Genome Sequence of Uric Acid-Fermenting Eubacterium angustum DSM 1989T (MK-1).

Eubacterium angustum DSM 1989T (MK-1) is a strictly anaerobic and uric acid-, xanthine-, and guanine-fermenting organism isolated from sewage sludge. The draft genome consists of one circular chromosome (2.4 Mb) and harbors 2,397 predicted protein-encoding genes.

The Complete Genome Sequence of Clostridium aceticum: a Missing Link between Rnf- and Cytochrome-Containing Autotrophic Acetogens.

UNLABELLED: Clostridium aceticum was the first isolated autotrophic acetogen, converting CO2 plus H2 or syngas to acetate. Its genome has now been completely sequenced and consists of a 4.2-Mbp chromosome and a small circular plasmid of 5.7 kbp. Sequence analysis revealed major differences from other autotrophic acetogens. C. aceticum contains an Rnf complex for energy conservation (via pumping protons or sodium ions). Such systems have also been found in C. ljungdahlii and Acetobacterium woodii. However, C. aceticum also contains a cytochrome, as does Moorella thermoacetica, which has been proposed to be involved in the generation of a proton gradient. Thus, C. aceticum seems to represent a link between Rnf- and cytochrome-containing autotrophic acetogens. In C. aceticum, however, the cytochrome is probably not involved in an electron transport chain that leads to proton translocation, as no genes for quinone biosynthesis are present in the genome. IMPORTANCE: Autotrophic acetogenic bacteria are receiving more and more industrial focus, as CO2 plus H2 as well as syngas are interesting new substrates for biotechnological processes. They are both cheap and abundant, and their use, if it results in sustainable products, also leads to reduction of greenhouse gases. Clostridium aceticum can use both gas mixtures, is phylogenetically not closely related to the commonly used species, and may thus become an even more attractive workhorse. In addition, its energy metabolism, which is characterized here, and the ability to synthesize cytochromes might offer new targets for improving the ATP yield by metabolic engineering and thus allow use of C. aceticum for production of compounds by pathways that currently present challenges for energy-limited acetogens.

Complete Genome Sequence of Amino Acid-Utilizing Eubacterium acidaminophilum al-2 (DSM 3953).

Eubacterium acidaminophilum is a strictly anaerobic, Gram-positive, rod-shaped bacterium which belongs to cluster XI of the Clostridia. It ferments amino acids by a Stickland reaction. The genome harbors a chromosome (2.25 Mb) and a megaplasmid (0.8 Mb). It contains several gene clusters coding for selenocysteine-containing, glycine-derived, and amino acid-degrading reductases.

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

BACKGROUND: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed. RESULTS: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms. CONCLUSIONS: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.

Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

The transcription of reductive dehalogenase homologous (rdh) genes of "Dehalococcoides" sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3- and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of the transcription of 29 rdhA genes was indicated in response to 1,2,3- and 1,2,4-TCB added after a substrate depletion period of 72 h. The transcription of the remaining three rdhA genes additionally was detected using specific primers. While most rdhA genes were upregulated similarly in cultures after induction with 1,2,3-TCB or 1,2,4-TCB, three rdhA genes responded differentially to 1,2,3- and 1,2,4-TCB, as revealed by the comparison of t-RFLP profiles. The enhanced transcription of cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TCB, while the transcription of cbdbA1624 was strongly induced by 1,2,4-TCB. Comparison of t-RFLP profiles obtained from cDNA and genomic DNA indicated a particularly high induction of the transcription of cbrA (=cbdbA84) by both TCBs. As indicated by reverse transcription-quantitative PCR, the transcription of these plus two other rdhA genes (cbdbA1588 and cbdbA1618) increased within 48 to 72 h by one or two orders of magnitude. Subsequently, transcript levels slowly decreased and approached initial transcript levels several days after complete dehalogenation. Finally, cbrA was transcribed to a level of 22 transcripts per cbrA gene, suggesting that cbrA mRNA could be an appropriate biomarker for the investigation of the natural dechlorination potential at chlorobenzene-contaminated sites.

Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase cultures.

Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

Factors and selenocysteine insertion sequence requirements for the synthesis of selenoproteins from a gram-positive anaerobe in Escherichia coli.

Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of (75)Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNA(Sec)) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of (75)Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.

Tungsten, the surprisingly positively acting heavy metal element for prokaryotes.

