Design and testing of 'genome-proxy' microarrays to profile marine microbial communities

Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a 'genome-proxy' microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to 13 of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual open reading frame, and distributed along each approximately 40-160 kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having 相似文献   

Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre

High-throughput identification of rRNA gene-containing clones in large insert metagenomic libraries is difficult, because of the high background of host ribosomal RNA (rRNA) and rRNA genes. To address this challenge, a membrane hybridization method was developed to identify all bacterial small subunit rRNA-containing fosmid clones of microbial community DNA from seven different depths in the North Pacific Subtropical Gyre. Out of 101,376 clones screened, 751 rDNA-containing clones were identified that grouped in ∼60 different clades. Several rare sequences only remotely related to known groups were detected, including a Wolbachia -related sequence containing a putative intron or intervening sequence, as well as seven sequences from Order Myxococcales not previously detected in pelagic habitats. Stratified, depth-specific population structure was evident within both cultured and uncultured lineages. Conversely, some eurybathyal members of the genera Alcanivorax and Rhizobium shared identical small subunit ribosomal DNA sequences that were distributed from surface waters to the 4000 m depth. Comparison with similar analyses in Monterey Bay microbial communities revealed previously recognized, as well as some distinctive, depth-stratified partitioning that distinguished coastal from open ocean bacterioplankton populations. While some bias was evident in fosmid clone recovery in a few particular lineages, the overall phylogenetic group recovery and distributions were consistent with previous studies, as well as with direct shotgun sequence data from the same source DNA.     

Two nucleotide substitutions in the A-site of yeast 18S rRNA affect translation and differentiate the interaction of ribosomes with aminoglycoside antibiotics

Two mutations in the A-site of 18S rRNA of Saccharomyces cerevisiae were investigated. The first, A1491G (rdn15), creates in yeast the same C1409-G1491 base pair as in Escherichia coli and has behaved as an antisuppressor in genetic studies. Ribosomes from rdn15 are error-restrictive but their peptidyltransferase activity remains unchanged. The second mutation, U1495C (rdnhyg1), was initially isolated as a hygromycin-resistant mutation in Tetrahymena thermophila. We show that rdnhyg1 ribosomes are slightly error prone. Mutation rdnhyg1 does not affect catalytic activity, but it affects translocation, confirming the importance of nucleotide 1495 in the ratchet-like movement of the two subunits during translation. Paromomycin, an aminoglycoside antibiotic that binds to the ribosomal A-site, induces translational misreading and causes sensitivity to yeast cells. Mutation rdn15 is shown to be highly sensitive to both effects of paromomycin, while mutation rdnhyg1 is relatively resistant. Tobramycin, another aminoglycoside, does not affect the growth of yeast cells. Like paromomycin, however, it increases the error rate in rdn15 ribosomes relative to wild-type and decreases it in rdnhyg1 ribosomes. These mutations help define the role of two crucial sites in ribosome function and distinguish the modes of action of two aminoglycosides, a useful fact in the search for new strategies in drug design.     

Individual genome assembly from complex community short-read metagenomic datasets

Assembling individual genomes from complex community metagenomic data remains a challenging issue for environmental studies. We evaluated the quality of genome assemblies from community short read data (Illumina 100 bp pair-ended sequences) using datasets recovered from freshwater and soil microbial communities as well as in silico simulations. Our analyses revealed that the genome of a single genotype (or species) can be accurately assembled from a complex metagenome when it shows at least about 20 × coverage. At lower coverage, however, the derived assemblies contained a substantial fraction of non-target sequences (chimeras), which explains, at least in part, the higher number of hypothetical genes recovered in metagenomic relative to genomic projects. We also provide examples of how to detect intrapopulation structure in metagenomic datasets and estimate the type and frequency of errors in assembled genes and contigs from datasets of varied species complexity.     

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.     

