Vibrio navarrensis is an aquatic bacterium recently shown to be associated with human illness. We report the first genome sequences of three V. navarrensis strains obtained from clinical and environmental sources. Preliminary analyses of the sequences reveal that V. navarrensis contains genes commonly associated with virulence in other human pathogens. Copyright © 2014 Gladney et al.
BackgroundThe chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as `SMRT (Single Molecule, Real-Time) sequencing¿, has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots.ResultsThe sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently.ConclusionsBecause of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate `finished¿ genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.
TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.
We report here the sequencing and analysis of the genome of the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. This microbe is a model for studies of its carboxydotrophic life style under anaerobic condition, based on its ability to utilize carbon monoxide (CO) as the sole carbon substrate and water as the electron acceptor, yielding CO2 and H2 as the end products. The CO-oxidation reaction is known to be catalyzed by two enzyme complexes, the CO dehydrogenase and hydrogenase. As expected, analysis of the genome of Rx. gelatinosus CBS reveals the presence of genes encoding both enzyme complexes. The CO-oxidation reaction is CO-inducible, which is consistent with the presence of two putative CO-sensing transcription factors in its genome. Genome analysis also reveals the presence of two additional hydrogenases, an uptake hydrogenase that liberates the electrons in H2 in support of cell growth, and a regulatory hydrogenase that senses H2 and relays the signal to a two-component system that ultimately controls synthesis of the uptake hydrogenase. The genome also contains two sets of hydrogenase maturation genes which are known to assemble the catalytic metallocluster of the hydrogenase NiFe active site. Collectively, the genome sequence and analysis information reveals the blueprint of an intricate network of signal transduction pathways and its underlying regulation that enables Rx. gelatinosus CBS to thrive on CO or H2 in support of cell growth.
Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 ß-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different ß-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.
Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
Pseudomonas rhizosphaerae IH5(T) (=DSM 16299(T)), isolated from the rhizospheric soil of grass growing in Spain, has been reported as a novel species of the genus Pseudomonas harboring insoluble phosphorus solubilizing activity. To understanding the multifunctional biofertilizer better, we report the complete genome sequence of P. rhizosphaerae IH5(T). Copyright © 2014 Elsevier B.V. All rights reserved.
The incorrect alignments are a severe problem in variant calling, and remain as a challenge computational issue in Bioinformatics field. Although there have been some methods utilizing the re-alignment approach to tackle the misalignments, a standalone re-alignment tool for long sequencing reads is lacking. Hence, we present a standalone tool to correct the misalignments, called ProbAlign. It can be integrated into the pipelines of not only variant calling but also other genomic applications. We demonstrate the use of re-alignment in two diverse and important genomics fields: variant calling and viral quasispecies reconstruction. First, variant calling results in the Pacific Biosciences SMRT re-sequencing data of NA12878 show that false positives can be reduced by 43.5%, and true positives can be increased by 24.8% averagely, after re-alignment. Second, results in reconstructing a 5-virus-mix show that the viral population can be completely unraveled, and also the estimation of quasispecies frequencies has been improved, after re-alignment. ProbAlign is freely available in the PyroTools toolkit (https://github.com/homopolymer/PyroTools).
In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated Klebsiella sp. strain BRL6-2 on minimal media with alkali lignin as the sole carbon source. This organism was isolated anaerobically from tropical forest soils collected from the Bisley watershed at the Ridge site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are characterized by cycles of iron oxidation and reduction. Genome sequencing was targeted because of its ability to grow on lignin anaerobically and lignocellulolytic activity via in vitro enzyme assays. The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of genes encoding lignocellulolytic carbohydrate active enzymes. The genome revealed four putative peroxidases including glutathione and DyP-type peroxidases, and a complete protocatechuate pathway encoded in a single gene cluster. Physiological studies revealed Klebsiella sp. strain BRL6-2 to be relatively stress tolerant to high ionic strength conditions. It grows in increasing concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44 mM and NaCl up to 1.5 M.
