Transfer RNAs with novel cloverleaf structures.

We report the identification of novel tRNA species with 12-base pair amino-acid acceptor branches composed of longer acceptor stem and shorter T-stem. While canonical tRNAs have a 7/5 configuration of the branch, the novel tRNAs have either 8/4 or 9/3 structure. They were found during the search for selenocysteine tRNAs in terabytes of genome, metagenome and metatranscriptome sequences. Certain bacteria and their phages employ the 8/4 structure for serine and histidine tRNAs, while minor cysteine and selenocysteine tRNA species may have a modified 8/4 structure with one bulge nucleotide. In Acidobacteria, tRNAs with 8/4 and 9/3 structures may function as missense and nonsense suppressor tRNAs and/or regulatory noncoding RNAs. In δ-proteobacteria, an additional cysteine tRNA with an 8/4 structure mimics selenocysteine tRNA and may function as opal suppressor. We examined the potential translation function of suppressor tRNA species in Escherichia coli; tRNAs with 8/4 or 9/3 structures efficiently inserted serine, alanine and cysteine in response to stop and sense codons, depending on the identity element and anticodon sequence of the tRNA. These findings expand our view of how tRNA, and possibly the genetic code, is diversified in nature.

Metagenomic investigation of the geologically unique Hellenic Volcanic Arc reveals a distinctive ecosystem with unexpected physiology.

Hydrothermal vents represent a deep, hot, aphotic biosphere where chemosynthetic primary producers, fuelled by chemicals from Earth's subsurface, form the basis of life. In this study, we examined microbial mats from two distinct volcanic sites within the Hellenic Volcanic Arc (HVA). The HVA is geologically and ecologically unique, with reported emissions of CO2 -saturated fluids at temperatures up to 220°C and a notable absence of macrofauna. Metagenomic data reveals highly complex prokaryotic communities composed of chemolithoautotrophs, some methanotrophs, and to our surprise, heterotrophs capable of anaerobic degradation of aromatic hydrocarbons. Our data suggest that aromatic hydrocarbons may indeed be a significant source of carbon in these sites, and instigate additional research into the nature and origin of these compounds in the HVA. Novel physiology was assigned to several uncultured prokaryotic lineages; most notably, a SAR406 representative is attributed with a role in anaerobic hydrocarbon degradation. This dataset, the largest to date from submarine volcanic ecosystems, constitutes a significant resource of novel genes and pathways with potential biotechnological applications.

Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium.

Nitrogen (N(2))-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N(2)-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.

Facile Recoding of Selenocysteine in Nature.

Selenocysteine (Sec or U) is encoded by UGA, a stop codon reassigned by a Sec-specific elongation factor and a distinctive RNA structure. To discover possible code variations in extant organisms we analyzed 6.4 trillion base pairs of metagenomic sequences and 24 903 microbial genomes for tRNA(Sec) species. As expected, UGA is the predominant Sec codon in use. We also found tRNA(Sec) species that recognize the stop codons UAG and UAA, and ten sense codons. Selenoprotein synthesis programmed by UAG in Geodermatophilus and Blastococcus, and by the Cys codon UGU in Aeromonas salmonicida was confirmed by metabolic labeling with (75) Se or mass spectrometry. Other tRNA(Sec) species with different anticodons enabled E. coli to synthesize active formate dehydrogenase H, a selenoenzyme. This illustrates the ease by which the genetic code may evolve new coding schemes, possibly aiding organisms to adapt to changing environments, and show the genetic code is much more flexible than previously thought.

ARBitrator: a software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank.

MOTIVATION: Studies of the biochemical functions and activities of uncultivated microorganisms in the environment require analysis of DNA sequences for phylogenetic characterization and for the development of sequence-based assays for the detection of microorganisms. The numbers of sequences for genes that are indicators of environmentally important functions such as nitrogen (N2) fixation have been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Information's GenBank database is problematic because of annotation errors, nomenclature variation and paralogues; moreover, GenBank's structure and tools are not conducive to searching solely by function. For some genes, such as the nifH gene commonly used to assess community potential for N2 fixation, manual collection and curation are becoming intractable because of the large number of sequences in GenBank and the large number of highly similar paralogues. If analysis is to keep pace with sequence discovery, an automated retrieval and curation system is necessary. RESULTS: ARBitrator uses a two-step process composed of a broad collection of potential homologues followed by screening with a best hit strategy to conserved domains. 34 420 nifH sequences were identified in GenBank as of November 20, 2012. The false-positive rate is ∼0.033%. ARBitrator rapidly updates a public nifH sequence database, and we show that it can be adapted for other genes. AVAILABILITY AND IMPLEMENTATION: Java source and executable code are freely available to non-commercial users at http://pmc.ucsc.edu/∼wwwzehr/research/database/. CONTACT: zehrj@ucsc.edu SUPPLEMENTARY INFORMATION: SUPPLEMENTARY INFORMATION is available at Bioinformatics online.

nifH pyrosequencing reveals the potential for location-specific soil chemistry to influence N2 -fixing community dynamics.

