Gene replacement in Penicillium roqueforti.

Most cheese-making filamentous fungi lack suitable molecular tools to improve their biotechnology potential. Penicillium roqueforti, a species of high industrial importance, would benefit from functional data yielded by molecular genetic approaches. This work provides the first example of gene replacement by homologous recombination in P. roqueforti, demonstrating that knockout experiments can be performed in this fungus. To do so, we improved the existing transformation method to integrate transgenes into P. roqueforti genome. In the meantime, we cloned the PrNiaD gene, which encodes a NADPH-dependent nitrate reductase that reduces nitrate to nitrite. Then, we performed a deletion of the PrNiaD gene from P. roqueforti strain AGO. The ΔPrNiaD mutant strain is more resistant to chlorate-containing medium than the wild-type strain, but did not grow on nitrate-containing medium. Because genomic data are now available, we believe that generating selective deletions of candidate genes will be a key step to open the way for a comprehensive exploration of gene function in P. roqueforti.

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3.

Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK3. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV-visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.

[Nitric oxide system in blood of newborn infants at elevated risk of perinatal pathology].

The study was designed to evaluate activity of the blood nitric oxide (NO) system in newborns at high risk of perinatal pathology. The observed rise in NO production should be regarded as a compensatory and adaptive reaction facilitating the maintenance of microcirculation and hemodynamics in organs and their systems of newborn infants. The inter-relation and inter-dependence between NO levels and enzymatic activities (NOS, HP, O2-, ONOO-, and SOD) make up an integral pathogenetic mechanism determining severity and progress of affection of vitally important organs and systems as well as associated clinical symptoms in full-term and premature infants at high perinatal risk.

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei.

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO (3) (-) , NO (2) (-) or NH (4) (+) ) for growth. Different approaches were considered to study the effect of NH (4) (+) on nitrogen assimilation in Hfx. mediterranei cells grown in KNO(3) medium. The NH (4) (+) addition to KNO(3) medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH (4) (+) media. Both activities increased when NH (4) (+) was removed from culture, showing that the negative effect of NH (4) (+) on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO (3) (-) and NH (4) (+) , NH (4) (+) was preferentially consumed, but NO (3) (-) uptake was not completely inhibited by NH (4) (+) at prolonged time scale. The addition of MSX to NH (4) (+) or NO (3) (-) cultures results in an increase of Nas and NiR activities, suggesting that NH (4) (+) assimilation, rather than NH (4) (+ ) per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei.

[Properties of nitrate reductase from Fusarium oxysporum 11dn1 fungi grown under aerobic and anaerobic conditions].

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.

Enhanced nitric oxide production is closely associated with serum lipid concentrations in adolescents.

BACKGROUND: To investigate whether nitric oxide (NO) production is associated with serum lipid concentrations and body mass index (BMI), we measured serum nitrate and nitrites (NOx) concentrations, serum lipid profiles, and anthropometric parameters in 319 adolescents. METHODS: Serum NOx concentrations were determined using the Griess reaction. Serum concentrations of triglyceride, total cholesterol, and low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by standard enzymatic procedures. RESULTS: Subjects with increased serum cholesterol or triglyceride concentrations exhibited remarkably high NOx levels. Total cholesterol and triglyceride averaged 161.5+/-27.4 and 205.9+/-107.8 mg/dl in males with NOx >92.8 micromol/l (upper 20%), which were significantly above the values (132.4+/-17.2 and 58.1+/-20.3 mg/dl) in those with NOx <15.6 micromol/l (lower 20%). The prevalences of male adolescents with increased concentrations of cholesterol and triglyceride were significantly higher in the subjects with NOx > or =51.2 micromol/l than in those with NOx <51.2 micromol/l (8.9% and 22.2% vs. 1.6% and 2.3%, p<0.05, respectively). Correlation coefficients of serum lipid concentrations and anthropometric parameters vs. serum NOx concentrations were higher in males than in females for cholesterol (r=0.28 vs. 0.23), triglyceride (r=0.51 vs. 0.42), HDL-C (r=-0.25 vs. -0.16), and BMI (r=0.39 vs. 0.27). CONCLUSIONS: NO production is closely associated with serum lipid concentrations in adolescents, and these associations are stronger in males than in females.

Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:Nitrate reductase.

The cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase (EC 1.6.6.3) was overexpressed in Escherichia coli with a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochrome b reductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase of N. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductase kcat as wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochrome b reductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2' phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).

A conserved cysteine in molybdenum oxotransferases.

