A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.     

Promoted electron transfer of mitoxantrone binding with DNA by cytochrome c

A promoted electron transfer of an antitumor drug, mitoxantrone (MTX), intercalating into DNA duplex was successfully obtained upon addition of cytochromes c (cyt. c) in NaAc-HAc buffer solution (pH 4.5). The experimental results suggested that co-existence of MTX and cyt. c in the DNA helix is an important factor for accelerated electron transfer of MTX, where the promoter, cyt. c, operated smoothly through the DNA bridge. The UV/Vis spectroscopic experiments further confirmed the interaction process. Furthermore, a possible mechanism of such reaction was also discussed in this paper.     

Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus

The purine nucleoside phosphorylase from Thermus thermophilus crystallized in space group P4(3)2(1)2 with the unit cell dimensions a = 131.9 A and c = 169.9 A and one biologically active hexamer in the asymmetric unit. The structure was solved by the molecular replacement method and refined at a 1.9A resolution to an r(free) value of 20.8%. The crystals of the binary complex with sulfate ion and ternary complexes with sulfate and adenosine or guanosine were also prepared and their crystal structures were refined at 2.1A, 2.4A and 2.4A, respectively. The overall structure of the T.thermophilus enzyme is similar to the structures of hexameric enzymes from Escherichia coli and Sulfolobus solfataricus, but significant differences are observed in the purine base recognition site. A base recognizing aspartic acid, which is conserved among the hexameric purine nucleoside phosphorylases, is Asn204 in the T.thermophilus enzyme, which is reminiscent of the base recognizing asparagine in trimeric purine nucleoside phosphorylases. Isothermal titration calorimetry measurements indicate that both adenosine and guanosine bind the enzyme with nearly similar affinity. However, the functional assays show that as in trimeric PNPs, only the guanosine is a true substrate of the T.thermophilus enzyme. In the case of adenosine recognition, the Asn204 forms hydrogen bonds with N6 and N7 of the base. While in the case of guanosine recognition, the Asn204 is slightly shifted together with the beta(9)alpha(7) loop and predisposed to hydrogen bond formation with O6 of the base in the transition state. The obtained experimental data suggest that the catalytic properties of the T.thermophilus enzyme are reminiscent of the trimeric rather than hexameric purine nucleoside phosphorylases.     

Electron flow through metalloproteins

Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.     

Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Structure of Thermus thermophilus 2-Keto-3-deoxygluconate kinase: evidence for recognition of an open chain substrate

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate (KDGP). The genome sequence of Thermus thermophilus HB8 contains an open reading frame that has a 30% identity to Escherichia coli KDGK. The KDGK activity of T.thermophilus protein (TtKDGK) has been confirmed, and its crystal structure has been determined by the molecular replacement method and refined with two crystal forms to 2.3 angstroms and 3.2 angstroms, respectively. The enzyme is a hexamer organized as a trimer of dimers. Each subunit is composed of two domains, a larger alpha/beta domain and a smaller beta-sheet domain, similar to that of ribokinase and adenosine kinase, members of the PfkB family of carbohydrate kinases. Furthermore, the TtKDGK structure with its KDG and ATP analogue was determined and refined at 2.1 angstroms. The bound KDG was observed predominantly as an open chain structure. The positioning of ligands and the conservation of important catalytic residues suggest that the reaction mechanism is likely to be similar to that of other members of the PfkB family, including ribokinase. In particular, the Asp251 is postulated to have a role in transferring the gamma-phosphate of ATP to the 5'-hydroxyl group of KDG.     

Electron transfer from cytochrome c to cupredoxins

Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (ΔG ⁰) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k _obs). The influence of the ΔG ⁰ value for ET from PA to PC or RC on the k _obs value was examined using a series of designed PA proteins exhibiting a variety of E _m values, which afford the ΔG ⁰ variation range of 58–399 meV. The plots of the k _obs values obtained against the ΔG ⁰ values for both PA–PC and PA–RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E _m value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reaction of mercurials with the CuA center in the soluble fragment of cytochrome ba 3 subunit II from Thermus thermophilus

 Optical absorption, EPR and electrospray ionization mass spectrometries were used to characterize a stoichiometric reaction between mercurials and the soluble ba ₃–Cu_A protein from Thermus thermophilus. Either one Hg(II) or two RHg(II)ions react(s) to destroy the unique spectral properties of the Cu_A center. EPR spectra of the resulting product indicate that one Cu from the binuclear Cu_A center is released into the medium as a Type 2 Cu(II) while the other remains EPR silent. Mass spectra indicate that either one Hg(II) or two RHg(II) ions remain(s) bound to the protein along with one Cu, which is assumed to be a Cu(I) ion. The latter is slowly released from the protein under aerobic conditions as additional Type 2 Cu(II), and this process is catalyzed by fungal laccase, which serves as a strong one-electron oxidant. Received: 23 July 1996 / Accepted: 27 September 1996     

Crystal structure of a lysine biosynthesis enzyme,LysX, from Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the alpha-aminoadipate pathway, which uses alpha-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of alpha-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0A and 2.38A resolutions, respectively. LysX is composed of three alpha+beta domains, each composed of a four to five-stranded beta-sheet core flanked by alpha-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as D-Ala:D-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity.     

