Change in food ingestion induces rapid shifts in the diversity of microbiota associated with cutaneous mucus of Atlantic salmon Salmo salar.

To test the hypothesis that a switch in diet might cause changes in the abundance and composition of mucous-dwelling microorganisms, a short-term experiment was conducted with Atlantic salmon Salmo salar. Fish were fed on three different diets: pelleted S. salar feed, macroinvertebrates or pellets supplemented with an antibiotic. A fourth group of fish was deprived of food throughout the trial. Seven days after manipulating diets, significant differences were found in microbial density and community composition (quantified by different morphologically distinct colonies), particularly between fed and unfed animals. Moreover, food deprivation caused a rapid decrease in the number of epidermal mucous cells of the lateral skin, which may indicate a decrease in mucous secretion and explain differences in the diversity of mucous-dwelling microbiota observed in the fish. This is the first report of an effect of feeding regime on the abundance of microbial communities associated with cutaneous mucus of fishes.

DFT Calculations of Isotropic Hyperfine Coupling Constants of Nitrogen Aromatic Radicals: The Challenge of Nitroxide Radicals.

The performance of DFT methodology to predict with accuracy the isotropic hyperfine coupling constants (hfccs) of aromatic radicals containing (14)N nucleus is investigated by an extensive study in which 165 hfccs, belonging to 38 radical species, are obtained from calculations with B3LYP and PBE0 functionals combined with 6-31G*, N07D, TZVP, and EPR-III basis sets, and are compared to the reported experimental data. The results indicate that the selection of the basis set is of fundamental importance in the calculation of (14)N hfccs, whereas there is not so great an influence on the accurate computation of that parameter for (1)H nuclei. The values of the calculated (14)N coupling constants of aromatic nitroxide radicals using DFT methodology are noticeably lower than the experimental ones. A very simple relation to predict these hfccs with high accuracy is proposed on the basis of the present results, as an interesting alternative to the highly computationally demanding integrated approaches so far used.

Density functional theory study of 14N isotropic hyperfine coupling constants of organic radicals.

Nitrogen hyperfine coupling constants (hfccs) of organic radicals have been calculated by density functional theory (DFT) methodology. The capability of the B3LYP functional, combined with 6-31G*, TZVP and EPR-III basis sets, to reproduce experimental nitrogen coupling constant data has been analyzed for 109 neutral, cationic and anionic radicals, all of them containing at least one nitrogen atom. The results indicate that the selection of the basis set plays an important role in the accuracy of DFT calculations of hfccs, mainly in relation with the composition of the primitive functions and the quantum number of those functions. The main conclusion obtained is the high reliability of the scheme B3LYP/6-31G* for the prediction of nitrogen hfccs with very low computational cost.

Density functional theory predictions of isotropic hyperfine coupling constants.

The reliability of density functional theory (DFT) in the determination of the isotropic hyperfine coupling constants (hfccs) of the ground electronic states of organic and inorganic radicals is examined. Predictions using several DFT methods and 6-31G, TZVP, EPR-III and cc-pVQZ basis sets are made and compared to experimental values. The set of 75 radicals here studied was selected using a wide range of criteria. The systems studied are neutral, cationic, anionic; doublet, triplet, quartet; localized, and conjugated radicals, containing 1H, 9Be, 11B, 13C, 14N, 17O, 19F, 23Na, 25Mg, 27Al, 29Si, 31P, 33S, and 35Cl nuclei. The considered radicals provide 241 theoretical hfcc values, which are compared with 174 available experimental ones. The geometries of the studied systems are obtained by theoretical optimization using the same functional and basis set with which the hfccs were calculated. Regression analysis is used as a basic and appropriate methodology for this kind of comparative study. From this analysis, we conclude that DFT predictions of the hfccs are reliable for B3LYP/TZVP and B3LYP/EPR-III combinations. Both functional/basis set scheme are the more useful theoretical tools for predicting hfccs if compared to other much more expensive methods.

Theoretical isotropic hyperfine coupling constants of third-row nuclei (29Si, 31P, and 33S).

