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Asset Tag: NGS

July 7, 2019

Fe-S cluster assembly in oxymonads and related protists.

The oxymonad Monocercomonoides exilis was recently reported to be the first eukaryote that has completely lost the mitochondrial compartment. It was proposed that an important prerequisite for such a radical evolutionary step was the acquisition of the SUF Fe-S cluster assembly pathway from prokaryotes, making the mitochondrial ISC pathway dispensable. We have investigated genomic and transcriptomic data from six oxymonad species and their relatives, composing the group Preaxostyla (Metamonada, Excavata), for the presence and absence of enzymes involved in Fe-S cluster biosynthesis. None possesses enzymes of mitochondrial ISC pathway and all apparently possess the SUF pathway, composed of SufB, C, D, S, and U proteins, altogether suggesting that the transition from ISC to SUF preceded their last common ancestor. Interestingly, we observed that SufDSU were fused in all three oxymonad genomes, and in the genome of Paratrimastix pyriformis. The donor of the SUF genes is not clear from phylogenetic analyses, but the enzyme composition of the pathway and the presence of SufDSU fusion suggests Firmicutes, Thermotogae, Spirochaetes, Proteobacteria, or Chloroflexi as donors. The inventory of the downstream CIA pathway enzymes is consistent with that of closely related species that retain ISC, indicating that the switch from ISC to SUF did not markedly affect the downstream process of maturation of cytosolic and nuclear Fe-S proteins.

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July 7, 2019

The molecular basis for the intramolecular migration (NIH shift) of the carboxyl group during para-hydroxybenzoate catabolism.

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration, primarily during an enzymatic hydroxylation reaction. The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for 40 years. Here, we elucidate the molecular mechanism of the reaction in the conversion of para-hydroxybenzoate (PHB) to gentisate (GA, 2, 5-dihydroxybenzoate). Three genes (phgABC) from the PHB utilizer Brevibacillus laterosporus PHB-7a encode enzymes (p-hydroxybenzoyl-CoA ligase, p-hydroxybenzoyl-CoA hydroxylase and gentisyl-CoA thioesterase, respectively) catalyzing the conversion of PHB to GA via a route involving CoA thioester formation, hydroxylation concomitant with a 1, 2-shift of the acetyl CoA moiety and thioester hydrolysis. The shift of the carboxyl group was established rigorously by stable isotopic experiments with heterologously expressed phgABC, converting 2, 3, 5, 6-tetradeutero-PHB and [carboxyl-13 C]-PHB to 3, 4, 6-trideutero-GA and [carboxyl-13 C]-GA respectively. This is distinct from the NIH shifts of hydrogen and aceto substituents, where a single oxygenase catalyzes the reaction without the involvement of a thioester. The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the CoA thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.© 2018 John Wiley & Sons Ltd.

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July 7, 2019

Genomic features of the Helicobacter pylori strain PMSS1 and its virulence attributes as deduced from its in vivo colonisation patterns.

The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI-encoded type-4-secretion-system (T4SS) or not. Only a few cagPAI-positive strains have been successfully adapted for long-term infection of mice, including the pre-mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin-associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co-factor required for the translocation of CagA into the host cell and activation of NF-?B via the T4SS. Long-term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re-isolated at four months post-infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re-established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.© 2018 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

