Development of molecular driven screening for desulfurizing microorganisms targeting the dszB desulfinase gene.

COnsensus DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) were developed for the detection of the dszB desulfinase gene (2'-hydroxybiphenyl-2-sulfinate desulfinase; EC 3.13.1.3) by polymerase chain reaction (PCR), which allow to reveal larger diversity than traditional primers. The new developed primers were used as molecular monitoring tool to drive a procedure for the isolation of desulfurizing microorganisms. The primers revealed a large dszB gene diversity in environmental samples, particularly in diesel-contaminated soil that served as inoculum for enrichment cultures. The isolation procedure using the dibenzothiophene sulfone (DBTO2) as sole sulfur source reduced drastically the dszB gene diversity. A dszB gene closely related to that carried by Gordonia species was selected. The desulfurization activity was confirmed by the production of desulfurized 2-hydroxybiphenyl (2-HBP). Metagenomic 16S rRNA gene sequencing showed that the Gordonia genus was represented at low abundance in the initial bacterial community. Such observation highlighted that the culture medium and conditions represent the bottleneck for isolating novel desulfurizing microorganisms. The new developed primers constitute useful tool for the development of appropriate cultural-dependent procedures, including medium and culture conditions, to access novel desulfurizing microorganisms useful for the petroleum industry.

Identification of interferon-γ-inducible-lysosomal thiol reductase (GILT) gene from Mefugu (Takifugu obscures) and its immune response to LPS challenge.

Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a Mefugu cDNA (ToGILT) encodes a deduced protein of 242 amino acids with a putative molecular weight of 28.6 kDa. It contains typical features of GILT proteins including the signature sequence CQHGX2ECX2NX4C, CXXC motif and other five cysteines. Genomic analysis revealed that ToGILT gene exhibited a similar exon-intron organization to human and mouse GILT. Phylogenetic analysis showed that ToGILT derived from a common ancestor with other vertebrate GILT proteins. The ToGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in spleen and kidney after LPS induction. These results suggest that ToGILT may be involved in the immune response to bacteria challenge in Takifugu obscurus.

Phylogenetics and enzymology of plant quiescin sulfhydryl oxidase.

Quiescin Sulfhydryl Oxidase (QSOX), a catalyst of disulfide bond formation, is found in both plants and animals. Mammalian, avian, and trypanosomal QSOX enzymes have been studied in detail, but plant QSOX has yet to be characterized. Differences between plant and animal QSOXs in domain composition and active-site sequences raise the question of whether these QSOXs function by the same mechanism. We demonstrate that Arabidopsis thaliana QSOX produced in bacteria is folded and functional as a sulfhydryl oxidase but does not exhibit the interdomain electron transfer observed for its animal counterpart. Based on this finding, further exploration into the respective roles of the redox-active sites in plant QSOX and the reason for their concatenation is warranted.

Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers.

BACKGROUND: The sulfate assimilation pathway is present in photosynthetic organisms, fungi, and many bacteria, providing reduced sulfur for the synthesis of cysteine and methionine and a range of other metabolites. In photosynthetic eukaryotes sulfate is reduced in the plastids whereas in aplastidic eukaryotes the pathway is cytosolic. The only known exception is Euglena gracilis, where the pathway is localized in mitochondria. To obtain an insight into the evolution of the sulfate assimilation pathway in eukaryotes and relationships of the differently compartmentalized isoforms we determined the locations of the pathway in lineages for which this was unknown and performed detailed phylogenetic analyses of three enzymes involved in sulfate reduction: ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate reductase (APR) and sulfite reductase (SiR). RESULTS: The inheritance of ATPS, APR and the related 3'-phosphoadenosine 5'-phosphosulfate reductase (PAPR) are remarkable, with multiple origins in the lineages that comprise the opisthokonts, different isoforms in chlorophytes and streptophytes, gene fusions with other enzymes of the pathway, evidence a eukaryote to prokaryote lateral gene transfer, changes in substrate specificity and two reversals of cellular location of host- and endosymbiont-originating enzymes. We also found that the ATPS and APR active in the mitochondria of Euglena were inherited from its secondary, green algal plastid. CONCLUSION: Our results reveal a complex history for the enzymes of the sulfate assimilation pathway. Whilst they shed light on the origin of some characterised novelties, such as a recently described novel isoform of APR from Bryophytes and the origin of the pathway active in the mitochondria of Euglenids, the many distinct and novel isoforms identified here represent an excellent resource for detailed biochemical studies of the enzyme structure/function relationships.

Molecular and morphological characterization of the association between bacterial endosymbionts and the marine nematode Astomonema sp. from the Bahamas.