In a previous paper (Hermosilla, L.; Calle, P.; Garcia de la Vega, J. M.; Sieiro, C. J. Phys. Chem. A 2005, 109, 1114), an adequate computational protocol for the calculation of isotropic hyperfine coupling constants (hfcc's) was proposed. The main conclusion concerns the reliability of the scheme B3LYP/TZVP//B3LYP/6-31G* in the predictions of hfcc's with low computational cost. In the present study, we gain insight into the behavior of the above functional/basis set scheme on nuclei of the third row, for which few systematic studies have been carried out up to the present date. The systems studied are neutral, cationic, anionic, localized, and conjugated radicals, containing (29)Si, (31)P, and (33)S nuclei. After carrying out a regression analysis, we conclude that density functional theory (DFT) predictions on the hfcc's of the third-row nuclei are reliable for B3LYP/TZVP by using an optimized geometry with B3LYP/6-31G* combination. By comparison with other much more computationally demanding schemes, namely, B3LYP/cc-pVTZ and B3LYP/cc-pVQZ, we conclude that the B3LYP functional in conjunction with the TZVP basis set is the most useful computational protocol for the assignment of experimental hfcc's, not only for nuclei of first and second rows, but also for those of the third row.

Cloning and expression of clt genes encoding milk-clotting proteases from Myxococcus xanthus 422.

The screening of a gene library of the milk-clotting strain Myxococcus xanthus 422 constructed in Escherichia coli allowed the description of eight positive clones containing 26 open reading frames. Only three of them (cltA, cltB, and cltC) encoded proteins that exhibited intracellular milk-clotting ability in E. coli, Saccharomyces cerevisiae, and Pichia pastoris expression systems.

Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422.

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 degrees C. The enzyme was completely inactivated by heating for 12 min at 65 degrees C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.

Genetic basis of microbial carotenogenesis.

The synthesis of carotenoids begins with the formation of a phytoene from geranylgeranyl pyrophosphate, a well conserved step in all carotenogenic organisms and catalyzed by a phytoene synthase, an enzyme encoded by the crtB ( spy) genes. The next step is the dehydrogenation of the phytoene, which is carried out by phytoene dehydrogenase. In organisms with oxygenic photosynthesis, this enzyme, which accomplishes two dehydrogenations, is encoded by the crtP genes. In organisms that lack oxygenic photosynthesis, dehydrogenation is carried out by an enzyme completely unrelated to the former one, which carries out four dehydrogenations and is encoded by the crtI genes. In organisms with oxygenic photosynthesis, dehydrogenation of the phytoene is accomplished by a zeta-carotene dehydrogenase encoded by the crtQ ( zds) genes. In many carotenogenic organisms, the process is completed with the cyclization of lycopene. In organisms exhibiting oxygenic photosynthesis, this step is performed by a lycopene cyclase encoded by the crtL genes. In contrast, anoxygenic photosynthetic and non-photosynthetic organisms use a different lycopene cyclase, encoded by the crtY ( lyc) genes. A third and unrelated type of lycopene beta-cyclase has been described in certain bacteria and archaea. Fungi differ from the rest of non-photosynthetic organisms in that they have a bifunctional enzyme that displays both phytoene synthase and lycopene cyclase activity. Carotenoids can be modified by oxygen-containing functional groups, thus originating xanthophylls. Only two enzymes are necessary for the conversion of beta-carotene into astaxanthin, using several ketocarotenoids as intermediates, in both prokaryotes and eukaryotes. These enzymes are a beta-carotene hydroxylase ( crtZ genes) and a beta-carotene ketolase, encoded by the crtW (bacteria) or bkt (algae) genes.

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous.

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.

Heterologous expression of the Saccharomyces cerevisiae PGU1 gene in Schizosaccharomyces pombe yields an enzyme with more desirable properties for the food industry.

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme. The K(m) value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.

Characterization of a broad pH range protease of Candida caseinolytica.

