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Previous in vitro experiments withDesulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c 3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c 3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c 3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

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A novel anaerobic, thermophilic, xylanolytic, motile rod-shaped bacterium with a sheath-like outer structure (toga) was isolated from a Mexican oil well in the Gulf of Mexico. Strain MET12 T was a Gram-negative bacterium, reducing elemental sulfur, thiosulfate and sulfite to hydrogen sulfide. Its optimum growth conditions were 55 6C, pH 6?6, 3 % NaCl and 0?15 % MgCl 2 .6H 2 O. The DNA G+C content was 36?1 mol%. Phylogenetically, strain MET12T was related to members of genus Petrotoga, with similarities to Petrotoga mobilis, Petrotoga sibirica, Petrotoga miotherma and Petrotoga olearia varying from 97?6 to 98?8 %. However DNA-DNA relatedness values between these species and strain MET12 T were lower than 70 %. As strain MET12 T (=DSM 14811 T =CIP 107371 T ) was genomically and phenotypically different from existing Petrotoga species, it is proposed as the type strain of a novel species, Petrotoga mexicana sp. nov.Anaerobes have always been considered as the dominant micro-organisms of petroleum reservoirs (Magot et al., 2000). Among them, fermentative bacteria constitute an important microbial community of the oilfield environment. Thermophilic isolates are the most studied, probably because (i) most oil reservoirs occur at depths where the temperature is high and (ii) thermophiles possess thermostable enzymes of great interest in industrial processes. Stetter et al. (1993) provided evidence of the presence of Thermotoga strains, members of the order Thermotogales, in oilfields. The isolation of Thermotoga elfii, Thermotoga subterranea and Thermotoga hypogea from such ecosystems was reported shortly afterwards (Fardeau et al., 1997a;Jeanthon et al., 1995;Ravot et al., 1995a). Microbial studies on various hot oil reservoirs throughout the world have provided evidence of the importance of micro-organisms morphologically and physiologically close to other members of Thermotogales, namely Fervidobacterium, Thermosipho, Geotoga and Petrotoga (Grassia et al., 1996;Magot et al., 2000). However, no Fervidobacterium species originating from oilfield reservoirs has been characterized so far. Davey et al. (1993) first reported the presence of microorganisms belonging to the genera Geotoga and Petrotoga in petroleum reservoirs located in Oklahoma and Texas.Three novel species were fully characterized: Geotoga petraea, Geotoga subterranea and Petrotoga miotherma. Later, Petrotoga mobilis was isolated from a North Sea oilproduction well (Lien et al., 1998). Petrotoga olearia and Petrotoga sibirica were recently isolated from a continental petroleum reservoir in Western Siberia (L'Haridon et al., 2002), from which L' Haridon et al. (2001) also isolated a novel species of the genus Thermosipho. Members of the genus Petrotoga are thermophilic heterotrophs that grow in a broad range of salt concentrations (up to 10 % NaCl). During our microbial studies of oil waters originating from offshore reservoirs located in the Gulf of Mexico, we isolated a novel thermophilic strain phylogenetically related to the genus Petrotoga, which we...

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).

This paper reports the characterization of a Halomonas sp. strain (named HTB24) isolated from olive-mill wastewater and capable of transforming tyrosol into hydroxytyrosol (HT) and 3,4-dihydroxyphenylacetic acid (DHPA) in hypersaline conditions. This is the first time that a halophile has been shown to perform such reactions. The potent natural antioxidant HT was obtained through a C3 hydroxylation on the ring cycle, whereas DHPA was synthesized via the 4-hydroxyphenylacetic acid (HPA) pathway, which has been well described from other bacterial sources. HT was produced first, and then DHPA was detected in the medium accompanied by traces of HPA. HPA involved another pathway resulting from the activity of an aryl-dehydrogenase, which is suggested to be responsible for both tyrosol and hydroxytyrosol oxidation. Maximal HT content (2.30 mM) and maximal DHPA (5.15+/-0.42 mM) were obtained from a culture inoculated in the presence of 20 mM tyrosol and 0.5 g L(-1) yeast extract. Following this, DHPA was quickly degraded into 5-carboxymethyl-2-hydroxymuconic semialdehyde by a 2,3-dioxygenase, finally resulting in succinate and pyruvate. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain HTB24, with a G+C content of 55.3 mol%, is closely related to Halomonas neptunia DSM 15720(T), 'Halomonas alkaliantarctica' DSM 15686(T) and Halomonas boliviensis DSM 15516(T).