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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 26 April 2011, Vol.108(17), pp.7200-5
    Description: Defining bacterial species remains a challenging problem even for the model bacterium Escherichia coli and has major practical consequences for reliable diagnosis of infectious disease agents and regulations for transport and possession of organisms of economic importance. E. coli traditionally is thought to live within the gastrointestinal tract of humans and other warm-blooded animals and not to survive for extended periods outside its host; this understanding is the basis for its widespread use as a fecal contamination indicator. Here, we report the genome sequences of nine environmentally adapted strains that are phenotypically and taxonomically indistinguishable from typical E. coli (commensal or pathogenic). We find, however, that the commensal genomes encode for more functions that are important for fitness in the human gut, do not exchange genetic material with their environmental counterparts, and hence do not evolve according to the recently proposed fragmented speciation model. These findings are consistent with a more stringent and ecologic definition for bacterial species than the current definition and provide means to start replacing traditional approaches of defining distinctive phenotypes for new species with omics-based procedures. They also have important implications for reliable diagnosis and regulation of pathogenic E. coli and for the coliform cell-counting test.
    Keywords: Evolution, Molecular ; Models, Biological ; Escherichia Coli -- Genetics ; Genome, Bacterial -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 14 August 2012, Vol.109(33), pp.13272-7
    Description: Deep sequencing has enabled the investigation of a wide range of environmental microbial ecosystems, but the high memory requirements for de novo assembly of short-read shotgun sequencing data from these complex populations are an increasingly large practical barrier. Here we introduce a memory-efficient graph representation with which we can analyze the k-mer connectivity of metagenomic samples. The graph representation is based on a probabilistic data structure, a Bloom filter, that allows us to efficiently store assembly graphs in as little as 4 bits per k-mer, albeit inexactly. We show that this data structure accurately represents DNA assembly graphs in low memory. We apply this data structure to the problem of partitioning assembly graphs into components as a prelude to assembly, and show that this reduces the overall memory requirements for de novo assembly of metagenomes. On one soil metagenome assembly, this approach achieves a nearly 40-fold decrease in the maximum memory requirements for assembly. This probabilistic graph representation is a significant theoretical advance in storing assembly graphs and also yields immediate leverage on metagenomic assembly.
    Keywords: Computational Biology ; Genome, Bacterial -- Genetics ; Metagenome -- Genetics ; Sequence Analysis, DNA -- Methods
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 01 April 2014, Vol.111(13), pp.4904-9
    Description: The large volumes of sequencing data required to sample deeply the microbial communities of complex environments pose new challenges to sequence analysis. De novo metagenomic assembly effectively reduces the total amount of data to be analyzed but requires substantial computational resources. We combine two preassembly filtering approaches--digital normalization and partitioning--to generate previously intractable large metagenome assemblies. Using a human-gut mock community dataset, we demonstrate that these methods result in assemblies nearly identical to assemblies from unprocessed data. We then assemble two large soil metagenomes totaling 398 billion bp (equivalent to 88,000 Escherichia coli genomes) from matched Iowa corn and native prairie soils. The resulting assembled contigs could be used to identify molecular interactions and reaction networks of known metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes Orthology database. Nonetheless, more than 60% of predicted proteins in assemblies could not be annotated against known databases. Many of these unknown proteins were abundant in both corn and prairie soils, highlighting the benefits of assembly for the discovery and characterization of novelty in soil biodiversity. Moreover, 80% of the sequencing data could not be assembled because of low coverage, suggesting that considerably more sequencing data are needed to characterize the functional content of soil.
    Keywords: Biodiversity ; Soil ; Soil Microbiology ; Metagenome -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 2011, Vol.108(35), pp.14637-14642
    Description: High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. ; p. 14637-14642.
