Seasonality and disturbance: annual pattern and response of the bacterial and microbial eukaryotic assemblages in a freshwater ecosystem.

High-throughput pyrosequencing of SSU rDNA genes was used to obtain monthly snapshots of eukaryotic and bacterial diversity and community structure at two locations in Lake Texoma, a low salinity lake in the south central United States, over 1 year. The lake experienced two disturbance events (i) a localized bloom of Prymnesium parvum restricted to one of the locations that lasted from January to April, and (ii) a large (17 cm), global rain event in the beginning of May, overlaid onto seasonal environmental change. Eukaryotic species richness as well as both eukaryotic and bacterial community similarity exhibited seasonal patterns, including distinct responses to the rain event. The P. parvum bloom created a natural experiment in which to directly explore the effects of an Ecosystem Disruptive Algal Bloom (EDAB) on the microbial community separated from seasonal changes. Microbial species richness was unaffected by the bloom, however, the eukaryotic community structure (evenness) and the patterns of both eukaryotic and bacterial community similarity at bloom and non-bloom sites were statistically distinct during the 4 months of the bloom. These results indicate that physical and biological disturbances as well as seasonal environmental forces contribute to the structure of both the eukaryotic and bacterial communities.

Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimedium sagittatum (Sieb. Et Zucc.) Maxim.

BACKGROUND: Epimedium sagittatum (Sieb. Et Zucc.) Maxim, a traditional Chinese medicinal plant species, has been used extensively as genuine medicinal materials. Certain Epimedium species are endangered due to commercial overexploition, while sustainable application studies, conservation genetics, systematics, and marker-assisted selection (MAS) of Epimedium is less-studied due to the lack of molecular markers. Here, we report a set of expressed sequence tags (ESTs) and simple sequence repeats (SSRs) identified in these ESTs for E. sagittatum. RESULTS: cDNAs of E. sagittatum are sequenced using 454 GS-FLX pyrosequencing technology. The raw reads are cleaned and assembled into a total of 76,459 consensus sequences comprising of 17,231 contigs and 59,228 singlets. About 38.5% (29,466) of the consensus sequences significantly match to the non-redundant protein database (E-value < 1e-10), 22,295 of which are further annotated using Gene Ontology (GO) terms. A total of 2,810 EST-SSRs is identified from the Epimedium EST dataset. Trinucleotide SSR is the dominant repeat type (55.2%) followed by dinucleotide (30.4%), tetranuleotide (7.3%), hexanucleotide (4.9%), and pentanucleotide (2.2%) SSR. The dominant repeat motif is AAG/CTT (23.6%) followed by AG/CT (19.3%), ACC/GGT (11.1%), AT/AT (7.5%), and AAC/GTT (5.9%). Thirty-two SSR-ESTs are randomly selected and primer pairs are synthesized for testing the transferability across 52 Epimedium species. Eighteen primer pairs (85.7%) could be successfully transferred to Epimedium species and sixteen of those show high genetic diversity with 0.35 of observed heterozygosity (Ho) and 0.65 of expected heterozygosity (He) and high number of alleles per locus (11.9). CONCLUSION: A large EST dataset with a total of 76,459 consensus sequences is generated, aiming to provide sequence information for deciphering secondary metabolism, especially for flavonoid pathway in Epimedium. A total of 2,810 EST-SSRs is identified from EST dataset and approximately 1580 EST-SSR markers are transferable. E. sagittatum EST-SSR transferability to the major Epimedium germplasm is up to 85.7%. Therefore, this EST dataset and EST-SSRs will be a powerful resource for further studies such as taxonomy, molecular breeding, genetics, genomics, and secondary metabolism in Epimedium species.

Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer.

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.