Project description:Saccharomonospora azurea Runmao et al. 1987 is a member of the genus Saccharomonospora, which is in the family Pseudonocardiaceae and thus far poorly characterized genomically. Members of the genus Saccharomonospora are of interest because they originate from diverse habitats, such as leaf litter, manure, compost, the surface of peat, and moist and over-heated grain, and may play a role in the primary degradation of plant material by attacking hemicellulose. Next to S. viridis, S. azurea is only the second member in the genus Saccharomonospora for which a completely sequenced type strain genome will be published. Here we describe the features of this organism, together with the complete genome sequence with project status 'Improved high quality draft', and the annotation. The 4,763,832 bp long chromosome with its 4,472 protein-coding and 58 RNA genes was sequenced as part of the DOE funded Community Sequencing Program (CSP) 2010 at the Joint Genome Institute (JGI).
Project description:In the present study, the detection of a pantropic canine coronavirus (CCoV) strain in a dog with lethal diarrhoea is reported. RT-PCR and real-time RT-PCR assays were used for the detection, characterization and quantitation of CCoV. Sequence and phylogenetic analysis of the CCoV NA/09 revealed a high degree of sequence identity with the pantropic strain CB/05, indicating the presence of CB/05-like pantropic strains in Greece. The absence of the 38-nucleotide deletion in ORF3b, which is characteristic of CB/05, indicates the need to identify new genetic markers for pantropic variants of CCoV, probably in the spike-protein gene region.
Project description:Saccharomonospora cyanea Runmao et al. 1988 is a member of the genus Saccharomonospora in the family Pseudonocardiaceae that is moderately well characterized at the genome level thus far. Members of the genus Saccharomonospora are of interest because they originate from diverse habitats, such as soil, leaf litter, manure, compost, surface of peat, moist, over-heated grain, and ocean sediment, where they probably play a role in the primary degradation of plant material by attacking hemicellulose. Species of the genus Saccharomonospora are usually Gram-positive, non-acid fast, and are classified among the actinomycetes. S. cyanea is characterized by a dark blue (= cyan blue) aerial mycelium. After S. viridis, S. azurea, and S. marina, S. cyanea is only the fourth member in the genus for which a completely sequenced (non-contiguous finished draft status) type strain genome will be published. Here we describe the features of this organism, together with the draft genome sequence, and annotation. The 5,408,301 bp long chromosome with its 5,139 protein-coding and 57 RNA genes was sequenced as part of the DOE funded Community Sequencing Program (CSP) 2010 at the Joint Genome Institute (JGI).
Project description:Background: Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomics strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis (BSA) and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na+ from the soil. Methodology/Principal Findings: Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that Ts-1 and Tsu-1 accumulate higher shoot levels of Na+ than Col-0 and other accessions. We identify AtHKT1, known to encode a Na+ transporter, as being the causal locus driving elevated shoot Na+ in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence ~5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na+. Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions this novel AtHKT1 allele not only does not confer NaCl sensitivity, but co-segregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; www.purdue.edu/dp/ionomics). Conclusions/Significance: Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the causal locus driving the natural variation in shoot Na+ accumulation we observed in Ts-1 and Tsu-1, two coastal populations of Arabidopsis. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions, and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes. Keywords: genomics hybridization bulk segregant analysis
Project description:A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.
Project description:Halomonas sp. Y2 is a halotolerant alkaliphilic strain from Na+-rich pulp mill wastewater with high alkalinity (pH >11.0). Transcriptome analysis of this isolate revealed this strain may use various transport systems for pH homeostasis. In particular, the genes encoding four putative Na+/H+ antiporters were differentially expressed upon acidic or alkaline conditions. Further evidence, from heterologous expression and mutant studies, suggested that Halomonas sp. Y2 employs its Na+/H+ antiporters in a labor division way to deal with saline and alkaline environments. Ha-NhaD2 displayed robust Na+(Li+) resistance and high transport activities in Escherichia coli; a ?Ha-nhaD2 mutant exhibited growth inhibition at high Na+(Li+) concentrations at pH values of 6.2, 8.0, and 10.0, suggesting its physiological role in osmotic homeostasis. In contrast, Ha-NhaD1 showed much weaker activities in ion exporting and pH homeostasis. Ha-Mrp displayed a combination of properties similar to those of Mrp transporters from some Bacillus alkaliphiles and neutrophiles. This conferred obvious Na+(Li+, K+) resistance in E. coli-deficient strains, as those ion transport spectra of some neutrophil Mrp antiporters. Conversely, similar to the Bacillus alkaliphiles, Ha-Mrp showed central roles in the pH homeostasis of Halomonas sp. Y2. An Ha-mrp-disrupted mutant was seriously inhibited by high concentrations of Na+(Li+, K+) but only under alkaline conditions. Ha-NhaP was determined to be a K+/H+ antiporter and shown to confer strong K+ resistance both at acidic and alkaline stresses.
