We present the genome of Roseovarius mucosus strain SMR3, a marine bacterium isolated from the diatom Skeletonema marinoi strain RO5AC sampled from top layer sediments at 14 m depth. Its 4,381,426 bp genome consists of a circular chromosome and two circular plasmids and contains 4,178 coding sequences (CDSs). Copyright © 2017 Töpel et al.
Vibrio campbellii is widely distributed in the marine environment and is an important pathogen of aquatic organisms such as shrimp, fish, and mollusks. An isolate of V. campbellii carrying the pirAB(vp) gene, causing acute hepatopancreatic necrosis disease (AHPND), has been reported. There are no previous reports about the complete genome of V. campbellii causing AHPND (VCAHPND). To extend our understanding of the pathogenesis of VCAHPND at the genomic level, the genome of V. campbellii 20130629003S01 isolated from a shrimp with AHPND was sequenced and analysed.The complete genome sequence of V. campbellii 20130629003S01 was generated using the PacBio RSII platform with single molecule, real-time sequencing. The 20130629003S01 strain consists of two circular chromosomes (3,621,712 bp in chromosome 1 and 2,245,751 bp in chromosome 2) and four plasmids of 70,066, 204,531, 143,140, and 86,121 bp. The genome contains a total of 5855 protein coding genes, 134 tRNA genes and 37 rRNA genes. The average nucleotide identity value of 20130629003S01 and other reference V. campbellii strains was 97.46%, suggesting that they are closely related.The genome sequence of V. campbellii 20130629003S01 and its comparative analysis with other V. campbellii strains that we present here are important for a better understanding of the genomic characteristics of VCAHPND.
Bacillus subtilis is a Gram-positive bacterium that serves as an important experimental system. B. subtilis NCIB 3610 is an undomesticated strain that exhibits phenotypes lost from the more common domesticated laboratory strains. Here, we announce the complete genome sequence of DK1042, a genetically competent derivative of NCIB 3610. Copyright © 2017 Nye et al.
Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.
Structural rearrangements have long been recognized as an important source of genetic variation, with implications in phenotypic diversity and disease, yet their detailed evolutionary dynamics remain elusive. Here we use long-read sequencing to generate end-to-end genome assemblies for 12 strains representing major subpopulations of the partially domesticated yeast Saccharomyces cerevisiae and its wild relative Saccharomyces paradoxus. These population-level high-quality genomes with comprehensive annotation enable precise definition of chromosomal boundaries between cores and subtelomeres and a high-resolution view of evolutionary genome dynamics. In chromosomal cores, S. paradoxus shows faster accumulation of balanced rearrangements (inversions, reciprocal translocations and transpositions), whereas S. cerevisiae accumulates unbalanced rearrangements (novel insertions, deletions and duplications) more rapidly. In subtelomeres, both species show extensive interchromosomal reshuffling, with a higher tempo in S. cerevisiae. Such striking contrasts between wild and domesticated yeasts are likely to reflect the influence of human activities on structural genome evolution.
We sequenced the genomes of ten Salmonella enterica serovar Infantis containing blaCTX-M-65 isolated from chicken, cattle, and human sources collected between 2012 and 2015 in the United States through routine NARMS surveillance and product sampling programs. We also completely assembled the plasmids from four of the isolates. All isolates had a D87Y mutation in the gyrA gene and harbored between 7 and 10 resistance genes (aph (4)-Ia, aac (3)-IVa, aph(3′ )-Ic, blaCTX-M-65, fosA3, floR, dfrA14, sul1, tetA, aadA1) located in two distinct sites of a megaplasmid (~316-323kb) similar to that described in a blaCTX-M-65-positive S. Infantis isolated from a patient in Italy. High-quality single nucleotide polymorphism (hqSNP) analysis revealed that all U.S. isolates were closely related, separated by only 1 to 38 pairwise high quality SNPs, indicating a high likelihood that strains from humans, chicken, and cattle recently evolved from a common ancestor. The U.S. isolates were genetically similar to the blaCTX-M-65-positive S. Infantis isolate from Italy, with a separation of 34 to 47 SNPs. This is the first report of the blaCTX-M-65 gene and the pESI-like megaplasmid from S. Infantis in the United States, and illustrates the importance of applying a global One Health, human and animal perspective to combat antimicrobial resistance. Copyright © 2017 American Society for Microbiology.
Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production.In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon.Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.
Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline (tetA), sulfonamides (sulI), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor (csi). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 (csi and traI) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli. Copyright © 2017 American Society for Microbiology.
Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a “two-speed” genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit “two-speed” genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale.
Clostridium (C.) chauvoei is a Gram-positive, spore forming, anaerobic bacterium. It causes black leg in ruminants, a typically fatal histotoxic myonecrosis. High quality circular genome sequences were generated for the C. chauvoei type strain DSM 7528(T) (ATCC 10092(T)) and a field strain 12S0467 isolated in Germany. The origin of replication (oriC) was comparable to that of Bacillus subtilis in structure with two regions containing DnaA boxes. Similar prophages were identified in the genomes of both C. chauvoei strains which also harbored hemolysin and bacterial spore formation genes. A CRISPR type I-B system with limited variations in the repeat number was identified. Sporulation and germination process related genes were homologous to that of the Clostridia cluster I group but novel variations for regulatory genes were identified indicative for strain specific control of regulatory events. Phylogenomics showed a higher relatedness to C. septicum than to other so far sequenced genomes of species belonging to the genus Clostridium. Comparative genome analysis of three C. chauvoei circular genome sequences revealed the presence of few inversions and translocations in locally collinear blocks (LCBs). The species genome also shows a large number of genes involved in proteolysis, genes for glycosyl hydrolases and metal iron transportation genes which are presumably involved in virulence and survival in the host. Three conserved flagellar genes (fliC) were identified in each of the circular genomes. In conclusion this is the first comparative analysis of circular genomes for the species C. chauvoei, enabling insights into genome composition and virulence factor variation. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Plasmodium cynomolgi, a non-human primate malaria parasite species, has been an important model parasite since its discovery in 1907. Similarities in the biology of P. cynomolgi to the closely related, but less tractable, human malaria parasite P. vivax make it the model parasite of choice for liver biology and vaccine studies pertinent to P. vivax malaria. Molecular and genome-scale studies of P. cynomolgi have relied on the current reference genome sequence, which remains highly fragmented with 1,649 unassigned scaffolds and little representation of the subtelomeres. Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated a new reference genome sequence, PcyM, sourced from an Indian rhesus monkey. We compare the newly assembled genome sequence with those of several other Plasmodium species, including a re-annotated P. coatneyi assembly.The new PcyM genome assembly is of significantly higher quality than the existing reference, comprising only 56 pieces, no gaps and an improved average gene length. Detailed manual curation has ensured a comprehensive annotation of the genome with 6,632 genes, nearly 1,000 more than previously attributed to P. cynomolgi. The new assembly also has an improved representation of the subtelomeric regions, which account for nearly 40% of the sequence. Within the subtelomeres, we identified more than 1300 Plasmodium interspersed repeat ( pir) genes, as well as a striking expansion of 36 methyltransferase pseudogenes that originated from a single copy on chromosome 9.The manually curated PcyM reference genome sequence is an important new resource for the malaria research community. The high quality and contiguity of the data have enabled the discovery of a novel expansion of methyltransferase in the subtelomeres, and illustrates the new comparative genomics capabilities that are being unlocked by complete reference genomes.
An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.
This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.
Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68–69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201–366 contigs), sharing only 92.2–100% and 92.0–100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.
The genome of Fusarium oxysporum (Fo) consists of a set of eleven ‘core’ chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. A central part of this chromosome contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX 13. We show that SIX6 contributes to virulence of Forc. Through horizontal chromosome transfer (HCT) to a non-pathogenic strain, we also show that the accessory chromosome containing the SIX gene homologs is indeed a pathogenicity chromosome for cucurbit infection. Conversely, complete loss of virulence was observed in Forc016 strains that lost this chromosome. We conclude that also a non-wilt-inducing Fo pathogen relies on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Three out of nine HCT strains investigated have undergone large-scale chromosome alterations, reflecting the remarkable plasticity of Fo genomes.
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