    Keywords: Data Collection ; Habitats ; Genes ; Phylogeny ; Ribosomal Rna ; Bacterial Communities ; High-Throughput Nucleotide Sequencing ; Sequence Homology ; Nucleotide Sequences ; Sequence Alignment ; Community Structure ; Taxonomy ; Species Diversity
    ISSN: 0027-8424
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States, Feb 26, 2013, Vol.110(9), p.3435(6)
    Description: Antibiotic resistance genes (ARGs) are emerging contaminants posing a potential worldwide human health risk. Intensive animal husbandry is believed to be a major contributor to the increased environmental burden of ARGs. Despite the volume of antibiotics used in China, little information is available regarding the corresponding ARGs associated with animal farms. We assessed type and concentrations of ARGs at three stages of manure processing to land disposal at three large-scale (10,000 animals per year) commercial swine farms in China. In-feed or therapeutic antibiotics used on these farms include all major classes of antibiotics except vancomycins, High-capacity quantitative PCR arrays detected 149 unique resistance genes among all of the farm samples, the top 63 ARGs being enriched 192-fold (median) up to 28,000-fold (maximum) compared with their respective antibiotic-free manure or soil controls. Antibiotics and heavy metals used as feed supplements were elevated in the manures, suggesting the potential for coselection of resistance traits. The potential for horizontal transfer of ARGs because of transposon-specific ARGs is implicated by the enrichment of transposases--the top six alleles being enriched 189-fold (median) up to 90,000-fold in manure--as well as the high correlation ([r.sup.2] = 0.96) between ARG and transposase abundance. In addition, abundance of ARGs correlated directly with antibiotic and metal concentrations, indicating their importance in selection of resistance genes. Diverse, abundant, and potentially mobile ARGs in farm samples suggest that unmonitored use of antibiotics and metals is causing the emergence and release of ARGs to the environment. concentrated animal feeding operations | horizontal gene transfer | growth-promoting antibiotics | tetracycline doi/ 10.1073/pnas.1222743110
    Keywords: Microbial Drug Resistance -- Research ; Livestock Industry ; Agriculture ; Livestock Farms ; Genes ; Swine ; Pork Industry
    ISSN: 0027-8424
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  • 6
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 8 March 2011, Vol.108(10), pp.4170-4175
    Description: Ammonia oxidation is an essential part of the global nitrogen cycling and was long thought to be driven only by bacteria. Recent findings expanded this pathway also to the archaea. However, most questions concerning the metabolism of ammonia-oxidizing archaea, such as ammonia oxidation and potential CO₂ fixation, remain open, especially for terrestrial environments. Here, we investigated the activity of ammonia-oxidizing archaea and bacteria in an agricultural soil by comparison of RNA-and DNA-stable isotope probing (SIP). RNA-SIP demonstrated a highly dynamic and diverse community involved in CO₂ fixation and carbon assimilation coupled to ammonia oxidation. DNA-SIP showed growth of the ammonia-oxidizing bacteria but not of archaea. Furthermore, the analysis of labeled RNA found transcripts of the archaeal acetyl-CoA/propionyl-CoA carboxylase (accA/pccB) to be expressed and labeled. These findings strongly suggest that ammoniaoxidizing archaeal groups in soil autotrophically fix CO₂ using the 3-hydroxypropionate-4-hydroxybutyrate cycle, one of the two pathways recently identified for CO₂ fixation in Crenarchaeota. Catalyzed reporter deposition (CARD)-FISH targeting the gene encoding subunit A of ammonia monooxygenase (amoA) mRNA and 16S rRNA of archaea also revealed ammonia-oxidizing archaea to be numerically relevant among the archaea in this soil. Our results demonstrate a diverse and dynamic contribution of ammonia-oxidizing archaea in soil to nitrification and CO₂ assimilation and that their importance to the overall archaeal community might be larger than previously thought.
    Keywords: Biological sciences -- Biology -- Biological taxonomies ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biology -- Microbiology ; Biological sciences -- Agriculture -- Agricultural sciences ; Biological sciences -- Agriculture -- Agricultural sciences ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical reactions ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Chemistry -- Chemical compounds
    ISSN: 00278424
    E-ISSN: 10916490
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  • 7
    Language: English
    In: Applied and environmental microbiology, July 2015, Vol.81(13), pp.4536-45
    Description: The sequencing chips and kits of the Ion Torrent Personal Genome Machine (PGM), which employs semiconductor technology to measure pH changes in polymerization events, have recently been upgraded. The quality of PGM sequences has not been reassessed, and results have not been compared in the context of a gene-targeted microbial ecology study. To address this, we compared sequence profiles across available PGM chips and chemistries and with 454 pyrosequencing data by determining error types and rates and diazotrophic community structures. The PGM was then used to assess differences in nifH-harboring bacterial community structure among four corn-based cropping systems. Using our suggested filters from mock community analyses, the overall error rates were 0.62, 0.36, and 0.39% per base for chips 318 and 314 with the 400-bp kit and chip 318 with the Hi-Q chemistry, respectively. Compared with the 400-bp kit, the Hi-Q kit reduced indel rates by 28 to 59% and produced one to seven times more reads acceptable for downstream analyses. The PGM produced higher frameshift rates than pyrosequencing that were corrected by the RDP FrameBot tool. Significant differences among platforms were identified, although the diversity indices and overall site-based conclusions remained similar. For the cropping system analyses, a total of 6,182 unique NifH operational taxonomic units at 5% amino acid dissimilarity were obtained. The current crop type, as well as the crop rotation history, significantly influenced the composition of the soil diazotrophic community detected.