Project description:Abstract The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the non‐genetically modified Aspergillus oryzae strain NZYM‐NA by Novozymes A/S. It was considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: starch processing for the production of glucose and maltose syrups and other starch hydrolysates, distilled alcohol production, brewing processes, baking processes, cereal‐based processes, plant processing for production of dairy analogues and fruit and vegetable processing for juice production. Since residual amounts of food enzyme–total organic solids (TOS) are removed by the purification steps applied during the production of glucose syrups and distillation, dietary exposure was not calculated for these processes. For the remaining five food manufacturing processes, dietary exposure was estimated to be up to 0.134 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,862 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 13,896. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use (other than distilled alcohol production), the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.
Project description:The whole-cell voltage-clamp technique was used in rat cardiac myocytes to investigate the kinetics of ADP binding to phosphorylated states of Na,K-ATPase and its effects on presteady-state Na(+)-dependent charge movements by this enzyme. Ouabain-sensitive transient currents generated by Na,K-ATPase functioning in electroneutral Na(+)-Na(+) exchange mode were measured at 23 degrees C with pipette ADP concentrations ([ADP]) of up to 4.3 mM and extracellular Na(+) concentrations ([Na](o)) between 36 and 145 mM at membrane potentials (V(M)) from -160 to +80 mV. Analysis of charge-V(M) curves showed that the midpoint potential of charge distribution was shifted toward more positive V(M) both by increasing [ADP] at constant Na(+)(o) and by increasing [Na](o) at constant ADP. The total quantity of mobile charge, on the other hand, was found to be independent of changes in [ADP] or [Na](o). The presence of ADP increased the apparent rate constant for current relaxation at hyperpolarizing V(M) but decreased it at depolarizing V(M) as compared to control (no added ADP), an indication that ADP binding facilitates backward reaction steps during Na(+)-Na(+) exchange while slowing forward reactions. Data analysis using a pseudo three-state model yielded an apparent K(d) of approximately 6 mM for ADP binding to and release from the Na,K-ATPase phosphoenzyme; a value of 130 s(-1) for k(2), a rate constant that groups Na(+) deocclusion/release and the enzyme conformational transition E(1) approximately P --> E(2)-P; a value of 162 s(-1)M(-1) for k(-2), a lumped second-order V(M)-independent rate constant describing the reverse reactions; and a Hill coefficient of approximately 1 for Na(+)(o) binding to E(2)-P. The results are consistent with electroneutral release of ADP before Na(+) is deoccluded and released through an ion well. The same approach can be used to study additional charge-moving reactions and associated electrically silent steps of the Na,K-pump and other transporters.
Project description:Tolerance to high soil [Na(+)] involves processes in many different parts of the plant, and is manifested in a wide range of specializations at disparate levels of organization, such as gross morphology, membrane transport, biochemistry and gene transcription. Multiple adaptations to high [Na(+)] operate concurrently within a particular plant, and mechanisms of tolerance show large taxonomic variation. These mechanisms can occur in all cells within the plant, or can occur in specific cell types, reflecting adaptations at two major levels of organization: those that confer tolerance to individual cells, and those that contribute to tolerance not of cells per se, but of the whole plant. Salt-tolerant cells can contribute to salt tolerance of plants; but we suggest that equally important in a wide range of conditions are processes involving the management of Na(+) movements within the plant. These require specific cell types in specific locations within the plant catalysing transport in a coordinated manner. For further understanding of whole plant tolerance, we require more knowledge of cell-specific transport processes and the consequences of manipulation of transporters and signalling elements in specific cell types.