A dataset of 87 020 nifH reads and 16 782 unique nifH protein sequences obtained over 2 years from four locations across a gradient of agricultural soil types in Argentina were analysed to provide a detailed and comprehensive picture of the diversity, abundance and responses of the N2 -fixing community in relation to differences in soil chemistry and agricultural practices. Phylogenetic analysis revealed an expected high proportion of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria, mainly relatives to Bradyrhizobium and Methylosinus/Methylocystis, but a surprising paucity of Gammaproteobacteria. Analysis of variance and stepwise regression modelling suggested location and treatment-specific influences of soil type on diazotrophic community composition and organic carbon concentrations on nifH diversity. nifH gene abundance, determined by quantitative real-time polymerase chain reaction, was higher in agricultural soils than in non-agricultural soils, and was influenced by soil chemistry under intensive crop rotation but not under monoculture. At some locations, sustainable increased crop yields might be possible through the management of soil chemistry to improve the abundance and diversity of N2 -fixing bacteria.

SAR11 marine bacteria require exogenous reduced sulphur for growth.

Sulphur is a universally required cell nutrient found in two amino acids and other small organic molecules. All aerobic marine bacteria are known to use assimilatory sulphate reduction to supply sulphur for biosynthesis, although many can assimilate sulphur from organic compounds that contain reduced sulphur atoms. An analysis of three complete 'Candidatus Pelagibacter ubique' genomes, and public ocean metagenomic data sets, suggested that members of the ubiquitous and abundant SAR11 alphaproteobacterial clade are deficient in assimilatory sulphate reduction genes. Here we show that SAR11 requires exogenous sources of reduced sulphur, such as methionine or 3-dimethylsulphoniopropionate (DMSP) for growth. Titrations of the algal osmolyte DMSP in seawater medium containing all other macronutrients in excess showed that 1.5 x 10(8) SAR11 cells are produced per nanomole of DMSP. Although it has been shown that other marine alphaproteobacteria use sulphur from DMSP in preference to sulphate, our results indicate that 'Cand. P. ubique' relies exclusively on reduced sulphur compounds that originate from other plankton.

Misannotations of rRNA can now generate 90% false positive protein matches in metatranscriptomic studies.

In the course of analyzing 9,522,746 pyrosequencing reads from 23 stations in the Southwestern Pacific and equatorial Atlantic oceans, it came to our attention that misannotations of rRNA as proteins is now so widespread that false positive matching of rRNA pyrosequencing reads to the National Center for Biotechnology Information (NCBI) non-redundant protein database approaches 90%. One conserved portion of 23S rRNA was consistently misannotated often enough to prompt curators at Pfam to create a spurious protein family. Detailed examination of the annotation history of each seed sequence in the spurious Pfam protein family (PF10695, 'Cw-hydrolase') uncovered issues in the standard operating procedures and quality assurance programs of major sequencing centers, and other issues relating to the curation practices of those managing public databases such as GenBank and SwissProt. We offer recommendations for all these issues, and recommend as well that workers in the field of metatranscriptomics take extra care to avoid including false positive matches in their datasets.

Proteorhodopsin in the ubiquitous marine bacterium SAR11.

Proteorhodopsins are light-dependent proton pumps that are predicted to have an important role in the ecology of the oceans by supplying energy for microbial metabolism. Proteorhodopsin genes were first discovered through the cloning and sequencing of large genomic DNA fragments from seawater. They were later shown to be widely distributed, phylogenetically diverse, and active in the oceans. Proteorhodopsin genes have not been found in cultured bacteria, and on the basis of environmental sequence data, it has not yet been possible to reconstruct the genomes of uncultured bacterial strains that have proteorhodopsin genes. Although the metabolic effect of proteorhodopsins is uncertain, they are thought to function in cells for which the primary mode of metabolism is the heterotrophic assimilation of dissolved organic carbon. Here we report that SAR11 strain HTCC1062 ('Pelagibacter ubique'), the first cultivated member of the extraordinarily abundant SAR11 clade, expresses a proteorhodopsin gene when cultured in autoclaved seawater and in its natural environment, the ocean. The Pelagibacter proteorhodopsin functions as a light-dependent proton pump. The gene is expressed by cells grown in either diurnal light or in darkness, and there is no difference between the growth rates or cell yields of cultures grown in light or darkness.

