Analysis of the Desulfovibrio gigas transcriptional unit containing rubredoxin (rd) and rubredoxin-oxygen oxidoreductase (roo) genes and upstream ORFs

Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment Of D. gigas chromosomal DNA containing the roo gene and the rubredoxin gene was sequenced, Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. OFF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG; start codon of rubredoxin, The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative -10 and -35 regulator regions of a sigma (70)-type promoter, having similarity with E. coli sigma (70) promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study

Molecular cloning of the gene encoding flavoredoxin, a flavoprotein from Desulfovibrio gigas

Sulfate-reducing bacteria are rich in unique redox proteins and electron carriers that participate in a variety of essential pathways. Several studies have been carried out to characterize these proteins, but the structure and function of many are poorly understood. Many Desulfovibrio species can grow using hydrogen as the sole energy source, indicating that the oxidation of hydrogen with sulfite as the terminal electron acceptor is an energy-conserving mechanism. Flavoredoxin is an FMN-binding protein isolated from the sulfate-reducing bacteria Desulfovibrio gigas that participates in the reduction of bisulfite from hydrogen. Here we report the cloning and sequencing of the flavoredoxin gene. The derived amino acid sequence exhibits similarity to several flavoproteins which are members of a new family of flavin reductases suggested to bind FMN in a novel mode. (C) 2000 Academic Pres

Studies on the redox centers of the terminal oxidase from Desulfovibrio gigas and evidence for its interaction with rubredoxin

Abstract: Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at similar to 2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 +/- 15 mV for the quinone/semiquinone couple and at -130 +/- 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is -350 +/- 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented

The ‘strict’ anaerobe Desulfovibrio gigas contains a membrane-bound oxygen-reducing respiratory chain

AbstractSulfate-reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane-bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O2 min−1 mg protein−1, respectively). This ‘strict anaerobic’ bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought

Survey of Water Bugs in Bankim, a New Buruli Ulcer Endemic Area in Cameroon

Buruli ulcer is a debitliating human skin disease with an unknown transmission mode although epidemiological data link it with swampy areas. Data available suggest that aquatic insects play a role in the dissemination and/or transmission of this disease. However, their biodiversity and biology remain poorly documented. We conducted an entomological survey in Bankim, Cameroon, an area recently described as endemic for Buruli ulcer in order to identify the commonly occurring aquatic bugs and document their relative abundance, diversity, and spatial distribution. Collection of aquatic bugs was realized over a period of one month by daily direct capture in different aquatic environments (streams, ponds, and rivers) and through light traps at night. Globally, the data obtained showed the presence of five families (Belostomatidae, Naucoridae, Nepidae, Notonectidae, and Gerridae), their abundance, distribution and diversity varying according to the type of aquatic environments and light attraction

Desulfovibrio gigas neelaredoxin: a novel superoxide dismutase integrated in a putative oxygen sensory operon of an anaerobe

Neelaredoxin, a small non-heme blue iron protein from the sulfate-reducing bacterium Desulfovibrio gigas [Chen, L., Sharma, P., LeGall, J., Mariano, A.M., Teixeira M. and Xavier, A.V. (1994) Eur. J. Biochem. 226, 613±618] is shown to be encoded by a polycistronic unit which contains two additional open reading frames (ORF-1 and ORF-2) coding for chemotaxis-like proteins. ORF-1 has domains highly homologous with those structurally and functionally important in methyl-accepting chemotaxis proteins, including two putative transmembrane helices, potential methylation sites and the interaction domain with CheW proteins. Interestingly, ORF-2 encodes a protein having homologies with CheW proteins. Neelaredoxin is also shown to have significant superoxide dismutase activity (1200 U´mg±1), making it a novel type of iron superoxide dismutase. Analysis of genomic data shows that neelaredoxin-like putative polypeptides are present in strict anaerobic archaea, suggesting that this is a primordial superoxide dismutase. The three proteins encoded in this operon may be involved in the oxygen-sensing mechanisms of this anaerobic bacterium, indicating a possible transcriptional mechanism to sense and respond to potential stress agents

Amino acid sequence of rubredoxin from Desulfovibrio desulfuricans strain 27774

AbstractThe amino acid sequence of a rubredoxin from Desulfovibrio desulfuricans (strain 27774) has been determined. Comparison with rubredoxins from other species reveals pervasive homology, including the regions known to provide the cysteine ligands to the iron atom in several rubredoxins. Neither an extra cysteinyl residue nor a unique histidyl residue in the new sequence is located in the sequence in such a way that, by homology, a functional role in the structure is suggested

Structure of a dioxygen reduction enzyme from Desulfovibrio gigas

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 Angstrom resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta -lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead