Infectious Bursal Disease Virus VP3 Upregulates VP1-Mediated RNA-Dependent RNA Replication

Genome replication is a critical step in virus life cycles. Here, we analyzed the role of the infectious bursal disease virus (IBDV) VP3, a major component of IBDV ribonucleoprotein complexes, on the regulation of VP1, the virus-encoded RNA-dependent RNA polymerase (RdRp). Data show that VP3, as well as a peptide mimicking its C-terminal domain, efficiently stimulates the ability of VP1 to replicate synthetic single-stranded RNA templates containing the 3′ untranslated regions (UTRs) from the IBDV genome segments.     

Development of a Mouse-Adapted Live Attenuated Influenza Virus That Permits In Vivo Analysis of Enhancements to the Safety of Live Attenuated Influenza Virus Vaccine

The live attenuated influenza virus vaccine (LAIV) is preferentially recommended for use in persons 2 through 49 years of age but has not been approved for children under 2 or asthmatics due to safety concerns. Therefore, increasing safety is desirable. Here we describe a murine LAIV with reduced pathogenicity that retains lethality at high doses and further demonstrate that we can enhance safety in vivo through mutations within NS1. This model may permit preliminary safety analysis of improved LAIVs.     

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.     

CO Dehydrogenase Genes Found in Metagenomic Fosmid Clones from the Deep Mediterranean Sea