AIMS: The study of a protease secreted by Candida caseinolytica for use in future industrial applications. METHODS AND RESULTS: Growth of Candida caseinolytica on a medium containing milk induced a rapid production of an extracellular enzyme able to hydrolyse casein. The crude extract was applied to both Sephacryl S-200 and DEAE-Biogel A columns, obtaining one peak of activity showing a molecular mass of approximately 30 kDa and three active peaks, respectively. These four peaks showed the same biochemical parameters. In all cases, an extremely broad pH range of action was determined. CONCLUSIONS: Candida caseinolytica secretes high levels of an extracellular protease when grown either in rotary shakers or in batch-fermenters. SIGNIFICANCE AND IMPACT OF THE STUDY: The biochemical properties of this enzyme suggest its possible industrial application in the brewing industry, in the formulation of certain type of detergents and in the fur and leather industries, among others.

Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis.

An alpha-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var. capitata) leaves. Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an alpha-xylosidase that we propose to call AtXYL1. It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM. AtXYL1 comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites. The protein encoded by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only alpha-xylosidases, but also alpha-glucosidases. The alpha-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide. The first eight leaves from Arabidopsis plants were harvested to analyze alpha-xylosidase activity and AtXYL1 expression levels. Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher. When this gene was introduced in a suitable expression vector and used to transform Saccharomyces cerevisiae, significantly higher alpha-xylosidase activity was detected in the yeast cells. alpha-Glucosidase activity was also increased in the transformed cells, although to a lesser extent. These results show that AtXYL1 encodes for an apoplastic alpha-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-alpha-D-glucoside.

Analysis of canthaxanthin and related pigments from Gordonia jacobaea mutants.

A collection of 43 mutant strains of the bacterium Gordonia jacobaea was obtained by means of ethyl methanesulfonate treatment, and the strains were selected for their different pigmentation with respect to the wild-type strain. None of the mutants showed auxotrophy. They all showed good genetic stability and a growth rate similar to that of the parental strain. Canthaxanthin and other carotenoids from these mutants were extracted with acetone and ethanol and separated by high-performance liquid chromatography (HPLC). These HPLC analyses, together with spectrophotometric detection at 480 nm, revealed variations in the pigment contents of the different mutant strains.

ESR-spin trapping study on the sonochemistry of liquids in the presence of oxygen. Evidence for the superoxide radical anion formation.

The sonolysis of water and some organic liquids such as ethylene glycol, methanol and chloroform in the presence of oxygen, at 20 and 475 kHz ultrasound frequencies has been investigated by the ESR-spin trapping technique. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO), 3,3,5,5-tetramethylpyrroline-N-oxide (TMPO) and N-tert-butyl-alpha-phenyl nitrone (PBN) were able to trap superoxide radical anion, generated as the result of the sonication of the organic media. The addition of superoxide dismutase (SOD) resulted in a dramatic decrease of the ESR signal intensity of the superoxide radical adduct. In addition, the thermolysis of the liquids under ultrasound was shown by ESR detection of the spin adducts of the radicals formed by homolytic fragmentation. Occasionally, the nature of the detected spin adduct was dependent on the sonication time or on the frequency of the ultrasonic radiation. Experiments carried out in the presence of 2-methyl-2-nitrosopropane (MNP) resulted in the detection of radicals originating from thermal decomposition of the spin trap, showing its lability under ultrasonic radiation.

Isolation and taxonomic study of a new canthaxanthin-containing bacterium, Gordonia jacobaea MV-1 sp. nov.

This article describes the isolation and taxonomic study of a coryneform isolate of a new Gordonia species (G. jacobaea), strain MV-1, which accumulates several carotenoids, including the ketocarotenoid trans-canthaxanthin. Identification of this new isolate by morphobiochemical methods did not allow unambiguous taxon assignment, but sequencing of the 16S rRNA gene clearly pointed to the genus Gordonia, Gordonia sputi being the closest fit. Differences in certain transversions/transitions in otherwise very well-conserved sequences of the described Gordonia species supported the proposal of this new taxon. The fact that both the best growth and best pigmentation were obtained with glucose, an inexpensive carbon source and at an industrially suitable temperature, suggests that this new bacterial strain may have good potential for the industrial production of canthaxanthin.

