SummaryThis paper describes the genome sequence of M. thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the WoodLjungdahl pathway of CO and CO 2 fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into three mol of acetate and to grow autotrophically using H 2 and CO as electron donors and CO 2 as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO 2 . Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols, and aromatic compounds) and electron acceptors (CO 2 , nitrate, nitrite, thiosulfate, dimethylsulfoxide, and aromatic carboxyl groups). The genome consists of a single circular 2628784 bp chromosome encoding 2615 open reading frames, which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. Two thousand three hundred eighty-four (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in ortholog clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.

A five-gene "oxidative stress protection" cluster has recently been described from the strictly anaerobic, acetogenic bacterium, Moorella thermoacetica [Das, A., et al. (2001) J. Bacteriol. 183, 1560-1567]. Within this cluster are two cotranscribed genes, fprA (for A-type flavoprotein) and hrb (for high molecular weight rubredoxin) whose encoded proteins have no known functions. Here we show that FprA and Hrb are expressed in M. thermoacetica under normal anaerobic growth conditions and report characterizations of the recombinant FprA and Hrb. FprA contains flavin mononucleotide (FMN) and a non-heme diiron site. Mössbauer spectroscopy shows that the irons of the diferric site are antiferromagnetically coupled, implying a single-atom, presumably solvent, bridge between the irons. Hrb contains FMN and a rubredoxin-like [Fe(SCys)4] site. NADH does not directly reduce either the FMN or the diiron site in FprA, whereas Hrb functions as an efficient NADH:FprA oxidoreductase. Substitution of zinc for iron in Hrb completely abolished this activity. The observation that homologues of FprA from other organisms show O2 and/or anaerobic NO consumption activity prompted an examination of these activities for M. thermoacetica FprA. The Hrb/FprA combination does indeed have both NADH:O2 and NADH:NO oxidoreductase activities. The NO reductase activity, however, was significantly more efficient due to a lower Km for NO (4 M) and to progressive and irreversible inactivation of FprA during O2 reductase turnover but retention of activity during NO reductase turnover. Substitution of zinc for iron in FprA completely abolished these reductase activities. The stoichiometry of 1 mol of NADH oxidized:2 mol of NO consumed implies reduction to N2O. Fits of an appropriate rate law to the kinetics data are consistent with a mechanism in which 2NO's react at each FprA active site in the committed step. Expression of FprA in an Escherichia coli strain deficient in NO reductase restored the anaerobic growth phenotype of cultures exposed to otherwise toxic levels of exogenous NO. The accumulated results indicate that Hrb/FprA is fully capable of functioning in nitrosative stress protection in M. thermoacetica.

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The gram-positive, thermophilic, acetogenic bacterium Moorella thermoacetica can reduce CO 2 to acetate via the Wood-Ljungdahl (acetyl coenzyme A synthesis) pathway. This report demonstrates that, despite its classification as a strict anaerobe, M. thermoacetica contains a membrane-bound cytochrome bd oxidase that can catalyze reduction of low levels of dioxygen. Whole-cell suspensions of M. thermoacetica had significant endogenous O 2 uptake activity, and this activity was increased in the presence of methanol or CO, which are substrates in the Wood-Ljungdahl pathway. Cyanide and azide strongly (ϳ70%) inhibited both the endogenous and CO/methanol-dependent O 2 uptake. UV-visible light absorption and electron paramagnetic resonance spectra of n-dodecyl-␤-maltoside extracts of M. thermoacetica membranes showed the presence of a cytochrome bd oxidase complex containing cytochrome b 561 , cytochrome b 595 , and cytochrome d (chlorin). Subunits I and II of the bd oxidase were identified by N-terminal amino acid sequencing. The M. thermoacetica cytochrome bd oxidase exhibited cyanide-sensitive quinol oxidase activity. The M. thermoacetica cytochrome bd (cyd) operon consists of four genes, encoding subunits I and II along with two ABC-type transporter proteins, homologs of which in other bacteria are required for assembly of the bd complex. The level of this cyd operon transcript was significantly increased when M. thermoacetica was grown in the absence of added reducing agent (cysteine ؉ H 2 S). Expression of a 35-kDa cytosolic protein, identified as a cysteine synthase (CysK), was also induced by the nonreducing growth conditions. The combined evidence indicates that cytochrome bd oxidase and cysteine synthase protect against oxidative stress and contribute to the limited dioxygen tolerance of M. thermoacetica.Dioxygen is the preferred electron sink in the respiratory chain of most aerobic bacteria (58). Cytochrome oxidases are the membrane-bound, terminal components of the dioxygendependent respiratory chain, which reduces dioxygen to water with formation of pH and potential gradients (6, 45). Two main types of respiratory cytochrome oxidases are known in bacteria: the heme/copper oxidases (cytochrome aa 3 , cytochrome caa 3 oxidase, and cytochrome bo oxidase), and the heme-only cytochrome bd quinol oxidase, which is associated with microaerobic dioxygen respiration (24,56,59,62). Cytochrome bd oxidases purified from aerobic bacteria have been characterized as 1:1 heterodimers of two integral membrane proteins referred to as subunits I and II (35,55). The heterodimers contain three heme components: a low-spin heme, b 558 , and two high-spin hemes, b 595 and d. In the genomes of Escherichia coli (31) and Azotobacter vinelandii (41), subunits I and II are encoded by cydA and cydB, respectively, of the cytochrome bd (cyd) operon. Two ABC-type transporter proteins, CydD and CydC, are typically required for assembly of the cytochrome bd complex in both gram-positive and gram-negative aerobic bacteria (14, 60). Aerobic r...