The effects of multiple ancestral residues on the Thermus thermophilus 3-isopropylmalate dehydrogenase

Previously, we showed that mutants of Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) each containing a residue (ancestral residue) that had been predicted to exist in a postulated common ancestor protein often have greater thermal stabilities than does the contemporary wild-type enzyme. In this study, the combined effects of multiple ancestral residues were analyzed. Two mutants, containing multiple mutations, Sup3mut (Val181Thr/Pro324Thr/Ala335Glu) and Sup4mut (Leu134Asn/Val181Thr/Pro324Thr/Ala335Glu) were constructed and show greater thermal stabilities than the wild-type and single-point mutant IPMDHs do. Most of the mutants have similar or improved catalytic efficiencies at 70 degrees C when compared with the wild-type IPMDH.     

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Background

In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.

Results

We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.

Conclusions

We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.     

Molecular characterization of a novel thermostable mannose-6-phosphate isomerase from Thermus thermophilus

Mannose-6-phosphate isomerase catalyzes the interconversion of mannose-6-phosphate and fructose-6-phosphate. The gene encoding a putative mannose-6-phosphate isomerase from Thermus thermophilus was cloned and expressed in Escherichia coli. The native enzyme was a 29 kDa monomer with activity maxima for mannose 6-phosphate at pH 7.0 and 80 °C in the presence of 0.5 mM Zn²⁺ that was present at one molecule per monomer. The half-lives of the enzyme at 65, 70, 75, 80, and 85 °C were 13, 6.5, 3.7, 1.8, and 0.2 h, respectively. The 15 putative active-site residues within 4.5 Å of the substrate mannose 6-phosphate in the homology model were individually replaced with other amino acids. The sequence alignments, activities, and kinetic analyses of the wild-type and mutant enzymes with amino acid changes at His50, Glu67, His122, and Glu132 as well as homology modeling suggested that these four residues are metal-binding residues and may be indirectly involved in catalysis. In the model, Arg11, Lys37, Gln48, Lys65 and Arg142 were located within 3 Å of the bound mannose 6-phosphate. Alanine substitutions of Gln48 as well as Arg142 resulted in increase of K_m and dramatic decrease of k_cat, and alanine substitutions of Arg11, Lys37, and Lys65 affected enzyme activity. These results suggest that these 5 residues are substrate-binding residues. Although Trp13 was located more than 3 Å from the substrate and may not interact directly with substrate or metal, the ring of Trp13 was essential for enzyme activity.     

Aeropyrum pernix K1, a strictly aerobic and hyperthermophilic archaeon,has two terminal oxidases,cytochrome ba3 and cytochrome aa3

Aeropyrum pernix K1 is a strictly aerobic and hyperthermophilic archaeon that thrives even at 100 degrees C. The archaeon is quite interesting with respect to the evolution of aerobic electron transport systems and the thermal stability of the respiratory components. An isolated membrane fraction was found to oxidize bovine cytochrome c.The activity was solubilized in the presence of detergents and separated into two fractions by successive chromatography. Two cytochrome oxidases, designated as CO-1 and CO-2, were further purified. CO-1 was a ba(3)-type cytochrome containing at least two subunits. Chemically digested fragments of CO-1 revealed a peptide with a sequence identical to a part of a putative cytochrome oxidase subunit I encoded by the gene ape1623. CO-2, an aa(3)-type cytochrome, was present in lower amounts than CO-1 and was immunologically identified as a product of aoxABC gene (DDBJ accession no. AB020482). Both cytochromes reacted with carbon monoxide. The apparent K(m) values of CO-1 and CO-2 for oxygen were 5.5 and 32 micro M, respectively, at 25 degrees C. The terminal oxidases CO-1 and CO-2 phylogenetically correspond to the SoxB and SoxM branches, respectively, of the heme-copper oxidase tree.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Thermus oshimai JL-2 and T. thermophilus JL-18 genome analysis illuminates pathways for carbon,nitrogen, and sulfur cycling

The complete genomes of Thermus oshimai JL-2 and T. thermophilus JL-18 each consist of a circular chromosome, 2.07 Mb and 1.9 Mb, respectively, and two plasmids ranging from 0.27 Mb to 57.2 kb. Comparison of the T. thermophilus JL-18 chromosome with those from other strains of T. thermophilus revealed a high degree of synteny, whereas the megaplasmids from the same strains were highly plastic. The T. oshimai JL-2 chromosome and megaplasmids shared little or no synteny with other sequenced Thermus strains. Phylogenomic analyses using a concatenated set of conserved proteins confirmed the phylogenetic and taxonomic assignments based on 16S rRNA phylogenetics. Both chromosomes encode a complete glycolysis, tricarboxylic acid (TCA) cycle, and pentose phosphate pathway plus glucosidases, glycosidases, proteases, and peptidases, highlighting highly versatile heterotrophic capabilities. Megaplasmids of both strains contained a gene cluster encoding enzymes predicted to catalyze the sequential reduction of nitrate to nitrous oxide; however, the nitrous oxide reductase required for the terminal step in denitrification was absent, consistent with their incomplete denitrification phenotypes. A sox gene cluster was identified in both chromosomes, suggesting a mode of chemolithotrophy. In addition, nrf and psr gene clusters in T. oshmai JL-2 suggest respiratory nitrite ammonification and polysulfide reduction as possible modes of anaerobic respiration.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.