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Objective To identify the frequencies (F) of ferredoxin and nitroreductase mutations on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazole-resistant giardiasis. Methods Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran. DNAs were extracted and amplified by targeting ferredoxin and GlNR genes. The amplicons were directly sequenced to determine gene mutations. Results The various amino acid substitutions (F: 20%, Haplotype diversity: 0.891, Tajima's D: −0.440 13) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates. Only two haplotypes (F: 5%, HD: 0.345; Tajima's D: 0.778 15) characterized in metronidazole-resistant isolates of GlNR, however, no point mutations was found in symptomatic isolates. Conclusions Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity. This leads to decrease in metronidazole drug delivery into the cells. Mutations in these isolates may lead to their resistance to metronidazole. No to low synonymous mutations of GlNR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

Increased hydrogen (H2) levels are known to inhibit H2 formation in Caldicellulosiruptor saccharolyticus. To investigate this organism's strategy for dealing with elevated H2 levels the effect of the hydrogen partial pressure ( P H 2 ) on fermentation performance was studied by growing cultures under high and low P H 2 in a glucose limited chemostat setup. Transcriptome analysis revealed the upregulation of genes involved in the disposal of reducing equivalents under high P H 2 , like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings are in line with the observed shift in fermentation profiles from acetate production to the production of acetate, lactate and ethanol under high P H 2 . Moreover, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. In addition, presented transcription data provide evidence for the involvement of the redox sensing Rex protein in gene regulation under high P H 2 cultivation conditions.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Oxidative decarboxylation of pyruvate is a fundamental reaction of living organisms in general, leading to energy conservation. In some anaerobic or microaerophilic eukaryotic or prokaryotic organisms pyruvate decarboxylation is carried out by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFO). PFO contains Fe-S clusters and thiamin pyrophosphate cofactor (TPP). In the reaction catalyzed by PFO, from pyruvate and Co-A arise acetyl-CoA, CO2, and two electrons are released. Those electrons are accepted by low molecular carrier proteins. Most frequently these proteins are ferredoxins or flavodoxins such as in nitrogen fixating bacteria. PFO can perform a reversible reaction. Trichomonads are mostly parasitic or endosymbiotic organisms with mitochondria-like organelles, hydrogenosomes. These organelles possess PFO which is one of the key enzymes in the metabolism of Trichomonas vaginalis hydrogenosomes. PFO of T. vaginalis, a sexually transmitted pathogen of man, plays also a role in a term of medical importance. PFO is, by a universally accepted concept, one of the key proteins acting in the activation of antimicrobial drugs against trichomoniasis 5-nitroimidazoles, including metronidazole. In the genome of T. vaginalis seven PFO genes were identified. They were named PFO A, B1, B2, C, D, E and...

Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10(-7) M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tung state-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AlDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AlDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AlDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.

Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 mu M tungstate contained a benzylviologen-linked aldehyde oxidoreductase, The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000, It contained 0.68 a 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit, After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained, Acetahdehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent K-m values of 12.5, 10.8, and 20 mu M, respectively, The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent K-m, 0.55 mM), The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate, In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97, The dithionite-reduced enzyme exhibited a broad signal at low temperature,vith g = 2.04 and 1.92; this is indicative of a [4Fe-4S](1+) cluster interacting with a second paramagnet, possibly the S = 1 system of W(TV), Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea, The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10(-7) M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tung state-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AlDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AlDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AlDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.

The iron-sulfur clusters of a pyruvate:ferredoxin oxidoreductase isolated from a methanogenic archaeon, Methanosarcina barkeri (Fusaro), have been unambiguously identified for the first time. In agreement with the estimated iron and sulfur contents (Bock and Schonheit, Eur. J. Biochem. , 237 (1996) 35–44), the enzyme is shown to contain three [4Fe-4S] 2+/1+ clusters, which in the reduced state give a complex EPR spectrum resulting from three distinct centres, magnetically interacting. The catalytic cycle of the enzyme was studied by visible and EPR spectroscopies. A thiamine diphosphate based radical is also an intermediate in the M. barkeri enzyme catalytic cycle. However, under anaerobic conditions, the enzyme or Clostridium pasteurianum ferredoxin iron-sulfur clusters are reduced only in the presence of both substrates, pyruvate and coenzyme A.