Spatial patterns and light-driven variation of microbial population gene expression in surface waters of the oligotrophic open ocean.

Because bacterioplankton production rates do not vary strongly across vast expanses of the ocean, it is unclear how variability in community structure corresponds with functional variability in the open ocean. We surveyed community transcript functional profiles at eight locations in the open ocean, in both the light and in the dark, using the genomic subsystems approach, to understand variability in gene expression patterns in surface waters. Metatranscriptomes from geographically distinct areas and collected during the day and night shared a large proportion of metabolic functional similarity (74%) at the finest metabolic resolution possible. The variability between metatranscriptomes could be explained by phylogenetic differences between libraries (Mantel test, P < 0.0001). Several key gene expression pathways, including Photosystem I, Photosystem II and ammonium uptake, demonstrated the most variability both geographically and between light and dark. Libraries were dominated by transcripts of the cyanobacterium Prochlorocococcus marinus, where most geographical and diel variability between metatranscriptomes reflected between-station differences in cyanobacterial phototrophic metabolism. Our results demonstrate that active genetic machinery in surface waters of the ocean is dominated by photosynthetic microorganisms and their site-to-site variability, while variability in the remainder of assemblages is dependent on local taxonomic composition.

Unicellular cyanobacteria with a new mode of life: the lack of photosynthetic oxygen evolution allows nitrogen fixation to proceed.

Some unicellular N(2)-fixing cyanobacteria have recently been found to lack a functional photosystem II of photosynthesis. Such organisms, provisionally termed UCYN-A, of the oceanic picoplanktion are major contributors to the global marine N-input by N(2)-fixation. Since their photosystem II is inactive, they can perform N(2)-fixation during the day. UCYN-A organisms cannot be cultivated as yet. Their genomic analysis indicates that they lack genes coding for enzymes of the Calvin cycle, the tricarboxylic acid cycle and for the biosynthesis of several amino acids. The carbon source in the ocean that allows them to thrive in such high abundance has not been identified. Their genomic analysis implies that they metabolize organic carbon by a new mode of life. These unicellular N(2)-fixing cyanobacteria of the oceanic picoplankton are evolutionarily related to spheroid bodies present in diatoms of the family Epithemiaceae, such as Rhopalodia gibba. More recently, spheroid bodies were ultimately proven to be related to cyanobacteria and to express nitrogenase. They have been reported to be completely inactive in all photosynthetic reactions despite the presence of thylakoids. Sequence data show that R. gibba and its spheroid bodies are an evolutionarily young symbiosis that might serve as a model system to unravel early events in the evolution of chloroplasts. The cell metabolism of UCYN-A and the spheroid bodies may be related to that of the acetate photoassimilating green alga Chlamydobotrys.

Unique glycine-activated riboswitch linked to glycine-serine auxotrophy in SAR11.

The genome sequence of the marine bacterium 'Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that 'Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 microM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 x 10(6) cells ml(-1) to 8.16 x 10(6) cells ml(-1) (R(2) = 0.992). Serine was capable of substituting for glycine at 1.5 microM. 'Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that 'Cand. P. ubique' is therefore a glycine-serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.

The small genome of an abundant coastal ocean methylotroph.

OM43 is a clade of uncultured beta-proteobacteria that is commonly found in environmental nucleic acid sequences from productive coastal ocean ecosystems, and some freshwater environments, but is rarely detected in ocean gyres. Ecological studies associate OM43 with phytoplankton blooms, and evolutionary relationships indicate that they might be methylotrophs. Here we report on the genome sequence and metabolic properties of the first axenic isolate of the OM43 clade, strain HTCC2181, which was obtained using new procedures for culturing cells in natural seawater. We found that this strain is an obligate methylotroph that cannot oxidize methane but can use the oxidized C1 compounds methanol and formaldehyde as sources of carbon and energy. Its complete genome is 1304 428 bp in length, the smallest yet reported for a free-living cell. The HTCC2181 genome includes genes for xanthorhodopsin and retinal biosynthesis, an auxiliary system for producing transmembrane electrochemical potentials from light. The discovery that HTCC2181 is an extremely simple specialist in C1 metabolism suggests an unanticipated, important role for oxidized C1 compounds as substrates for bacterioplankton productivity in coastal ecosystems.

