Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.     

The Upper Eocene South Pyrenean Coastal deposits (Liedena sandstone, navarre): Sedimentary facies, benthic formanifera and avian ichnology

Summary During the 1960's and the 1970's the Liedena Sandstone was a type deposit for “flysch-like facies” (sandstone and lutite alternations) of coastal sedimentary systems. However, the depositional system of these beds was never accurately defined. The sedimentological analysis along 100 km of outcrops in the western part of the South Pyrenean Zone (Navarre) allows these peculiar facies to be assigned to a mixed intertidal flat. Furthermore, sandy beach facies, different types of heterolithic, backbarrier deposits and conglomeratic, fluviatile facies have been recognized associated with these intriguing deposits. Generally, a northwestward-facing barrier-island system or wave-dominated delta was the likely depositional environment. The benthic foraminiferal assemblage in the intertidal deposits exhibits the typical characteristics of a marginal marine environment: extremely high dominance of one species (Pararotalia inermis), low species diversity, and a hyaline dominance with discrete amounts of miliolids. Furthermore, the most abundant species indicates that the Liedena Sandstone was deposited during the Late Eocene. Abundant footprints of aquatic birds are known in the tidal flat deposits. Six morphotypes have been distinguished: two (types 1 and 2) are ciconiforme-like; type 1 is here assigned to a new ichnotaxon,Leptoptilostipus pyrenaicus and is one of the oldest occurrences of Ciconiiforme-like ischmites in the fossil record. Two other morphotypes (5 and 6) are similar to those of the Charadriiformes and are refeered to asCharadriipeda. Finally, the affinities of the two remainder morphotypes (3 and 4) are unclear, they could have been made by Charadriiformes. Synsedimentary tectonic activity controlled the evolution of the depositional system, as the area of deposition of the Liedena Sandstone was progressively incorporated into the active thrust sheets of the Pyrenean Orogen during the Late Eocene. The structural uplift and the large amount of sediments derived from the adjacent highlands induced progradation of the depositional system and the definitive retreat of the sea from the South Pyrenean Zone.     

URGORRI COMPLANATUS GEN. ET SP. NOV. (CRYPTOPHYCEAE), A RED‐TIDE‐FORMING SPECIES IN BRACKISH WATERS1

The morphology, ultrastructure, phylogeny, and ecology of a new red‐tide‐forming cryptomonad, Urgorri complanatus Laza‐Martínez gen. et sp. nov., is described. U. complanatus has been collected in southwestern European estuaries, blooming in the inner reaches of several of them. The estuarine character of the species is also supported by its in vitro salinity preferences, showing a maximum growth rate at 10 psu. U. complanatus is a distinctive species and can be easily distinguished by LM from other known brackish and marine species. Cells are dorsoventrally flattened. The plastid has two anterior lobes. One pyrenoid is located in each of the lobes, and a third one on the posterior part. Thylakoids are arranged in pairs and do not penetrate pyrenoids. The plastid is reddish due to the presence of the phycoerythrin Cr‐PE545. An orange discoidal eyespot lies beneath the nucleus, in the posterior ventral face of the plastid. A long furrow runs from the vestibulum, and a gullet is lacking. The periplast is composed of an inner sheet. The nuclear 18S rDNA based molecular analysis reveals U. complanatus is not related to any of the main cryptomonad lineages. Based on ultrastructural and pigment data, the most probable relatives are those merged under the family Geminigeraceae. Its lack of derived characters, together with the presence of characters proposed in previous studies to be primitive, suggests Urgorri could be considered representative of the cryptophycean ancestral character state.     

Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase

AAO (aryl-alcohol oxidase) provides H?O? in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr?2. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His??2 (and CHO His???), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.     

Flow regulation by dams affects ecosystem metabolism in Mediterranean rivers

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