The history and changing function of tungsten as the heaviest element in biological systems is given. It starts from an inhibitory element/anion, especially for the iron molybdenum-cofactor (FeMoCo)-containing enzyme nitrogenase involved in dinitrogen fixation, as well as for the many "metal binding pterin" (MPT)-, also known as tricyclic pyranopterin- containing classic molybdoenzymes, such as the sulfite oxidase and the xanthine dehydrogenase family of enzymes. They are generally involved in the transformation of a variety of carbon-, nitrogen- and sulfur-containing compounds. But tungstate can serve as a potential positively acting element for some enzymes of the dimethyl sulfoxide (DMSO) reductase family, especially for CO(2)-reducing formate dehydrogenases (FDHs), formylmethanofuran dehydrogenases and acetylene hydratase (catalyzing only an addition of water, but no redox reaction). Tungsten even becomes an essential element for nearly all enzymes of the aldehyde oxidoreductase (AOR) family. Due to the close chemical and physical similarities between molybdate and tungstate, the latter was thought to be only unselectively cotransported or cometabolized with other tetrahedral anions, such as molybdate and also sulfate. However, it has now become clear that it can also be very selectively transported compared to molybdate into some prokaryotic cells by two very selective ABC-type of transporters that contain a binding protein TupA or WtpA. Both proteins exhibit an extremely high affinity for tungstate (K(D) < 1 nM) and can even discriminate between tungstate and molybdate. By that process, tungsten finally becomes selectively incorporated into the few enzymes noted above.

Peroxidase activity of selenoprotein GrdB of glycine reductase and stabilisation of its integrity by components of proprotein GrdE from Eubacterium acidaminophilum.

The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that organism. We purified this peroxidase and found that it was identical with the substrate-specific "protein B"-complex of glycine reductase. The "protein B"-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)(-1) with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT- and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted for a peroxidase activity of about 0.5 U (mg protein)(-1) in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.

Pseudonocardia tetrahydrofuranoxydans sp. nov.

A Gram-positive, rod-shaped, non-endospore-forming but mycelium-forming actinobacterium (strain K1(T)) was isolated from an enrichment culture containing tetrahydrofuran (THF) as the sole source of carbon. On the basis of its G+C content (71.3 mol%) and of 16S rRNA gene sequence similarity studies, strain K1(T) was shown to belong to the family Pseudonocardiaceae, most closely related to Pseudonocardia hydrocarbonoxydans (99.3 %), P. benzenivorans (98.8 %) and P. sulfidoxydans (98.3 %). The 16S rRNA gene sequence similarity to other Pseudonocardia species was less than 97 %. Chemotaxonomic data [major menaquinone MK-8(H(4)); major fatty acids C(16 : 0) iso, C(15 : 0) iso and C(17 : 1)omega6c] supported the affiliation of strain K1(T) to the genus Pseudonocardia. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain K1(T) from the three species P. benzenivorans, P. sulfidoxydans and P. hydrocarbonoxydans, although all four organisms utilized THF. Strain K1(T) represents a novel species, for which the name Pseudonocardia tetrahydrofuranoxydans sp. nov. is proposed, with the type strain K1(T) (=DSM 44239(T)=CIP 109050(T)).

Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system.

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.

Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5.

The P450mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fdmor) and cytochrome P450 (P450mor). In this study, we purified Fdmor and P450mor as recombinant proteins as well as FdRmor, which has been isolated previously. Kinetic investigations of the redox couple FdRmor/Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fdmor in this reaction, compared with the NADH-dependent reduction of cytochrome c. The quite low Km (5.3 +/- 0.3 nm) of FdRmor for Fdmor, measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form, which has previously not been possible for wild-type P450mor. The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the Kd values determined for these substances suggested that P450mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdRmor, Fdmor and P450mor were used to reconstitute the homologous P450-containing mono-oxygenase, which was shown to convert morpholine.

Glycine reductase mechanism.

The ability of some anaerobic bacteria to conserve energy via a soluble substrate level phosphorylation system by reducing glycine to acetyl-phosphate has been an intriguing mechanism for about half a century. The genes implicated in this system have been sequenced and form an operon structure with those of the thioredoxin system. The deduced proteins exhibit high degrees of similarity with glycine reductase from other bacteria. Faster progress in understanding the exact mechanisms is hampered, for example, by some unique reactions involving selenoethers and redox active selenocysteines, which do not allow an easy heterologous formation in Escherichia coli. Further major obstacles are the processing of a substrate-specific pro-protein to a new carbonyl/pyruvoyl group in one of the two peptides formed that stabilize the substrate-binding selenoprotein, which contains an additional rather unstable carbonyl group.