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July 7, 2019

Measuring the mappability spectrum of reference genome assemblies

The ability to infer actionable information from genomic variation data in a resequencing experiment relies on accurately aligning the sequences to a reference genome. However, this accuracy is inherently limited by the quality of the reference assembly and the repetitive content of the subject’s genome. As long read sequencing technologies become more widespread, it is crucial to investigate the expected improvements in alignment accuracy and variant analysis over existing short read methods. The ability to quantify the read length and error rate necessary to uniquely map regions of interest in a sequence allows users to make informed decisions regarding experiment design and provides useful metrics for comparing the magnitude of repetition across different reference assemblies. To this end we have developed NEAT-Repeat, a toolkit for exhaustively identifying the minimum read length required to uniquely map each position of a reference sequence given a specified error rate. Using these tools we computed the -mappability spectrum” for ten reference sequences, including human and a range of plants and animals, quantifying the theoretical improvements in alignment accuracy that would result from sequencing with longer reads or reads with less base-calling errors. Our inclusion of read length and error rate builds upon existing methods for mappability tracks based on uniqueness or aligner-specific mapping scores, and thus enables more comprehensive analysis. We apply our mappability results to whole-genome variant call data, and demonstrate that variants called with low mapping and genotype quality scores are disproportionately found in reference regions that require long reads to be uniquely covered. We propose that our mappability metrics provide a valuable supplement to established variant filtering and annotation pipelines by supplying users with an additional metric related to read mapping quality. NEAT-Repeat can process large and repetitive genomes, such as those of corn and soybean, in a tractable amount of time by leveraging efficient methods for edit distance computation as well as running multiple jobs in parallel. NEAT-Repeat is written in Python 2.7 and C++, and is available at https://github.com/zstephens/neat-repeat.

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July 7, 2019

Emergence of gyrovirus 3 in commercial broiler chickens with transmissible viral proventriculitis.

Gyrovirus 3 (GyV3) has been identified in faeces from children with acute gastroenteritis. However, whether GyV3 is prevalent in poultry has not been determined to date. To the best of our knowledge, this study is the first to isolate GyV3 from commercial broiler chickens with transmissible viral proventriculitis (TVP) in China. The complete genome of the virus shares 98.4% sequence identity with the FecGy strain that causes acute gastroenteritis in children. Epidemiological investigation from 2013 to 2017 revealed that the infection rate of GyV3 reached 12.5% (42/336) in commercial broiler chickens with TVP, indicating that the infection of GyV3 was ubiquitous in chickens. The emergence of GyV3 in commercial broiler chickens should be highly concerning for public health.© 2018 Blackwell Verlag GmbH.

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July 7, 2019

The complete genomic sequence of a novel cold-adapted bacterium, Planococcus maritimus Y42, isolated from crude oil-contaminated soil.

Planococcus maritimus Y42, isolated from the petroleum-contaminated soil of the Qaidam Basin, can use crude oil as its sole source of carbon and energy at 20 °C. The genome of P. maritimus strain Y42 has been sequenced to provide information on its properties. Genomic analysis shows that the genome of strain Y42 contains one circular DNA chromosome with a size of 3,718,896 bp and a GC content of 48.8%, and three plasmids (329,482; 89,073; and 12,282 bp). Although the strain Y42 did not show a remarkably higher ability in degrading crude oil than other oil-degrading bacteria, the existence of strain Y42 played a significant role to reducing the overall environmental impact as an indigenous oil-degrading bacterium. In addition, genome annotation revealed that strain Y42 has many genes responsible for hydrocarbon degradation. Structural features of the genomes might provide a competitive edge for P. maritimus strain Y42 to survive in oil-polluted environments and be worthy of further study in oil degradation for the recovery of crude oil-polluted environments.

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July 7, 2019

One complete and three draft genome sequences of four Brochothrix thermosphacta strains, CD 337, TAP 175, BSAS1 3 and EBP 3070.

Brochothrix thermosphacta is one of the dominant bacterial species associated with spoilage of chilled meat and seafood products through the production of various metabolites responsible for off-odors. However, metabolic pathways leading to meat and seafood spoilage are not all well known. The production of spoiling molecules seems to depend both on strains and on food matrix. Several B. thermosphacta genome sequences have been reported, all issued from meat isolates. Here, we report four genome sequences, one complete and three as drafts. The four B. thermosphacta strains CD 337, TAP 175, BSAS1 3, and EBP 3070 were isolated from different ecological niches (seafood or meat products either spoiled or not and bovine slaughterhouse). These strains known as phenotypically and genetically different were selected to represent intraspecies diversity. CD 337 genome is 2,594,337 bp long, complete and circular, containing 2593 protein coding sequences and 28 RNA genes. TAP 175, BSAS1 3, and EBP 3070 genomes are arranged in 57, 83, and 71 contigs, containing 2515, 2668, and 2611 protein-coding sequences, respectively. These genomes were compared with two other B. thermosphacta complete genome sequences. The main genome content differences between strains are phages, plasmids, restriction/modification systems, and cell surface functions, suggesting a similar metabolic potential but a different niche adaptation capacity.