Strains of a pink-pigmented Methylobacterium sp. are effective nitrogen- (N2) fixing microsymbionts of species of the African crotalarioid genus Listia. Strain WSM2598 is an aerobic, motile, Gram-negative, non-spore-forming rod isolated in 2002 from a Listia bainesii root nodule collected at Estcourt Research Station in South Africa. Here we describe the features of Methylobacterium sp. WSM2598, together with information and annotation of a high-quality draft genome sequence. The 7,669,765 bp draft genome is arranged in 5 scaffolds of 83 contigs, contains 7,236 protein-coding genes and 18 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 G enomic E ncyclopedia for B acteria and A rchaea- R oot N odule B acteria (GEBA-RNB) project.
More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). We have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences.Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes had quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes.The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.
Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency. Copyright © 2014 Kanesaki et al.
Leptospira santarosai serovar Shermani is the most frequently encountered serovar, and it causes leptospirosis and tubulointerstitial nephritis in Taiwan. This study aims to complete the genome sequence of L. santarosai serovar Shermani and analyze the transcriptional responses of L. santarosai serovar Shermani to renal tubular cells. To assemble this highly repetitive genome, we combined reads that were generated from four next-generation sequencing platforms by using hybrid assembly approaches to finish two-chromosome contiguous sequences without gaps by validating the data with optical restriction maps and Sanger sequencing. Whole-genome comparison studies revealed a 28-kb region containing genes that encode transposases and hypothetical proteins in L. santarosai serovar Shermani, but this region is absent in other pathogenic Leptospira spp. We found that lipoprotein gene expression in both L. santarosai serovar Shermani and L. interrogans serovar Copenhageni were upregulated upon interaction with renal tubular cells, and LSS19962, a L. santarosai serovar Shermani-specific gene within a 28-kb region that encodes hypothetical proteins, was upregulated in L. santarosai serovar Shermani-infected renal tubular cells. Lipoprotein expression during leptospiral infection might facilitate the interactions of leptospires within kidneys. The availability of the whole-genome sequence of L. santarosai serovar Shermani would make it the first completed sequence of this species, and its comparison with that of other Leptospira spp. may provide invaluable information for further studies in leptospiral pathogenesis.
The DNA Data Bank of Japan Center (DDBJ Center; http://www.ddbj.nig.ac.jp) maintains and provides public archival, retrieval and analytical services for biological information. Since October 2013, DDBJ Center has operated the Japanese Genotype-phenotype Archive (JGA) in collaboration with our partner institute, the National Bioscience Database Center (NBDC) of the Japan Science and Technology Agency. DDBJ Center provides the JGA database system which securely stores genotype and phenotype data collected from individuals whose consent agreements authorize data release only for specific research use. NBDC has established guidelines and policies for sharing human-derived data and reviews data submission and usage requests from researchers. In addition to the JGA project, DDBJ Center develops Semantic Web technologies for data integration and sharing in collaboration with the Database Center for Life Science. This paper describes the overview of the JGA project, updates to the DDBJ databases, and services for data retrieval, analysis and integration. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
The Campylobacter lari group is a phylogenetic clade within the epsilon subdivision of the Proteobacteria and is part of the thermotolerant Campylobacter spp., a division within the genus that includes the human pathogen Campylobacter jejuni. The C. lari group is currently composed of five species (C. lari, Campylobacter insulaenigrae, Campylobacter volucris, Campylobacter subantarcticus, and Campylobacter peloridis), as well as a group of strains termed the urease-positive thermophilic Campylobacter (UPTC) and other C. lari-like strains. Here we present the complete genome sequences of 11 C. lari group strains, including the five C. lari group species, four UPTC strains, and a lari-like strain isolated in this study. The genome of C. lari subsp. lari strain RM2100 was described previously. Analysis of the C. lari group genomes indicates that this group is highly related at the genome level. Furthermore, these genomes are strongly syntenic with minor rearrangements occurring only in 4 of the 12 genomes studied. The C. lari group can be bifurcated, based on the flagella and flagellar modification genes. Genomic analysis of the UPTC strains indicated that these organisms are variable but highly similar, closely related to but distinct from C. lari. Additionally, the C. lari group contains multiple genes encoding hemagglutination domain proteins, which are either contingency genes or linked to conserved contingency genes. Many of the features identified in strain RM2100, such as major deficiencies in amino acid biosynthesis and energy metabolism, are conserved across all 12 genomes, suggesting that these common features may play a role in the association of the C. lari group with coastal environments and watersheds. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2014. This work is written by US Government employees and is in the public domain in the US.
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