The use of carbon monoxide (CO) as a biological energy source is widespread in microbes. In recent years, the role of CO oxidation in superficial ocean waters has been shown to be an important energy supplement for heterotrophs (carboxydovores). The key enzyme CO dehydrogenase was found in both isolates and metagenomes from the ocean''s photic zone, where CO is continuously generated by organic matter photolysis. We have also found genes that code for both forms I (low affinity) and II (high affinity) in fosmids from a metagenomic library generated from a 3,000-m depth in the Mediterranean Sea. Analysis of other metagenomic databases indicates that similar genes are also found in the mesopelagic and bathypelagic North Pacific and on the surfaces of this and other oceanic locations (in lower proportions and similarities). The frequency with which this gene was found indicates that this energy-generating metabolism would be at least as important in the bathypelagic habitat as it is in the photic zone. Although there are no data about CO concentrations or origins deep in the ocean, it could have a geothermal origin or be associated with anaerobic metabolism of organic matter. The identities of the microbes that carry out these processes were not established, but they seem to be representatives of either Bacteroidetes or Chloroflexi.Carbon monoxide (CO) oxidation is a source of energy for a wide diversity of prokaryotes and is an important process within the global carbon cycle. There is a wide diversity of CO oxidation pathways among both archaea and bacteria (27, 28), and their wide distribution attests to both the ecological importance and ancient origin of CO oxidation. Most of these pathways are anaerobic (31, 40) and have been reported in both archaea and bacteria. However, aerobic CO oxidation is found only in a few groups of bacteria, specifically in many Actinobacteria and Proteobacteria spp. and in at least one Firmicutes sp. (for examples, see references 16, 17, 26, 35, 46, and 47). Classically, aerobic oxidation of CO has been known to be carried out in soils where, in addition to geological or anthropogenic emissions, there are local biological sources connected to plant roots and animals (15, 18, 19). However, more recently, the relevance of CO oxidation processes in the marine environment has also become clear, mostly from evidence from the fields of genomics and metagenomics (26, 42, 43).The aerobic oxidation of CO is very amenable to genomic analysis, since the genes involved are very characteristic, and their presence in marine bacterial genomes and in metagenomic databases can be considered diagnostic. The genes required for aerobic CO oxidation were first described in detail in chemolithoautotrophic Oligotropha carboxidovorans OM5 (10, 35, 36). The enzyme CO dehydrogenase (CODH) catalyzes the oxidation of CO and water to produce carbon dioxide, two electrons, and two protons (8, 11). The electrons are transferred to an electron transfer chain and used to generate a proton gradient across the membrane. Three genes, coxL, coxM, and coxS (for large, medium, and small subunits, respectively), encode the polypeptides for the CODH enzyme. Two heterotrimers, each composed of one CoxL, CoxM, and CoxS subunit, combine to form a functional aerobic CODH enzyme. The large subunit contains the molybdenum cofactor, the medium subunit binds flavin adenine dinucleotide, and the small subunit has two iron-sulfur clusters (13). In addition to these three genes, a number of other accessory genes have also been identified (CoxB, CoxC, CoxH, CoxD, CoxE, CoxF, CoxG, CoxI, and CoxK) that are believed to be required in the processes of regulation, posttranslational modification, and anchorage of the CODH complex to the cytoplasmic membrane. A number of these accessory genes are membrane-bound proteins themselves (CoxB, CoxC, CoxH, and CoxK), containing several transmembrane helices, and indeed, in O. carboxidovorans OM5, the CODH enzyme itself has been observed to associate with the inner cytoplasmic membrane.Based on sequence differences, genome organization, and catalytic properties, there are two types of aerobic molybdenum-based CODH (the anaerobic enzymes are a different class of genes) (20). Both forms can be readily differentiated from other molybdenum hydroxylases by phylogenetic analysis. Form I CODH (also called OMS, named after Oligotropha, Mycobacterium, and Pseudomonas) has been conclusively proven by mutagenesis experiments and X-ray crystallography (8, 32, 35) to be the key enzyme in aerobic CO oxidation by carboxydotrophic bacteria, i.e., those that can grow on CO as the sole carbon and energy source (at >10% CO concentration). The reaction mechanism has also been clearly defined. Form I CODH large-subunit CoxL can be readily diagnosed by its characteristic catalytic site motif AYXCSFR. Moreover, in all the organisms in which form I CODH genes have been identified so far, the genomic organization of the three subunits is always M→S→L. The organization of the accessory genes, however, may vary from organism to organism.There is much less known about the other form, form II CODH (or BMS, after Bradyrhizobium, Mesorhizobium, and Sinorhizobium), which was first described in Bradyrhizobium japonicum USDA 110 (23), a gram-negative bacterial strain and a nitrogen-fixing symbiont of soybeans. Form II CODH enables these bacteria to grow, albeit slowly, in the presence of CO as the sole carbon and energy source, but the rate of CO oxidation by form II CODH of B. japonicum USDA 110 is 10 to 1,000 times lower than that for form I CODH in O. carboxidovorans OM5 and Pseudomonas carboxydohydrogena OM5. The catalytic site of the form II CoxL large subunit is AYRGAGR. The genome organization of the form II subunits is S→L→M, different from that of form I. The number of accessory genes present along with these genes is also variable (20). Form II is often found as a paralogous copy of three subunits of form I, but without the accompanying set of CODH-related genes. This is not surprising, since in most cases, the genes appear to be already associated with the form I cluster elsewhere in the genome, like in Rhodothermus marinus DSM 4252, Dinoroseobacter shibae DFL 12, and Bradyrhizobium sp. strain BTAi1.The discovery of the role played by CODH in marine waters is relatively recent. First, it was found in the genome of Silicibacter pomeroyi DSS-3, a marine alphaproteobacterium of the Roseobacter cluster (26), which was concomitantly proven to be able to oxidize CO at low concentrations (as should be expected in marine waters). Later on the process, it was also found in metagenomic studies of surface waters for the Sargasso Sea metagenome project (26, 45). It has been proposed that many heterotrophic bacteria in surface waters are lithoheterotrophs and take advantage of the CO released by organic matter photolysis as an alternative energy source to supplement the scarce dissolved organic matter in a way akin to the photoheterotrophy mediated by proteorhodopsin or anoxygenic photosynthesis.We recently found evidence of a CODH presence deep in the Mediterranean Sea by the end sequencing of fosmids from a metagenomic library from a 3,000-m depth in the Ionian Sea (southeast of Sicily, Italy) (24). Here we present the analysis of nine fully sequenced fosmids that were chosen on the basis of the presence of CODH cluster genes at their ends. The results confirm the presence of complete CODH clusters, including one that has the gene sequence and cluster structure of a form I CODH. Although the source of CO deep in the ocean is unclear, the frequency in which these genes were found and the retrieval of similar sequences from the deep-ocean metagenomic database of the Hawaii Ocean Time-Series (HOT) station (7, 21) point toward an important contribution of this lithotrophic metabolism deep in the ocean, similarly relevant to that found in the surface waters.     

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.     

Detection of a Salmonella enterica serovar California strain spreading in spanish feed mills and genetic characterization with DNA microarrays

We performed an epidemiological study on Salmonella isolated from raw plant-based feed in Spanish mills. Overall, 32 different Salmonella serovars were detected. Despite its rare occurrence in humans and animals, Salmonella enterica serovar California was found to be the predominant serovar in Spanish feed mills. Different typing techniques showed that isolates of this serovar were genetically closely related, and comparative genomic hybridization using microarray technology revealed 23 S. enterica serovar Typhimurium LT2 gene clusters that are absent from serovar California.