Use of a PGU1 recombinant Saccharomyces cerevisiae strain in oenological fermentations.

AIM: The aim of this work was the construction of an oenological Saccharomyces cerevisiae strain able to overexpress the PGU1 gene in order to be used in trial fermentations. METHODS AND RESULTS: The recombinant strain is able to secrete an active endopolygalacturonase into the medium leaving its fermentation ability essentially unchanged. Wines obtained with the recombinant strain and the untransformed counterpart did not differ in their physicochemical parameters or major sensory characteristics. The time needed for wine filtration was dramatically reduced in wines elaborated with the PGU1 recombinant strain, and was comparable to the filtration time shown by wines elaborated from must supplemented with fungal pectolytic enzymes. CONCLUSIONS: The oenological strain constructed in this work secretes an endopolygalacturonase into the wine in an efficient manner, resulting in an improvement in wine filtration but preserving wine typicality and keeping the methanol levels unchanged. SIGNIFICANCE AND IMPACT OF THE STUDY: The PGU1 recombinant strains could be used in oenological fermentations as an alternative to commercial pectolytic enzymes of fungal origin.

Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations.

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.

Production of pectic enzymes in yeasts.

When grown in the appropriate medium, several yeast species produce pectinases able to degrade pectic substances. It is mainly exocellular endopolygalacturonases that break pectins or pectate down by hydrolysis of alpha-1,4-glycosidic linkages in a random way. Biochemical characterisation of these enzymes has shown that they have an optimal pH in the acidic region and an optimal temperature between 40 and 55 degrees C. Their production by yeasts is a constitutive feature and is repressed by the glucose concentration and aeration. Pectic substances and their hydrolysis products are used as carbon sources by a limited number of yeasts and hence these enzymes must be involved in the colonisation of different parts of plants, including fruits. The first yeast pectic enzyme (encoded by the PSE3 gene) was cloned from Tichosporon penicillatum. Recently, a polygalacturonase-encoding gene from Saccharomyces cerevisiae has been cloned and overexpressed in several strains and the gene for an extracellular endopolygalacturonase from Kluyveromyces marxianus has also been described. Taking all the results together, the idea is now emerging that this type of yeast enzyme could offer an alternative to fungal enzymes for industrial applications.

Cloning, molecular characterization, and expression of an endo-polygalacturonase-encoding gene from Saccharomyces cerevisiae IM1-8b.

A structural polygalacturonase-encoding gene (PGU1) from Saccharomyces cerevisiae IM1-8b was cloned and sequenced. The predicted protein comprises 361 amino acids, with a signal peptide between residues 1 and 18 and two potential glycosylation points in residues 318 and 330. The putative active site is a conserved histidine in position 222. This polygalacturonase showed 54% homology with the fungal ones and only 24% homology with their plant and bacterial counterparts. The gene is present in a single gene copy per haploid genome and it is detected in all strains, regardless of their phenotype. The expression of PGU1 gene in several strains of S. cerevisiae revealed that the polygalacturonase activity depended on the plasmid used and also on the genetic background of each strain but in all cases the enzymatic activity increased.

Genetic determination of polygalacturonase production in wild-type and laboratory strains of Saccharomyces cerevisiae.

The genetic determination of polygalacturonase (PG) production in Saccharomyces cerevisiae was studied by biochemical and classical genetic techniques. Crosses of PG+ strains with PG- strains showed that in the haploid wild-type-derived strain, two structural genes were involved in the production of a hydrolysis halo on plates with polygalacturonic acid. However, in the case of PG+ laboratory strain IM1-8b, the phenotype was controlled by only one structural gene although the analysis of PG- IM1-8b mutants demonstrated the existence of at least two complementation groups. All these genetic results were assessed biochemically by means of cation-exchange chromatography. Two enzymes were separated in the wild-type strain, and only one in the laboratory strain. The three enzymes had different Km values, molecular masses, and optimal pHs for activity.