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July 7, 2019

PacBio genome sequences of Escherichia coli serotype O157:H7, diffusely adherent E. coli, and Salmonella enterica strains, all carrying plasmids with an mcr-1 resistance gene.

We report here Illumina-corrected PacBio whole-genome sequences of an Escherichia coli serotype O157:H7 strain (2017C-4109), an E. coli serotype O[undetermined]:H2 strain (2017C-4173W12), and a Salmonella enterica subsp. enterica serovar Enteritidis strain (2017K-0021), all of which carried the mcr-1 resistance gene on an IncI2 or IncX4 plasmid. We also determined that pMCR-1-CTSe is identical to a previously published plasmid, pMCR-1-CT.

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July 7, 2019

Complete genome sequence of the Arcobacter mytili type strain LMG 24559

Multiple Arcobacter species have been recovered from fresh and con- taminated waters, marine environments, and shellfish. Arcobacter mytili was recov- ered in 2006 from mussels collected from the Ebro River delta in Catalonia, Spain. This study describes the complete whole-genome sequence of the A. mytili type strain LMG 24559 (=F2075T=CECT 7386T).

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July 7, 2019

Recombination hotspots in an extended human pseudoautosomal domain predicted from double-strand break maps and characterized by sperm-based crossover analysis.

The human X and Y chromosomes are heteromorphic but share a region of homology at the tips of their short arms, pseudoautosomal region 1 (PAR1), that supports obligate crossover in male meiosis. Although the boundary between pseudoautosomal and sex-specific DNA has traditionally been regarded as conserved among primates, it was recently discovered that the boundary position varies among human males, due to a translocation of ~110 kb from the X to the Y chromosome that creates an extended PAR1 (ePAR). This event has occurred at least twice in human evolution. So far, only limited evidence has been presented to suggest this extension is recombinationally active. Here, we sought direct proof by examining thousands of gametes from each of two ePAR-carrying men, for two subregions chosen on the basis of previously published male X-chromosomal meiotic double-strand break (DSB) maps. Crossover activity comparable to that seen at autosomal hotspots was observed between the X and the ePAR borne on the Y chromosome both at a distal and a proximal site within the 110-kb extension. Other hallmarks of classic recombination hotspots included evidence of transmission distortion and GC-biased gene conversion. We observed good correspondence between the male DSB clusters and historical recombination activity of this region in the X chromosomes of females, as ascertained from linkage disequilibrium analysis; this suggests that this region is similarly primed for crossover in both male and female germlines, although sex-specific differences may also exist. Extensive resequencing and inference of ePAR haplotypes, placed in the framework of the Y phylogeny as ascertained by both Y microsatellites and single nucleotide polymorphisms, allowed us to estimate a minimum rate of crossover over the entire ePAR region of 6-fold greater than genome average, comparable with pedigree estimates of PAR1 activity generally. We conclude ePAR very likely contributes to the critical crossover function of PAR1.

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July 7, 2019

Chromosomal Sil system contributes to silver resistance in E. coli ATCC 8739.

The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO3. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO3R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO3R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.

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July 7, 2019

Mitochondrial genomes of two diplectanids (Platyhelminthes: Monogenea) expose paraphyly of the order Dactylogyridea and extensive tRNA gene rearrangements.