The amino acid sequences of peptides derived from rat hepatic sulfite oxidase have been determined by a combination of amino acid analysis and Edman degradation of the purified protein. The data obtained showed the rat liver enzyme contained 3 cysteine residues which was confirmed by thiol modification studies using 4,4'-dithiodipyridine of the native enzyme. Combining these data with that previously published for chicken liver sulfite oxidase (Neame, P. J., and Barber, M. J. (1989) J. Biol. Chem. 264, 20894-20901) indicates that 2 cysteines (Cys186 and Cys430, based upon the numbering for the chicken sequence) are conserved in both chicken and rat liver enzymes with all the cysteine residues being present in the molybdenum-containing domain. Further comparison of the sequences of the molybdenum domains of rat and chicken liver sulfite oxidase with the amino acid sequences published for the molybdenum domains of a variety of assimilatory nitrate reductases suggests that only a single cysteine residue (Cys186) is conserved in all these enzymes, indicating that it may play a role in the binding of Mo-pterin to the protein.

Mutations in the Escherichia coli fnr and tgt genes: control of molybdate reductase activity and the cytochrome d complex by fnr.

In eubacteria, the tRNA transglycosylase (Tgt) in specific tRNAs exchanges a guanine in the anticodon for 7-aminomethyl-7-deazaguanine, which is finally converted to queuosine. The tgt gene of Escherichia coli has been mapped at 9 min on the genome, and mutant pairs containing an intact or mutated tgt allele were obtained after transduction of the tgt locus by P1 bacteriophages into a genetically defined E. coli strain (S. Noguchi, Y. Nishimura, Y. Hirota, and S. Nishimura, J. Biol. Chem. 257:6544-6550, 1982). These tgt mutants grew anerobically with fumarate as an electron acceptor, while nitrate or trimethylamine N-oxide could not be reduced. Furthermore, molybdate reductase activity was almost lacking and the characteristic absorption maxima, corresponding to cytochrome a1 and the cytochrome d complex, were not detectable in low-temperature reduced-minus-oxidized difference spectra in anaerobically grown cells. Transduction of the mutated tgt locus into another E. coli recipient resulted in tgt mutants without anaerobic defects. Transformation of the original tgt mutants with an fnr gene-containing plasmid reversed the anaerobic defects. Clearly, the original tgt mutants harbor a second mutation, affecting the anaerobic regulator protein Fnr. The results suggest that fnr is involved in anaerobic control of components of the cytochrome d complex and of the redox system that transfers electrons to molybdate. F' plasmids containing a fused lacI-lacZ gene with the nonsense codon UAG at different positions in the lacI part were transferred to E. coli strains with a mutated or nonmutated tgt locus but intact in fnr. A twofold increase in the frequency of incorrect readthrough of the UAG codon, dependent on the codon context, was observed in the tgt mutant and is suggested to be caused by a tRNA(Tyr) with G in place of queuosine.

Cloning of nitrate reductase genes from the cyanobacterium Anacystis nidulans.

Anacystis nidulans, a non-nitrogen-fixing cyanobacterium, can fulfill its nitrogen requirement by the assimilation of nitrate. The first step in the pathway, the reduction of nitrate to nitrite, is catalyzed by the molybdo-protein nitrate reductase. In this study, newly developed techniques for gene cloning in A. nidulans R2 were used for the isolation of two genes involved in nitrate reduction. One gene was cloned by complementation of the corresponding mutant; the other gene was picked up from a cosmid gene library by using a restriction fragment containing the transposon-inactivated gene as a probe. Both genes were unlinked single-copy chromosomal genes. Transformation studies provided evidence for the existence of a third locus involved in nitrate reduction.

A common pathway for the activation of several molybdoenzymes in Escherichia coli K12.

Three molybdoenzymes, nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase which form part of different systems, have been studied in a parental strain of Escherichia coli K12. When the organism is grown in the presence of 10 mM tungstate, these three enzymes are present in an inactive form which may be activated in vivo by the addition of 1 mM sodium molybdate. The mixing of soluble fractions from chlA and chlB mutants grown under the appropriate conditions leads to the activation of nitrate reductase, formate benzyl-viologen oxidoreductase and trimethylamine-N-oxide reductase. The activation of each enzyme is maximal when the mutants are grown under conditions that lead to the induction of that enzyme in the wild-type strain. The employment of purified proteins, the association factor FA and the Protein PA, which are presumed to be the products of the chlA and chlB genes, has shown that these proteins are responsible for the activation of the three enzymes during the complementation process.