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In JoVE (8)
- أخذ عينات مياه البحر وكوكتيل
- كبيرة الحجم (20L +) الترشيح لعينة من ماء البحر الساحلية
- حجم صغير (1 - 3L) الترشيح للعينات مياه البحر الساحلية
- استخراج الحمض النووي من 0.22 ميكرومتر Sterivex فلاتر والسيزيوم كلوريد التدرج الكثافة الطرد المركزي
- مكتبة كبيرة إدراج الجينوم البيئية الإنتاج
- استخراج عالية الوزن الجزيئي الحمض النووي الجينوم من التربة والرواسب
- التعبير عن البروتينات المؤتلف في الخميرة Methylotrophic Pichia pastoris
- شاشة إنتاجية عالية للنشاط Biomining سلولاز من مكتبات Metagenomic
Other Publications (19)
- Nature Neuroscience
- Applied and Environmental Microbiology
- Applied and Environmental Microbiology
- Applied and Environmental Microbiology
- Science (New York, N.Y.)
- Journal of Bacteriology
- Science (New York, N.Y.)
- PLoS Biology
- Biochemical and Biophysical Research Communications
- Proceedings of the National Academy of Sciences of the United States of America
- Photochemistry and Photobiology
- Environmental Microbiology
- Environmental Microbiology
- Science (New York, N.Y.)
- Environmental Microbiology
- Methods in Enzymology
- Current Opinion in Biotechnology
- FEMS Microbiology Letters
- Astrobiology
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Articles by Steven J. Hallam in JoVE
JoVE General
أخذ عينات مياه البحر وكوكتيل
Department of Microbiology and Immunology, University of British Columbia - UBC
هذا الفيديو وثائق أساليب جمع عينات من المياه الساحلية البحرية وتجهيزها لتطبيقات مختلفة بما في ذلك المصب تركيز الكتلة الحيوية ، وحمض النووي تنقية ، ووفرة الخلية ، والمغذيات وتحليل الغاز التتبع.
JoVE General
كبيرة الحجم (20L +) الترشيح لعينة من ماء البحر الساحلية
Department of Microbiology and Immunology, University of British Columbia - UBC
هذا الكتاب وثائق الفيديو الكبيرة (L ≥ 20) ترشيح الكتلة الحيوية الميكروبية ، وتتراوح ما بين 0.22μm 2.7μm في القطر ، من عمود الماء.
JoVE General
حجم صغير (1 - 3L) الترشيح للعينات مياه البحر الساحلية
Department of Microbiology and Immunology, University of British Columbia - UBC
هذا الكتاب وثائق فيديو صغيرة (~ 1 L) ترشيح الكتلة الحيوية الميكروبية من عمود الماء.
JoVE General
استخراج الحمض النووي من 0.22 ميكرومتر Sterivex فلاتر والسيزيوم كلوريد التدرج الكثافة الطرد المركزي
Department of Microbiology and Immunology, University of British Columbia - UBC
نحن تصف طريقة لاستخراج الحمض النووي عالية الوزن الجزيئي الجينومية من الكتلة الحيوية العوالق ركزت على 0.22 ميكرون Sterivex المرشحات ، تليها السيزيوم كلوريد كثافة الطرد المركزي لتنقية الانحدار.
JoVE General
مكتبة كبيرة إدراج الجينوم البيئية الإنتاج
Department of Microbiology and Immunology, University of British Columbia - UBC
يوصف بناء مكتبة fosmid مع الحمض النووي الجيني البيئية معزولة عن عمق التواصل الرأسي لالمضيق البحري ميتة موسميا. يتم اختيار مكتبة استنساخ الناتج إلى 384 لوحات وكذلك المحفوظة في التسلسل الوظيفي المصب والفرز من تطبيق نظام اختيار مستعمرة الآلي.
JoVE General
استخراج عالية الوزن الجزيئي الحمض النووي الجينوم من التربة والرواسب
Department of Microbiology and Immunology, University of British Columbia - UBC
ووصف المنهجية لعزل الوزن الجزيئي عالية وعالية الجودة من الحمض النووي الجينومي المجتمع الميكروبي في التربة.