Marine nematode worms without a mouth or functional gut are found worldwide in intertidal sandflats, deep-sea muds and methane-rich pock marks, and morphological studies show that they are associated with endosymbiotic bacteria. While it has been hypothesized that the symbionts are chemoautotrophic sulfur oxidizers, to date nothing is known about the phylogeny or function of endosymbionts from marine nematodes. In this study, we characterized the association between bacterial endosymbionts and the marine nematode Astomonema sp. from coral reef sediments in the Bahamas. Phylogenetic analysis of the host based on its 18S rRNA gene showed that Astomonema sp. is most closely related to non-symbiotic nematodes of the families Linhomoeidae and Axonolaimidae and is not closely related to marine stilbonematinid nematodes with ectosymbiotic sulfur-oxidizing bacteria. In contrast, phylogenetic analyses of the symbionts of Astomonema sp. using comparative 16S rRNA gene sequence analysis revealed that these are closely related to the stilbonematinid ectosymbionts (95-96% sequence similarity) as well as to the sulfur-oxidizing endosymbionts from gutless marine oligochaetes. The closest free-living relatives of these gammaproteobacterial symbionts are sulfur-oxidizing bacteria from the family Chromatiaceae. Transmission electron microscopy and fluorescence in situ hybridization showed that the bacterial symbionts completely fill the gut lumen of Astomonema sp., suggesting that these are their main source of nutrition. The close phylogenetic relationship of the Astomonema sp. symbionts to known sulfur-oxidizing bacteria as well as the presence of the aprA gene, typically found in sulfur-oxidizing bacteria, indicates that the Astomonema sp. symbionts use reduced sulfur compounds as an energy source to provide their hosts with nutrition.

Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in amphioxus Branchiostoma belcheri with implications for GILT in innate immune response.

An amphioxus cDNA, AmphiGILT, encoding GILT protein was isolated from the gut cDNA library of Branchiostoma belcheri. It codes for a deduced protein of 254 amino acids, which has all the main features typical of GILT proteins including the signature sequence CQHGX(2)CX(2)NX(4)C, CXXC motif and 11 conserved cysteines. Phylogenetic analysis showed that AmphiGILT and sea urchin GILT clubbed together and positioned at the base of vertebrate GILT clade, suggesting that both AmphiGILT and sea urchin GILT might share some characteristics of the archetype of vertebrate GILT proteins. The genomic DNA sequence of B. floridae contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. AmphiGILT was expressed in a tissue-specific manner with the most abundant mRNA in the digestive system including hepatic caecum and hind-gut. It was also found that mammalian IFN-gamma only exerted a slight effect on the expression of GILT gene in amphioxus, forming a contrast to the marked induction of human and mouse GILT expression by IFN-gamma. Taken the absence of the adaptive immune system including MHC class II molecules and lymphocytes into consideration, these results suggest that AmphiGILT is highly likely to play a role in the innate immune responses in amphioxus.

Enzymology and molecular biology of prokaryotic sulfite oxidation.

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite:acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate:phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate.

A novel organization of the dissimilatory sulfite reductase operon of Thermodesulforhabdus norvegica verified by RT-PCR.

The nucleotide sequence of the gene cluster for the dissimilatory sulfite reductase (Dsr) from the Gram-negative thermophilic sulfate reducer Thermodesulforhabdus norvegica was determined. The Dsr-encoding genes (dsrAB) were found to be located in an operon encompassing four other open reading frames in the following order: dsrD-dsrA-dsrB-dsrN-dsrC-fdx. Localization of these six genes in the same operon supports previous suggestions that dsrD, -C and -N play essential roles in the mechanism for reduction of sulfite to sulfide. Transcriptional analysis showed that these genes constitute a contiguous transcriptional unit of at least 5 kb. The phylogeny of Dsr is discussed.

The relationship between structure and function for the sulfite reductases.

The six-electron reductions of sulfite to sulfide and nitrite to ammonia, fundamental to early and contemporary life, are catalyzed by diverse sulfite and nitrite reductases that share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an Fe4S4 cluster. The recently determined crystallographic structure of the sulfite reductase hemoprotein from Escherichia coli complements extensive biochemical and spectroscopic studies in revealing structural features that are key for the catalytic mechanisms and in suggesting a common symmetric structural unit for this diverse family of enzymes.

Primary structure of the assimilatory-type sulfite reductase from Desulfovibrio vulgaris (Hildenborough): cloning and nucleotide sequence of the reductase gene.

The nucleotide sequence encoding the structural gene (651 bp) and flanking regions for the assimilatory-type sulfite reductase from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) was determined after cloning a 1.4 kb HindIII/SalI genomic fragment possessing the gene into Bluescript pBS(+)KS. The primary structure of the protein was deduced, and the molecular mass of the apoprotein was estimated as 24 kDa. The amino acid sequence of the polypeptide shows some similarities at putative [Fe4S4] cluster binding sites in comparison with the heme protein subunit of the larger Escherichia coli and Salmonella typhimurium sulfite reductases and spinach nitrite reductase. This is the first reported sequence of a member of a new class of low molecular weight assimilatory sulfite-reducing enzymes recently identified in a number of anaerobic bacteria [Moura, I., Lina, A. R., Moura, J. J. G., Xavier, A. V., Fauque, G., Peck, H. D., & Le Gall, J. (1986) Biochem. Biophys. Res. Commun. 141, 1032-1041].