Erratum to: The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

[This corrects the article DOI: 10.1186/s40793-015-0077-y.].

The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4).

The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provided via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation is followed by functional annotation including assignment of protein product names and connection to various protein family databases.

Toward a standard in structural genome annotation for prokaryotes.

BACKGROUND: In an effort to identify the best practice for finding genes in prokaryotic genomes and propose it as a standard for automated annotation pipelines, 1,004,576 peptides were collected from various publicly available resources, and were used as a basis to evaluate various gene-calling methods. The peptides came from 45 bacterial replicons with an average GC content from 31 % to 74 %, biased toward higher GC content genomes. Automated, manual, and semi-manual methods were used to tally errors in three widely used gene calling methods, as evidenced by peptides mapped outside the boundaries of called genes. RESULTS: We found that the consensus set of identical genes predicted by the three methods constitutes only about 70 % of the genes predicted by each individual method (with start and stop required to coincide). Peptide data was useful for evaluating some of the differences between gene callers, but not reliable enough to make the results conclusive, due to limitations inherent in any proteogenomic study. CONCLUSIONS: A single, unambiguous, unanimous best practice did not emerge from this analysis, since the available proteomics data were not adequate to provide an objective measurement of differences in the accuracy between these methods. However, as a result of this study, software, reference data, and procedures have been better matched among participants, representing a step toward a much-needed standard. In the absence of sufficient amount of exprimental data to achieve a universal standard, our recommendation is that any of these methods can be used by the community, as long as a single method is employed across all datasets to be compared.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

Comparative transcriptomics between Synechococcus PCC 7942 and Synechocystis PCC 6803 provide insights into mechanisms of stress acclimation.

Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 are model cyanobacteria from which the metabolism and adaptive responses of other cyanobacteria are inferred. Using stranded and 5' enriched libraries, we measured the gene expression response of cells transferred from reference conditions to stress conditions of decreased inorganic carbon, increased salinity, increased pH, and decreased illumination at 1-h and 24-h after transfer. We found that the specific responses of the two strains were by no means identical. Transcriptome profiles allowed us to improve the structural annotation of the genome i.e. identify possible missed genes (including anti-sense), alter gene coordinates and determine transcriptional units (operons). Finally, we predicted associations between proteins of unknown function and biochemical pathways by revealing proteins of known functions that are co-regulated with the unknowns. Future studies of these model organisms will benefit from the cataloging of their responses to environmentally relevant stresses, and improvements in their genome annotations found here.

Stop codon reassignments in the wild.

The canonical genetic code is assumed to be deeply conserved across all domains of life with very few exceptions. By scanning 5.6 trillion base pairs of metagenomic data for stop codon reassignment events, we detected recoding in a substantial fraction of the >1700 environmental samples examined. We observed extensive opal and amber stop codon reassignments in bacteriophages and of opal in bacteria. Our data indicate that bacteriophages can infect hosts with a different genetic code and demonstrate phage-host antagonism based on code differences. The abundance and diversity of genetic codes present in environmental organisms should be considered in the design of engineered organisms with altered genetic codes in order to preclude the exchange of genetic information with naturally occurring species.

The unique metabolism of SAR11 aquatic bacteria.

The deeply branching clade of abundant, globally distributed aquatic α-Proteobacteria known as "SAR11", are adapted to nutrient-poor environments such as the surface waters of the open ocean. Unknown prior to 1990, uncultured until 2002, members of the SAR11 clade can now be cultured in artificial, defined media to densities three orders of magnitude higher than in unamended natural media. Cultivation in natural and defined media has confirmed genomic and metagenomic predictions such as an inability to reduce sulfate to sulfide, a requirement for pyruvate, an ability to oxidize a wide variety of methylated and one-carbon compounds for energy, and an unusual form of conditional glycine auxotrophy. Here we describe the metabolism of the SAR11 type strain Candidatus "Pelagibacter ubique" str. HTCC1062, as revealed by genome-assisted studies of laboratory cultures. We also describe the discovery of SAR11 and field studies that have been done on natural populations.