellular protein which was enzymatically active and had low levels of contamination by the methanogenic cofactor, factor₄₂₀, were obtained.     

Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H₂-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [¹³C]propionate (<10 mM) and the oxidation of ~30 μmol of ¹³C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were ¹³C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” ¹³C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of ¹³C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [¹³C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Energetics of Syntrophic Propionate Oxidation in Defined Batch and Chemostat Cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H₂-consuming methanogens was <−20 kJ mol of CH₄⁻¹ and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >−10 kJ mol of propionate⁻¹. Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0.07 day⁻¹ and produced maximum biomass yields of 2.6 g mol of propionate⁻¹ and 7.6 g mol of CH₄⁻¹ for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP⁻¹. However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h⁻¹ mol of biomass C⁻¹) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

A Proteomic Investigation of Soluble Olfactory Proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.     

Deciphering Proteomic Signatures of Early Diapause in Nasonia

Insect diapause is an alternative life-history strategy used to increase longevity and survival in harsh environmental conditions. Even though some aspects of diapause are well investigated, broader scale studies that elucidate the global metabolic adjustments required for this remarkable trait, are rare. In order to better understand the metabolic changes during early insect diapause, we used a shotgun proteomics approach on early diapausing and non-diapausing larvae of the recently sequenced hymenopteran model organism Nasonia vitripennis. Our results deliver insights into the molecular underpinnings of diapause in Nasonia and corroborate previously reported diapause-associated features for invertebrates, such as a diapause-dependent abundance change for heat shock and storage proteins. Furthermore, we observed a diapause-dependent switch in enzymes involved in glycerol synthesis and a vastly changed capacity for protein synthesis and degradation. The abundance of structural proteins and proteins involved in protein synthesis decreased with increasing diapause duration, while the abundance of proteins likely involved in diapause maintenance (e.g. ferritins) increased. Only few potentially diapause-specific proteins were identified suggesting that diapause in Nasonia relies to a large extent on a modulation of pre-existing pathways. Studying a diapause syndrome on a proteomic level rather than isolated pathways or physiological networks, has proven to be an efficient and successful avenue to understand molecular mechanisms involved in diapause.     

Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H₂ uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (ΔG′) and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (ΔG′ = −17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (ΔG′ = −3.8 kJ/mol, v = 0.12 mM/h). The ΔG′ value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable ΔG′ resulted from the close coupling of the rates of butyrate and H₂ oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H₂ sink. When the specific activity of H₂ consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H₂ pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H₂ consumption, not the type of H₂ consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V_max.     

The Temperature Dependent Proteomic Analysis of Thermotoga maritima

Thermotoga maritima (T. maritima) is a typical thermophile, and its proteome response to environmental temperature changes has yet to be explored. This study aims to uncover the temperature-dependent proteins of T. maritima using comparative proteomic approach. T. maritima was cultured under four temperatures, 60°C, 70°C, 80°C and 90°C, and the bacterial proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, a total of 224 spots, either cytoplasm or membrane, were defined as temperature-dependent. Of these spots, 75 unique bacterial proteins were identified using MALDI TOF/TOF MS. As is well known, the chaperone proteins such as heat shock protein 60 and elongation factor Tu, were up-regulated in abundance due to increased temperature. However, several temperature-dependent proteins of T. maritima responded very differently when compared to responses of the thermophile T. tengcongensis. Intriguingly, a number of proteins involved in central carbohydrate metabolism were significantly up-regulated at higher temperature. Their corresponding mRNA levels were elevated accordingly. The increase in abundance of several key enzymes indicates that a number of central carbohydrate metabolism pathways of T. maritima are activated at higher temperatures.     

Proteomic Selection of Immunodiagnostic Antigens for Trypanosoma congolense

Animal African Trypanosomosis (AAT) presents a severe problem for agricultural development in sub-Saharan Africa. It is caused by several trypanosome species and current means of diagnosis are expensive and impractical for field use. Our aim was to discover antigens for the detection of antibodies to Trypanosoma congolense, one of the main causative agents of AAT. We took a proteomic approach to identify potential immunodiagnostic parasite protein antigens. One hundred and thirteen proteins were identified which were selectively recognized by infected cattle sera. These were assessed for likelihood of recombinant protein expression in E. coli and fifteen were successfully expressed and assessed for their immunodiagnostic potential by ELISA using pooled pre- and post-infection cattle sera. Three proteins, members of the invariant surface glycoprotein (ISG) family, performed favorably and were then assessed using individual cattle sera. One antigen, Tc38630, evaluated blind with 77 randomized cattle sera in an ELISA assay gave sensitivity and specificity performances of 87.2% and 97.4%, respectively. Cattle immunoreactivity to this antigen diminished significantly following drug-cure, a feature helpful for monitoring the efficacy of drug treatment.     

Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.Smith-Lemli-Opitz syndrome (SLOS)¹ is an autosomal recessive disorder associated with subnormal growth and failure to thrive, mental retardation and neurosensory deficits, and multiple congenital anomalies, including dysmorphologies (1, 2). Early epidemiological studies estimated the incidence of SLOS as 1 in 20,000 to 1 in 60,000 live births, primarily among Caucasians (1, 2). However, more recent studies suggest that the SLOS carrier frequency is ∼1 in 30 to 1 in 50; this predicts a much higher actual disease frequency, ranging from 1 in 1,590 to 1 in 17,000 (3, 4), making SLOS the fourth most common autosomal recessive human disease (after cystic fibrosis, phenylketonuria, and hemochromatosis). Mutation of the DHCR7 gene is the intrinsic cause of SLOS; this gene encodes the enzyme DHCR7 (3β-hydroxysterol-Δ7-reductase, a.k.a. 7-dehydrocholesterol reductase; EC1.3.1.21), which catalyzes the final step in the cholesterol biosynthetic pathway, reducing the Δ7 double bond and thus converting 7-dehydrocholesterol (7DHC) to cholesterol (4, 5). As a consequence, markedly reduced levels of cholesterol and aberrantly elevated levels of the cholesterol precursor 7DHC (and its epimer, 8DHC) are observed in the majority of affected SLOS patients (6, 7). Therefore, the clinical suspicion of SLOS is confirmed by elevated 7DHC in plasma or tissues, typically demonstrated via chromatographic methods (e.g. HPLC or GC/MS) (8, 9).Visual capacity may become compromised in SLOS patients because of a variety of congenital or postnatal pathologies, such as cataracts, aniridia, corneal endothelium defects, sclerocornea, electrophysiological defects in the retina, optic nerve abnormalities, or other ophthalmologic problems (10, 11). We currently lack full knowledge of the exact pathobiological mechanism underlying SLOS, but additional insights may be afforded by studies employing a rodent model of the disease in which rats are treated with AY9944 (trans-1,4-bis[2-chlorobenzylaminomethyl] cyclohexane dihydrochloride), a relatively selective inhibitor of DHCR7 (12–14). We previously described progressive retinal degeneration in this rat model of SLOS, which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina (which contains the photoreceptor nuclei), and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) (12, 13). Reduced rod outer segment membrane fluidity, primarily caused by a dramatic (30 to 40 mol%) decline in docosahexaenoic acid (22:6, n3) levels relative to age-matched controls, also was observed in the SLOS rat model by three postnatal months (15, 16). Retinal function and sterol steady-state in the same rat model of SLOS can be partially rescued using a high-cholesterol diet (2% by weight), although histological degeneration of the retina still occurs (17). However, the molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. Therefore, a comprehensive profiling of the retinal proteomes of AY9944-treated versus age-matched untreated control rats may contribute to further understanding of the underlying mechanisms responsible for the retinopathy associated with the SLOS model and, by extension, the human disease.Nevertheless, extensive and reliable expression profiling of the retinal proteome remains a prominent challenge, owing to the need to quantify data from multiple animals and a high percentage of integral membrane and membrane-associated proteins (18, 19). Label-free approaches can compare multiple replicates (20–22) with quantitative accuracy comparable to that attained with stable isotope-labeling methods (23–25). However, in order to achieve reliable relative quantification, highly quantitative and reproducible sample preparation and LC/MS analysis are required for relatively large-scale sample cohorts.In the present study, we performed a reproducible, well-controlled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n = 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples then underwent a reproducible precipitation/on-pellet-digestion procedure and long-column, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with 10 animals. The preparative and analytical procedures were carefully optimized and controlled to ensure optimal reproducibility. Two label-free approaches, the ion-current-based method and a spectral counting method, were compared in parallel. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis.