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Asset Tag: VM_MID_AM

September 22, 2019

Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes.

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

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September 22, 2019

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus.

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed “phototaxis,” enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

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September 21, 2019

Mistranslation drives the evolution of robustness in TEM-1 ß-lactamase.

How biological systems such as proteins achieve robustness to ubiquitous perturbations is a fundamental biological question. Such perturbations include errors that introduce phenotypic mutations into nascent proteins during the translation of mRNA. These errors are remarkably frequent. They are also costly, because they reduce protein stability and help create toxic misfolded proteins. Adaptive evolution might reduce these costs of protein mistranslation by two principal mechanisms. The first increases the accuracy of translation via synonymous “high fidelity” codons at especially sensitive sites. The second increases the robustness of proteins to phenotypic errors via amino acids that increase protein stability. To study how these mechanisms are exploited by populations evolving in the laboratory, we evolved the antibiotic resistance gene TEM-1 in Escherichia coli hosts with either normal or high rates of mistranslation. We analyzed TEM-1 populations that evolved under relaxed and stringent selection for antibiotic resistance by single molecule real-time sequencing. Under relaxed selection, mistranslating populations reduce mistranslation costs by reducing TEM-1 expression. Under stringent selection, they efficiently purge destabilizing amino acid changes. More importantly, they accumulate stabilizing amino acid changes rather than synonymous changes that increase translational accuracy. In the large populations we study, and on short evolutionary timescales, the path of least resistance in TEM-1 evolution consists of reducing the consequences of translation errors rather than the errors themselves.

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September 21, 2019

The kinetoplastid-infecting Bodo saltans virus (BsV), a window into the most abundant giant viruses in the sea.

Giant viruses are ecologically important players in aquatic ecosystems that have challenged concepts of what constitutes a virus. Herein, we present the giant Bodo saltans virus (BsV), the first characterized representative of the most abundant group of giant viruses in ocean metagenomes, and the first isolate of a klosneuvirus, a subgroup of the Mimiviridae proposed from metagenomic data. BsV infects an ecologically important microzooplankton, the kinetoplastid Bodo saltans. Its 1.39 Mb genome encodes 1227 predicted ORFs, including a complex replication machinery. Yet, much of its translational apparatus has been lost, including all tRNAs. Essential genes are invaded by homing endonuclease-encoding self-splicing introns that may defend against competing viruses. Putative anti-host factors show extensive gene duplication via a genomic accordion indicating an ongoing evolutionary arms race and highlighting the rapid evolution and genomic plasticity that has led to genome gigantism and the enigma that is giant viruses.© 2018, Deeg et al.

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September 21, 2019

Chromulinavorax destructans, a pathogenic TM6 bacterium with an unusual replication strategy targeting protist mitochondrion

Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts. Chromulinavorax destructans is an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate, Spumella elongata. Chromulinavorax destructans is characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential and C. destructans instead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.

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September 21, 2019

From the inside out: An epibiotic Bdellovibrio predator with an expanded genomic complement

Bdellovibrio and like organisms are abundant environmental predators of prokaryotes that show a diversity of predation strategies, ranging from intra-periplasmic to epibiotic predation. The novel epibiotic predator Bdellovibrio qaytius was isolated from a eutrophic freshwater pond in British Columbia, where it was a continual part of the microbial community. Bdellovibrio qaytius was found to preferentially prey on the beta-proteobacterium Paraburkholderia fungorum. Despite its epibiotic replication strategy, B. qaytius encodes a complex genomic complement more similar to periplasmic predators as well as several biosynthesis pathways not previously found in epibiotic predators. Bdellovibrio qaytius is representative of a widely distributed basal cluster within the genus Bdellovibrio, suggesting that epibiotic predation might be a common predation type in nature and ancestral to the genus.

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July 19, 2019

Comparison of single-molecule sequencing and hybrid approaches for finishing the genome of Clostridium autoethanogenum and analysis of CRISPR systems in industrial relevant Clostridia.

Clostridium autoethanogenum strain JA1-1 (DSM 10061) is an acetogen capable of fermenting CO, CO2 and H2 (e.g. from syngas or waste gases) into biofuel ethanol and commodity chemicals such as 2,3-butanediol. A draft genome sequence consisting of 100 contigs has been published.A closed, high-quality genome sequence for C. autoethanogenum DSM10061 was generated using only the latest single-molecule DNA sequencing technology and without the need for manual finishing. It is assigned to the most complex genome classification based upon genome features such as repeats, prophage, nine copies of the rRNA gene operons. It has a low G + C content of 31.1%. Illumina, 454, Illumina/454 hybrid assemblies were generated and then compared to the draft and PacBio assemblies using summary statistics, CGAL, QUAST and REAPR bioinformatics tools and comparative genomic approaches. Assemblies based upon shorter read DNA technologies were confounded by the large number repeats and their size, which in the case of the rRNA gene operons were ~5 kb. CRISPR (Clustered Regularly Interspaced Short Paloindromic Repeats) systems among biotechnologically relevant Clostridia were classified and related to plasmid content and prophages. Potential associations between plasmid content and CRISPR systems may have implications for historical industrial scale Acetone-Butanol-Ethanol (ABE) fermentation failures and future large scale bacterial fermentations. While C. autoethanogenum contains an active CRISPR system, no such system is present in the closely related Clostridium ljungdahlii DSM 13528. A common prophage inserted into the Arg-tRNA shared between the strains suggests a common ancestor. However, C. ljungdahlii contains several additional putative prophages and it has more than double the amount of prophage DNA compared to C. autoethanogenum. Other differences include important metabolic genes for central metabolism (as an additional hydrogenase and the absence of a phophoenolpyruvate synthase) and substrate utilization pathway (mannose and aromatics utilization) that might explain phenotypic differences between C. autoethanogenum and C. ljungdahlii.Single molecule sequencing will be increasingly used to produce finished microbial genomes. The complete genome will facilitate comparative genomics and functional genomics and support future comparisons between Clostridia and studies that examine the evolution of plasmids, bacteriophage and CRISPR systems.

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July 19, 2019

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens.

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens–Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n?=?44) and the complete gene of pvcsp (n?=?47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.

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July 19, 2019

The complex methylome of the human gastric pathogen Helicobacter pylori.

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.

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July 19, 2019

In vivo generation of DNA sequence diversity for cellular barcoding.

Heterogeneity is a ubiquitous feature of biological systems. A complete understanding of such systems requires a method for uniquely identifying and tracking individual components and their interactions with each other. We have developed a novel method of uniquely tagging individual cells in vivo with a genetic ‘barcode’ that can be recovered by DNA sequencing. Our method is a two-component system comprised of a genetic barcode cassette whose fragments are shuffled by Rci, a site-specific DNA invertase. The system is highly scalable, with the potential to generate theoretical diversities in the billions. We demonstrate the feasibility of this technique in Escherichia coli. Currently, this method could be employed to track the dynamics of populations of microbes through various bottlenecks. Advances of this method should prove useful in tracking interactions of cells within a network, and/or heterogeneity within complex biological samples.© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases.

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types.

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July 19, 2019

Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.

The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58×?coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of “finished grade” because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

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July 19, 2019

Detecting DNA modifications from SMRT sequencing data by modeling sequence context dependence of polymerase kinetic.

DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.

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July 19, 2019

Comprehensive methylome characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at single-base resolution.

In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N(6)-methyladenine (6 mA) and N(4)-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN(7)TAY-3’/3′-CTN(7)ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism.

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July 19, 2019

Characterizing and measuring bias in sequence data.

DNA sequencing technologies deviate from the ideal uniform distribution of reads. These biases impair scientific and medical applications. Accordingly, we have developed computational methods for discovering, describing and measuring bias.We applied these methods to the Illumina, Ion Torrent, Pacific Biosciences and Complete Genomics sequencing platforms, using data from human and from a set of microbes with diverse base compositions. As in previous work, library construction conditions significantly influence sequencing bias. Pacific Biosciences coverage levels are the least biased, followed by Illumina, although all technologies exhibit error-rate biases in high- and low-GC regions and at long homopolymer runs. The GC-rich regions prone to low coverage include a number of human promoters, so we therefore catalog 1,000 that were exceptionally resistant to sequencing. Our results indicate that combining data from two technologies can reduce coverage bias if the biases in the component technologies are complementary and of similar magnitude. Analysis of Illumina data representing 120-fold coverage of a well-studied human sample reveals that 0.20% of the autosomal genome was covered at less than 10% of the genome-wide average. Excluding locations that were similar to known bias motifs or likely due to sample-reference variations left only 0.045% of the autosomal genome with unexplained poor coverage.The assays presented in this paper provide a comprehensive view of sequencing bias, which can be used to drive laboratory improvements and to monitor production processes. Development guided by these assays should result in improved genome assemblies and better coverage of biologically important loci.

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