For establishment of the structure-activity relationship, 19 heterobicycle-coumarin conjugated compounds with the -SCH(2)- linker were synthesized and found to possess significant antiviral activities. Prominent examples included imidazopyridine-coumarin 12c, purine-coumarin 12d, and benzoxazole-coumarin 14c, which inhibited HCV replication at an EC(50) of 6.8, 2.0, and 12 microM, respectively. The heteroatoms in bicycles and the substituent effect on coumarin played essential roles.

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Abstract. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) is routinely used for the rapid detection of Avian influenza virus (AIV) in clinical samples, but inhibitory substances present in some clinical specimens can reduce or block PCR amplification. Most commercial RNA extraction kits have limited capacity to remove inhibitors from clinical samples, but using a modified commercial protocol (AmbionH MagMAX TM , Applied Biosystems, Foster City, CA) with an added high-salt wash of 2 M NaCl and 2 mM ethylenediamine tetra-acetic acid was shown to improve the ability of the kit to remove inhibitors from cloacal swabs and some tissues. Real-time RT-PCR was carried out in the presence of an internal positive control to detect inhibitors present in the purified RNA. Cloacal swabs from wild birds were analyzed by realtime RT-PCR comparing RNA extracted with the standard (MagMAX-S) and modified (MagMAX-M) protocols. Using the standard protocol on 2,668 samples, 18.4% of the samples had evidence of inhibitor(s) in the samples, but the modified protocol removed inhibitors from all but 21 (4.8%) of the problem samples. The modified protocol was also tested for RNA extraction from tissues using a TRIzol-MagMAX-M hybrid protocol. Tissues from chickens and ducks experimentally infected with high-pathogenicity Asian H5N1 AIV were analyzed by real-time RT-PCR, and the limit of detection of the virus was improved by 0.5-3.0 threshold cycle units with the RNA extracted by the MagMAX-M protocol. The MagMAX-M protocol reported in the present study can be useful in extracting high-quality RNA for accurate detection of AIV from cloacal swabs and tissues by real-time RT-PCR.

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.Several studies indicate that anaerobic and microaerophilic bacteria can tolerate varying degrees of O 2 exposure. Superoxide dismutase (SOD) and catalase, which are known to relieve oxidative stress in aerobes, are often absent in anaerobic bacteria. Recently, nonheme iron proteins such as rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo) (also known as desulfoferrodoxin) have been implicated in oxidative stress protection in anaerobes (1,23,27,33,42). So far, Rbr and Rbo or their genes have been found only in anaerobic or microaerophilic bacteria and archaea. The active sites of these proteins include a rubredoxin-type [Fe(SCys) 4 ] center in both Rbo and Rbr (3,5,11,12,22,23,35), a mononuclear [Fe(NHis) 4 SCys] center in Rbo (6), and a nonsulfur, oxo-bridged di-iron center in Rbr (8,17,19,23,34). The latter two sites in their reduced forms react rapidly with superoxide in the case of Rbo (27) and with hydrogen peroxide in the case of Rbr (7,8).Clostridium thermoaceticum is a thermophilic gram-positive, obligately anaerobic bacterium that produces acetate from virtually any carbon source, including sugars, aromatic compounds, and C 1 compounds (25,36,44). It is not known how this bacterium responds to oxygen toxicity during growth. Determinations of catalase and SOD activities in C. thermoaceticum have been inconclusive. In this study we show that C. thermoaceticum contains genes encoding Rbo and Rbr and that these two genes are present in a cluster with three additional genes encoding a rubredoxin (Rub), a high-molecular-weight rubredoxin (Hrb) and a type A flavoprotein (FprA). All these genes have been expressed in Escherichia coli, and recombinant Rbr, Rub, and FprA were purified...