Recent mitochondrial phylogenomics studies have reported a sister-group relationship of the orders Capsalidea and Dactylogyridea, which is inconsistent with previous morphology- and molecular-based phylogenies. As Dactylogyridea mitochondrial genomes (mitogenomes) are currently represented by only one family, to improve the phylogenetic resolution, we sequenced and characterized two dactylogyridean parasites, Lamellodiscus spari and Lepidotrema longipenis, belonging to a non-represented family Diplectanidae.The L. longipenis mitogenome (15,433 bp) contains the standard 36 flatworm mitochondrial genes (atp8 is absent), whereas we failed to detect trnS1, trnC and trnG in L. spari (14,614 bp). Both mitogenomes exhibit unique gene orders (among the Monogenea), with a number of tRNA rearrangements. Both long non-coding regions contain a number of different (partially overlapping) repeat sequences. Intriguingly, these include putative tRNA pseudogenes in a tandem array (17 trnV pseudogenes in L. longipenis, 13 trnY pseudogenes in L. spari). Combined nucleotide diversity, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently showed that nad2, nad5 and nad4 were the most variable PCGs, whereas cox1, cox2 and cytb were the most conserved. Phylogenomic analysis showed that the newly sequenced species of the family Diplectanidae formed a sister-group with the Dactylogyridae + Capsalidae clade. Thus Dactylogyridea (represented by the Diplectanidae and Dactylogyridae) was rendered paraphyletic (with high statistical support) by the nested Capsalidea (represented by the Capsalidae) clade.Our results show that nad2, nad5 and nad4 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in the Diplectanidae. The unique gene order pattern further suggests discontinuous evolution of mitogenomic gene order arrangement in the Class Monogenea. This first report of paraphyly of the Dactylogyridea highlights the need to generate more molecular data for monogenean parasites, in order to be able to clarify their relationships using large datasets, as single-gene markers appear to provide a phylogenetic resolution which is too low for the task.

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July 7, 2019

Genomic insights into date palm origins.

With the development of next-generation sequencing technology, the amount of date palm (Phoenix dactylifera L.) genomic data has grown rapidly and yielded new insights into this species and its origins. Here, we review advances in understanding of the evolutionary history of the date palm, with a particular emphasis on what has been learned from the analysis of genomic data. We first record current genomic resources available for date palm including genome assemblies and resequencing data. We discuss new insights into its domestication and diversification history based on these improved genomic resources. We further report recent discoveries such as the existence of wild ancestral populations in remote locations of Oman and high differentiation between African and Middle Eastern populations. While genomic data are consistent with the view that domestication took place in the Gulf region, they suggest that the process was more complex involving multiple gene pools and possibly a secondary domestication. Many questions remain unanswered, especially regarding the genetic architecture of domestication and diversification. We provide a road map to future studies that will further clarify the domestication history of this iconic crop.

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July 7, 2019

Salicibibacter kimchii gen. nov., sp. nov., a moderately halophilic and alkalitolerant bacterium in the family Bacillaceae, isolated from kimchi.

A moderately halophilic and alkalitolerant bacterial strain NKC1-1T was isolated from commercial kimchi in Korea. Strain NKC1-1T was Gram-stain-positive, aerobic, rod-shaped, non-motile, and contained diaminopimelic acid-type murein. Cell growth was observed in a medium containing 0-25% (w/v) NaCl (optimal at 10% [w/v]), at 20-40°C (optimal at 37°C) and pH 6.5-10.0 (optimal at pH 9.0). The major isoprenoid quinone of the isolate was menaquinone-7, and the major polar lipids were phosphatidylglycerol and unidentified phospholipids. Cell membrane of the strain contained iso-C17:0 and anteiso-C15:0 as the major fatty acids. Its DNA G + C content was 45.2 mol%. Phylogenetic analysis indicated the strain to be most closely related to Geomicrobium halophilum with 92.7-92.9% 16S rRNA gene sequence similarity. Based on polyphasic taxonomic evaluation with phenotypic, phylogenetic, and chemotaxonomic analyses, the strain represents a novel species in a new genus, for which the name Salicibibacter kimchii gen. nov., sp. nov. is proposed (= CECT 9537T; KCCM 43276T).

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