Tungsten-containing aldehyde oxidoreductase of Eubacterium acidaminophilum.

Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic conditions using a four-step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 +/- 0.1 iron, 1.1 +/- 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetaldehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N-terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a sigma 54-dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to sigma 54-dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression.

Properties of a trichlorodibenzo-p-dioxin-dechlorinating mixed culture with a Dehalococcoides as putative dechlorinating species.

An anaerobic mixed culture enriched over 16 transfers (1/10) from Saale river sediment reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) to di- and monochlorinated congeners in the presence of pyruvate (or lactate) and fumarate as cosubstrates. Besides TrCDD, tetrachloroethene and 1,2,3-trichlorobenzene were dechlorinated. Dioxin dehalogenation was sensitive to pasteurization, but was not remarkably influenced by inhibitors of methanogens, sulfate-reducing bacteria or Gram-positive bacteria. The rate of 1,3-dichlorodibenzo-p-dioxin formation increased with rising initial concentrations of 1,2,4-TrCDD (1-250 microM) from 0.05 to 5.4 micromol l(-1) day(-1). Two isolates, belonging to Sulfurospirillum and Trichococcus, did not show reductive dehalogenation. 16S rDNA-targeted methods further revealed the presence of Acetobacterium, Desulfitobacterium, Desulfuromonas and Dehalococcoides. Nested polymerase chain reaction (PCR) indicated the presence of Dehalococcoides in highest most probable number (MPN) dilutions that were positive for dioxin dechlorination.

Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum.

The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization. mop encodes a 69-amino-acid protein ( M(r) 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis. Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop. This result was obtained independent of the availability of tungstate in the growth medium. mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein. On the basis of gel filtration, the native protein was a homohexamer of 48 kDa. The specificity of oxyanion binding was examined by protein mobility shift assay. Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop. Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility. Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding.

Structural studies on a twin-arginine signal sequence.

Translocation of folded proteins across biological membranes can be mediated by the so-called 'twin-arginine translocation' (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.

Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1.

A gene cluster involved in the utilization of tetrahydrofuran by Pseudonocardia sp. strain K1 was cloned and sequenced. Analysis of a 9.2-kb DNA fragment revealed eight ORFs. The genes designated as thmADBC encode the components of a putative monooxygenase exhibiting a high similarity to different binuclear-iron-containing multicomponent monooxygenases. thmA encodes the derived 545-amino-acid oxygenase alpha-subunit, thmD the 360-amino-acid reductase component, thmB the 346-amino-acid oxygenase beta-subunit, and thmC the 117-amino-acid coupling protein. Upstream of the thm genes, an additional ORF ( sad) was identified coding for a protein with high similarity to various aldehyde dehydrogenases. A succinate semialdehyde dehydrogenase activity was specifically expressed in tetrahydrofuran-grown cells. N-terminal sequence analysis of the purified protein revealed that it is encoded by sad. Northern blot analysis indicated that transcription of the thm genes and sad was specifically induced during growth on tetrahydrofuran. Mono-, di- and polycistronic transcripts of these genes were detected. Primer-extension analysis identified transcriptional start sites 37, 61, and 41 bp upstream of the translation start of sad, thmA, and thmB, respectively. Additional ORFs were identified upstream ( orfY) and downstream ( orfZ and aldH) of the thm genes. Furthermore, the data indicated that the analyzed gene cluster was present as a single copy and located on a plasmid.

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs and PCDFs) are among the most notorious environmental pollutants. Some congeners, particularly those with lateral chlorine substitutions at positions 2, 3, 7 and 8, are extremely toxic and carcinogenic to humans. One particularly promising mechanism for the detoxification of PCDDs and PCDFs is microbial reductive dechlorination. So far only a limited number of phylogenetically diverse anaerobic bacteria have been found that couple the reductive dehalogenation of chlorinated compounds--the substitution of a chlorine for a hydrogen atom--to energy conservation and growth in a process called dehalorespiration. Microbial dechlorination of PCDDs occurs in sediments and anaerobic mixed cultures from sediments, but the responsible organisms have not yet been identified or isolated. Here we show the presence of a Dehalococcoides species in four dioxin-dechlorinating enrichment cultures from a freshwater sediment highly contaminated with PCDDs and PCDFs. We also show that the previously described chlorobenzene-dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 (ref. 3) is able to reductively dechlorinate selected dioxin congeners. Reductive dechlorination of 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) demonstrates that environmentally significant dioxins are attacked by this bacterium.