Strategic assay deployment as a method for countering analytical bottlenecks in high throughput process development: Case studies in ion exchange chromatography

High throughput approaches to facilitate the development of chromatographic separations have now been adopted widely in the biopharmaceutical industry, but issues of how to reduce the associated analytical burden remain. For example, acquiring experimental data by high level factorial designs in 96 well plates can place a considerable strain upon assay capabilities, generating a bottleneck that limits significantly the speed of process characterization. This article proposes an approach designed to counter this challenge; Strategic Assay Deployment (SAD). In SAD, a set of available analytical methods is investigated to determine which set of techniques is the most appropriate to use and how best to deploy these to reduce the consumption of analytical resources while still enabling accurate and complete process characterization. The approach is demonstrated by investigating how salt concentration and pH affect the binding of green fluorescent protein from Escherichia coli homogenate to an anion exchange resin presented in a 96‐well filter plate format. Compared with the deployment of routinely used analytical methods alone, the application of SAD reduced both the total assay time and total assay material consumption by at least 40% and 5%, respectively. SAD has significant utility in accelerating bioprocess development activities. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Microbial community structure and activity linked to contrasting biogeochemical gradients in bog and fen environments of the glacial lake agassiz peatland

The abundances, compositions, and activities of microbial communities were investigated at bog and fen sites in the Glacial Lake Agassiz Peatland of northwestern Minnesota. These sites contrast in the reactivity of dissolved organic matter (DOM) and the presence or absence of groundwater inputs. Microbial community composition was characterized using pyrosequencing and clone library construction of phylogenetic marker genes. Microbial distribution patterns were linked to pH, concentrations of dissolved organic carbon and nitrogen, C/N ratios, optical properties of DOM, and activities of laccase and peroxidase enzymes. Both bacterial and archaeal richness and rRNA gene abundance were >2 times higher on average in the fen than in the bog, in agreement with a higher pH, labile DOM content, and enhanced enzyme activities in the fen. Fungi were equivalent to an average of 1.4% of total prokaryotes in gene abundance assayed by quantitative PCR. Results revealed statistically distinct spatial patterns between bacterial and fungal communities. Fungal distribution did not covary with pH and DOM optical properties and was vertically stratified, with a prevalence of Ascomycota and Basidiomycota near the surface and much higher representation of Zygomycota in the subsurface. In contrast, bacterial community composition largely varied between environments, with the bog dominated by Acidobacteria (61% of total sequences), while the Firmicutes (52%) dominated in the fen. Acetoclastic Methanosarcinales showed a much higher relative abundance in the bog, in contrast to the dominance of diverse hydrogenotrophic methanogens in the fen. This is the first quantitative and compositional analysis of three microbial domains in peatlands and demonstrates that the microbial abundance, diversity, and activity parallel with the pronounced differences in environmental variables between bog and fen sites.     

Soil Microbial Community Responses to a Decade of Warming as Revealed by Comparative Metagenomics

Soil microbial communities are extremely complex, being composed of thousands of low-abundance species (<0.1% of total). How such complex communities respond to natural or human-induced fluctuations, including major perturbations such as global climate change, remains poorly understood, severely limiting our predictive ability for soil ecosystem functioning and resilience. In this study, we compared 12 whole-community shotgun metagenomic data sets from a grassland soil in the Midwestern United States, half representing soil that had undergone infrared warming by 2°C for 10 years, which simulated the effects of climate change, and the other half representing the adjacent soil that received no warming and thus, served as controls. Our analyses revealed that the heated communities showed significant shifts in composition and predicted metabolism, and these shifts were community wide as opposed to being attributable to a few taxa. Key metabolic pathways related to carbon turnover, such as cellulose degradation (∼13%) and CO₂ production (∼10%), and to nitrogen cycling, including denitrification (∼12%), were enriched under warming, which was consistent with independent physicochemical measurements. These community shifts were interlinked, in part, with higher primary productivity of the aboveground plant communities stimulated by warming, revealing that most of the additional, plant-derived soil carbon was likely respired by microbial activity. Warming also enriched for a higher abundance of sporulation genes and genomes with higher G+C content. Collectively, our results indicate that microbial communities of temperate grassland soils play important roles in mediating feedback responses to climate change and advance the understanding of the molecular mechanisms of community adaptation to environmental perturbations.