    Keywords: Biota ; Soil Microbiology ; Molecular Biology -- Methods ; Oxidoreductases -- Genetics ; Sequence Analysis, DNA -- Methods
    ISSN: 00992240
    E-ISSN: 1098-5336
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  • 8
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 5 April 2011, Vol.108(14), pp.5649-5654
    Description: Diatoms survive in dark, anoxic sediment layers for months to decades. Our investigation reveals a correlation between the dark survival potential of marine diatoms and their ability to accumulate NO₃⁻ intracellularly. Axenic strains of benthic and pelagic diatoms that stored 11-274 mM NO₃⁻ in their cells survived for 6-28 wk. After sudden shifts to dark, anoxic conditions, the benthic diatom Amphora coffeaeformis consumed 84-87% of its intracellular NO₃⁻ pool within 1 d. A stable-isotope labeling experiment proved that ¹⁵NO₃⁻ consumption was accompanied by the production and release of ¹⁵NH₄⁺, indicating dissimilatory nitrate reduction to ammonium (DNRA). DNRA is an anaerobic respiration process that is known mainly from prokaryotic organisms, and here shown as dissimilatory nitrate reduction pathway used by a eukaryotic phototroph. Similar to large sulfur bacteria and benthic foraminifera, diatoms may respire intracellular NO₃⁻ in sediment layers without O₂ and NO₃⁻. The rapid depletion of the intracellular NO₃⁻ storage, however, implies that diatoms use DNRA to enter a resting stage for long-term survival. Assuming that pelagic diatoms are also capable of DNRA, senescing diatoms that sink through oxygen-deficient water layers may be a significant NH₄⁺ source for anammox, the prevalent nitrogen loss pathway of oceanic oxygen minimum zones.
    Keywords: Biological sciences -- Biology -- Botany ; Physical sciences -- Earth sciences -- Geology ; Physical sciences -- Chemistry -- Chemical compounds ; Biological sciences -- Biology -- Cytology ; Physical sciences -- Earth sciences -- Hydrology ; Applied sciences -- Laboratory techniques -- Culture techniques ; Physical sciences -- Chemistry -- Chemical elements ; Biological sciences -- Biology -- Cytology ; Biological sciences -- Biology -- Microbiology ; Biological sciences -- Biology -- Microbiology
    ISSN: 00278424
    E-ISSN: 10916490
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  • 9
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 15 January 2013, Vol.110(3), pp.988-93
    Description: The Amazon rainforest is the Earth's largest reservoir of plant and animal diversity, and it has been subjected to especially high rates of land use change, primarily to cattle pasture. This conversion has had a strongly negative effect on biological diversity, reducing the number of plant and animal species and homogenizing communities. We report here that microbial biodiversity also responds strongly to conversion of the Amazon rainforest, but in a manner different from plants and animals. Local taxonomic and phylogenetic diversity of soil bacteria increases after conversion, but communities become more similar across space. This homogenization is driven by the loss of forest soil bacteria with restricted ranges (endemics) and results in a net loss of diversity. This study shows homogenization of microbial communities in response to human activities. Given that soil microbes represent the majority of biodiversity in terrestrial ecosystems and are intimately involved in ecosystem functions, we argue that microbial biodiversity loss should be taken into account when assessing the impact of land use change in tropical forests.
    Keywords: Agriculture ; Biodiversity ; Soil Microbiology ; Tropical Climate ; Bacteria -- Isolation & Purification
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 10
    Language: English
    In: Applied and environmental microbiology, November 2013, Vol.79(22), pp.6932-40
    Description: Understanding of functional diversity of microbial populations has lagged description of their molecular diversity. Differences in substrate specificity, kinetics, products, and regulation can dramatically influence phenotypic variation among closely related strains, features that are missed when the strains studied are the fastest-growing and most easily isolated from serial enrichments. To investigate the broader bacterial diversity underlying degradation of anthropogenic chemicals in nature, we studied the 3-chlorobenzoate (3-CBA) degradation rate in a collection of aerobic 3-CBA degraders previously isolated from undisturbed soils in two representative ecosystems: (i) Mediterranean sclerophyllous woodlands in California, Chile, South Africa, and Australia and (ii) boreal forests in Canada and Russia. The majority of isolates degraded 3-CBA slowly and did not completely mineralize 1.0 mM 3-CBA within 1 week. Those with intermediate degradation rates had incomplete degradation pathways and produced colored intermediates indicative of chlorocatechol, a product likely metabolized by other members of the community. About 10% of the isolates grew rapidly and mineralized greater than 90% of the 3-CBA, but because of population heterogeneity in soil, they are likely not large contributors to a soil's total transformation capacity. This suggests that xenobiotic degradation in nature is carried out by a community of cometabolic generalists and not by the efficient specialists that have been traditionally studied in the laboratory. A subset of 58 genotypically distinct strains able to degrade 〉80% of the 3-CBA was examined for their catabolic versatility using 45 different compounds: mono- and dichlorinated benzoates, phenols, anilines, toluenes, nitrobenzenes, chlorobenzenes, and 2,4-dichlorophenoxyacetic acid. The isolates degraded from 2 to more than 30 compounds with a median of 7, but there was no correlation to habitat of isolation or 3-CBA activity. However, these findings were indicative of finer-scale functional diversity.
    Keywords: Ecosystem ; Soil Microbiology ; Chlorobenzoates -- Analysis
    ISSN: 00992240
    E-ISSN: 1098-5336
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