JoVE General
التعبير عن البروتينات المؤتلف في الخميرة Methylotrophic Pichia pastoris
Department of Microbiology and Immunology, University of British Columbia - UBC
بروتوكول يصف تعبير البروتين باستخدام الخميرة methylotrophic
JoVE General
شاشة إنتاجية عالية للنشاط Biomining سلولاز من مكتبات Metagenomic
Microbiology and Immunology, University of British Columbia - UBC
يصف هذا البروتوكول شاشة إنتاجية عالية للنشاط cellulolytic من مكتبة metagenomic أعرب في القولونية. الشاشة هو حل قائم ومؤتمتة للغاية ، ويستخدم واحد في 384 وعاء الكيمياء microplates جيدا مع قراءات النهائي باعتبارها قياس الامتصاصية.
Other articles by Steven J. Hallam on PubMed
SYD-1, a Presynaptic Protein with PDZ, C2 and RhoGAP-like Domains, Specifies Axon Identity in C. Elegans
Axons are defined by the presence of presynaptic specializations at specific locations. We show here that loss-of-function mutations in the C. elegans gene syd-1 cause presynaptic specializations to form in the dendritic processes of GABA-expressing motor neurons during initial differentiation. At a later developmental stage, however, syd-1 is not required for the polarity respecification of a subset of these neurons. The SYD-1 protein contains PDZ, C2 and rho-GTPase activating protein (GAP)-like domains, and is localized to presynaptic terminals in mature neurons. A truncated SYD-1 that lacks the rhoGAP domain interferes with neurite outgrowth and guidance. Our data indicate that syd-1 may be involved in specifying axon identity during initial polarity acquisition.
Growth and Methane Oxidation Rates of Anaerobic Methanotrophic Archaea in a Continuous-flow Bioreactor
Anaerobic methanotrophic archaea have recently been identified in anoxic marine sediments, but have not yet been recovered in pure culture. Physiological studies on freshly collected samples containing archaea and their sulfate-reducing syntrophic partners have been conducted, but sample availability and viability can limit the scope of these experiments. To better study microbial anaerobic methane oxidation, we developed a novel continuous-flow anaerobic methane incubation system (AMIS) that simulates the majority of in situ conditions and supports the metabolism and growth of anaerobic methanotrophic archaea. We incubated sediments collected from within and outside a methane cold seep in Monterey Canyon, Calif., for 24 weeks on the AMIS system. Anaerobic methane oxidation was measured in all sediments after incubation on AMIS, and quantitative molecular techniques verified the increases in methane-oxidizing archaeal populations in both seep and nonseep sediments. Our results demonstrate that the AMIS system stimulated the maintenance and growth of anaerobic methanotrophic archaea, and possibly their syntrophic, sulfate-reducing partners. Our data demonstrate the utility of combining physiological and molecular techniques to quantify the growth and metabolic activity of anaerobic microbial consortia. Further experiments with the AMIS system should provide a better understanding of the biological mechanisms of methane oxidation in anoxic marine environments. The AMIS may also enable the enrichment, purification, and isolation of methanotrophic archaea as pure cultures or defined syntrophic consortia.
Identification of Methyl Coenzyme M Reductase A (mcrA) Genes Associated with Methane-oxidizing Archaea
Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 or ANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.
Environmental Acquisition of Thiotrophic Endosymbionts by Deep-sea Mussels of the Genus Bathymodiolus
Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally "leaky" system of vertical transmission could not be excluded.
Reverse Methanogenesis: Testing the Hypothesis with Environmental Genomics
Microbial methane consumption in anoxic sediments significantly impacts the global environment by reducing the flux of greenhouse gases from ocean to atmosphere. Despite its significance, the biological mechanisms controlling anaerobic methane oxidation are not well characterized. One current model suggests that relatives of methane-producing Archaea developed the capacity to reverse methanogenesis and thereby to consume methane to produce cellular carbon and energy. We report here a test of the "reverse-methanogenesis" hypothesis by genomic analyses of methane-oxidizing Archaea from deep-sea sediments. Our results show that nearly all genes typically associated with methane production are present in one specific group of archaeal methanotrophs. These genome-based observations support previous hypotheses and provide an informed foundation for metabolic modeling of anaerobic methane oxidation.
Histones in Crenarchaea
Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.
Community Genomics Among Stratified Microbial Assemblages in the Ocean's Interior
Microbial life predominates in the ocean, yet little is known about its genomic variability, especially along the depth continuum. We report here genomic analyses of planktonic microbial communities in the North Pacific Subtropical Gyre, from the ocean's surface to near-sea floor depths. Sequence variation in microbial community genes reflected vertical zonation of taxonomic groups, functional gene repertoires, and metabolic potential. The distributional patterns of microbial genes suggested depth-variable community trends in carbon and energy metabolism, attachment and motility, gene mobility, and host-viral interactions. Comparative genomic analyses of stratified microbial communities have the potential to provide significant insight into higher-order community organization and dynamics.
Pathways of Carbon Assimilation and Ammonia Oxidation Suggested by Environmental Genomic Analyses of Marine Crenarchaeota
Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems.
Archaeal Pre-mRNA Splicing: a Connection to Hetero-oligomeric Splicing Endonuclease
Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.
Genomic Analysis of the Uncultivated Marine Crenarchaeote Cenarchaeum Symbiosum
Crenarchaeota are ubiquitous and abundant microbial constituents of soils, sediments, lakes, and ocean waters. To further describe the cosmopolitan nonthermophilic Crenarchaeota, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although they were syntenic, overlapping a- and b-type ribotype genomes harbored significant variability. A single tiling path comprising the dominant a-type genotype was assembled and used to explore the genomic properties of C. symbiosum and its planktonic relatives. Of 2,066 ORFs, 55.6% matched genes with predicted function from previously sequenced genomes. The remaining genes partitioned between functional RNAs (2.4%) and hypotheticals (42%) with limited homology to known functional genes. The latter category included some genes likely involved in the archaeal-sponge symbiotic association. Conversely, 525 C. symbiosum ORFs were most highly similar to sequences from marine environmental genomic surveys, and they apparently represent orthologous genes from free-living planktonic Crenarchaeota. In total, the C. symbiosum genome was remarkably distinct from those of other known Archaea and shared many core metabolic features in common with its free-living planktonic relatives.
Photophysics and Multifunctionality of Hypericin-like Pigments in Heterotrich Ciliates: a Phylogenetic Perspective
In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.
Bacterial, Archaeal and Eukaryal Community Structures Throughout Soil Horizons of Harvested and Naturally Disturbed Forest Stands
Disturbances caused by timber harvesting have critical long-term effects on the forest soil microbiota and alter fundamental ecosystem services provided by these communities. This study assessed the effects of organic matter removal and soil compaction on microbial community structures in different soil horizons 13 years after timber harvesting at the long-term soil productivity site at Skulow Lake, British Columbia. A harvested stand was compared with an unmanaged forest stand. Ribosomal intergenic spacer profiles of bacteria, archaea and eukarya indicated significantly different community structures in the upper three soil horizons of the two stands, with differences decreasing with depth. Large-scale sequencing of the ribosomal intergenic spacers coupled to small-subunit ribosomal RNA genes allowed taxonomic identification of major microbial phylotypes affected by harvesting or varying among soil horizons. Actinobacteria and Gemmatimonadetes were the predominant phylotypes in the bacterial profiles, with the relative abundance of these groups highest in the unmanaged stand, particularly in the deeper soil horizons. Predominant eukaryal phylotypes were mainly assigned to known mycorrhizal and saprotrophic species of Basidiomycetes and Ascomycetes. Harvesting affected Basidiomycetes to a minor degree but had stronger effects on some Ascomycetes. Archaeal profiles had low diversity with only a few predominant crenarchaeal phylotypes whose abundance appeared to increase with depth. Detection of these effects 13 years after harvesting may indicate a long-term change in processes mediated by the microbial community with important consequences for forest productivity. These effects warrant more comprehensive investigation of the effects of harvesting on the structure of forest soil microbial communities and the functional consequences.
Phylogenetic Diversity of Transition and Anoxic Zone Bacterial Communities Within a Near-shore Anoxic Basin: Nitinat Lake
At two stations surveyed in Nitinat Lake, a approximately 200-m-deep anoxic tidal fjord, sulfide was detected as close as 15 m from the surface. Biological characterization, determined from small subunit ribosomal RNA gene sequencing, of the chemocline and anaerobic zone revealed many sequences related to sulfur-oxidizing bacteria, suggesting that sulfur cycling is a dominant process. gamma- and epsilon-Proteobacteria related to thiotrophic symbionts, as well as Chlorobium sp., dominated the transition zone. These are expected to play a role in dark and phototrophic CO(2) fixation, respectively. epsilon-Proteobacteria phylotype abundance increased with depth, eventually comprising 69-97% of all sequences recovered from the anoxic zone. The vast majority (74%) of these phylotypes were affiliated with a novel Acrobacter sp. group (NITEP5). Quantification of NITEP5 revealed that up to 2.8 x 10(5) cells ml(-1) were present in the anoxic zone. Surprisingly, although sequences related to known sulfate-reducing bacteria were recovered from the transition zone, quantification of the dsr gene and (35)SO(4)(2-) uptake tests suggest that sulfate-reduction within the water column is negligible. Overall, sequence diversity between different vertical zones was high, although the spatial segregation of gamma-Proteobacteria, Chlorobi, and epsilon-Proteobacteria did not appear to vary significantly between seasons.
Metagenome of a Versatile Chemolithoautotroph from Expanding Oceanic Dead Zones
Oxygen minimum zones, also known as oceanic "dead zones," are widespread oceanographic features currently expanding because of global warming. Although inhospitable to metazoan life, they support a cryptic microbiota whose metabolic activities affect nutrient and trace gas cycling within the global ocean. Here, we report metagenomic analyses of a ubiquitous and abundant but uncultivated oxygen minimum zone microbe (SUP05) related to chemoautotrophic gill symbionts of deep-sea clams and mussels. The SUP05 metagenome harbors a versatile repertoire of genes mediating autotrophic carbon assimilation, sulfur oxidation, and nitrate respiration responsive to a wide range of water-column redox states. Our analysis provides a genomic foundation for understanding the ecological and biogeochemical role of pelagic SUP05 in oxygen-deficient oceanic waters and its potential sensitivity to environmental changes.
Microbial Community Dynamics in a Seasonally Anoxic Fjord: Saanich Inlet, British Columbia
Dissolved oxygen concentration plays a major role in shaping biotic interactions and nutrient flows within marine ecosystems. Throughout the global ocean, regions of low dissolved oxygen concentration (hypoxia) are a common and expanding feature of the water column, with major feedback on productivity and greenhouse gas cycling. To better understand microbial diversity underlying biogeochemical transformations within oxygen-deficient oceanic waters, we monitored and quantified bacterial and archaeal community dynamics in relation to dissolved gases and nutrients during a seasonal stratification and deep water renewal cycle in Saanich Inlet, British Columbia, a seasonally anoxic fjord. A number of microbial groups partitioned within oxygen-deficient waters including Nitrospina and SAR324 affiliated with the delta-proteobacteria, SAR406 and gamma-proteobacteria related to thiotrophic gill symbionts of deep-sea clams and mussels. Microbial diversity was highest within the hypoxic transition zone decreasing dramatically within anoxic basin waters and temporal patterns of niche partitioning were observed along defined gradients of oxygen and phosphate. These results provide a robust comparative phylogenetic framework for inferring systems metabolism of nitrogen, carbon and sulfur cycling within oxygen-deficient oceanic waters and establish Saanich Inlet as a tractable model for studying the response of microbial communities to changing levels of water column hypoxia.
Molecular Tools for Investigating ANME Community Structure and Function
Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabolism. These include reactions catalyzed by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and methyl-coenzyme M reductase (MCR). The latter enzyme is central to C-H bond formation and cleavage underlying methanogenic and reverse methanogenic phenotypes. Here, we describe a set of novel tools for the detection and quantification of H4MPT-linked C1 transfer reactions mediated by uncultivated anaerobic methane-oxidizing archaea (ANME). These tools include polymerase chain reaction primers targeting ANME MCR subunit A subgroups and protein extraction methods from marine sediments compatible with high-resolution mass spectrometry for profiling community structure and functional dynamics.
The Art and Design of Functional Metagenomic Screens
This article summarizes general design principles for functional metagenomics. The focus is on Escherichia coli as an expression host, although alternative host-vector systems are discussed in relation to optimizing gene recovery in activity-based screens. Examples of DNA isolation and enrichment approaches, library construction and phenotypic read-out are described with special emphasis on the use of high throughput technologies for rapid isolation of environmental clones encoding phenotypic traits of interest.
V-REVCOMP: Automated High-throughput Detection of Reverse Complementary 16S RRNA Gene Sequences in Large Environmental and Taxonomic Datasets
Reverse complementary DNA sequences - sequences that are inadvertently given backwards with all purines and pyrimidines transposed - can affect sequence analysis detrimentally unless taken into account. We present an open-source, high-throughput software tool -v-revcomp (http://www.cmde.science.ubc.ca/mohn/software.html) - to detect and reorient reverse complementary entries of the small-subunit rRNA (16S) gene from sequencing datasets, particularly from environmental sources. The software supports sequence lengths ranging from full length down to the short reads that are characteristic of next-generation sequencing technologies. We evaluated the reliability of v-revcomp by screening all 406 781 16S sequences deposited in release 102 of the curated SILVA database and demonstrated that the tool has a detection accuracy of virtually 100%. We subsequently used v-revcomp to analyse 1 171 646 16S sequences deposited in the International Nucleotide Sequence Databases and found that about 1% of these user-submitted sequences were reverse complementary. In addition, a nontrivial proportion of the entries were otherwise anomalous, including reverse complementary chimeras, sequences associated with wrong taxa, nonribosomal genes, sequences of poor quality or otherwise erroneous sequences without a reasonable match to any other entry in the database. Thus, v-revcomp is highly efficient in detecting and reorienting reverse complementary 16S sequences of almost any length and can be used to detect various sequence anomalies.
Microbial Life in a Liquid Asphalt Desert
Pitch Lake in Trinidad and Tobago is a natural asphalt reservoir nourished by pitch seepage, a form of petroleum that consists of mostly asphaltines, from the surrounding oil-rich region. During upward seepage, pitch mixes with mud and gases under high pressure, and the lighter portion evaporates or is volatilized, which produces a liquid asphalt residue characterized by low water activity, recalcitrant carbon substrates, and noxious chemical compounds. An active microbial community of archaea and bacteria, many of them novel strains (particularly from the new Tar ARC groups), totaling a biomass of up to 10(7) cells per gram, was found to inhabit the liquid hydrocarbon matrix of Pitch Lake. Geochemical and molecular taxonomic approaches revealed diverse, novel, and deeply branching microbial lineages with the potential to mediate anaerobic hydrocarbon degradation processes in different parts of the asphalt column. In addition, we found markers for archaeal methane metabolism and specific gene sequences affiliated with facultative and obligate anaerobic sulfur- and nitrite-oxidizing bacteria. The microbial diversity at Pitch Lake was found to be unique when compared to microbial communities analyzed at other hydrocarbon-rich environments, which included Rancho Le Brea, a natural asphalt environment in California, USA, and an oil well and a mud volcano in Trinidad and Tobago, among other sites. These results open a window into the microbial ecology and biogeochemistry of recalcitrant hydrocarbon matrices and establish the site as a terrestrial analogue for modeling the biotic potential of hydrocarbon lakes such as those found on Saturn's largest moon Titan.
