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July 7, 2019

Genome-wide identification of the mutation underlying fleece variation and discriminating ancestral hairy species from modern woolly sheep.

The composition and structure of fleece variation observed in mammals is a consequence of a strong selective pressure for fiber production after domestication. In sheep, fleece variation discriminates ancestral species carrying a long and hairy fleece from modern domestic sheep (Ovis aries) owning a short and woolly fleece. Here, we report that the “woolly” allele results from the insertion of an antisense EIF2S2 retrogene (called asEIF2S2) into the 3′ UTR of the IRF2BP2 gene leading to an abnormal IRF2BP2 transcript. We provide evidence that this chimeric IRF2BP2/asEIF2S2 messenger 1) targets the genuine sense EIF2S2 RNA and 2) creates a long endogenous double-stranded RNA which alters the expression of both EIF2S2 and IRF2BP2 mRNA. This represents a unique example of a phenotype arising via a RNA-RNA hybrid, itself generated through a retroposition mechanism. Our results bring new insights on the sheep population history thanks to the identification of the molecular origin of an evolutionary phenotypic variation.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Molecular and genomic features of Mycobacterium bovis strain 1595 isolated from Korean cattle.

The aim of this study was to investigate the molecular characteristics and to conduct a comparative genomic analysis of Mycobacterium (M.) bovis strain 1595 isolated from a native Korean cow. Molecular typing showed that M. bovis 1595 has spoligotype SB0140 with mycobacterial interspersed repetitive units-variable number of tandem repeats typing of 4-2-5-3-2-7-5-5-4-3-4-3-4-3, representing the most common type of M. bovis in Korea. The complete genome sequence of strain 1595 was determined by single-molecule real-time technology, which showed a genome of 4351712 bp in size with a 65.64% G + C content and 4358 protein-coding genes. Comparative genomic analysis with the genomes of Mycobacterium tuberculosis complex strains revealed that all genomes are similar in size and G + C content. Phylogenetic analysis revealed all strains were within a 0.1% average nucleotide identity value, and MUMmer analysis illustrated that all genomes showed positive collinearity with strain 1595. A sequence comparison based on BLASTP analysis showed that M. bovis AF2122/97 was the strain with the greatest number of completely matched proteins to M. bovis 1595. This genome sequence analysis will serve as a valuable reference for improving understanding of the virulence and epidemiologic traits among M. bovis isolates in Korea.

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July 7, 2019

Comparative sequence analysis of multidrug-resistant IncA/C plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

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July 7, 2019

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation.

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

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July 7, 2019

Characterization of a PVL-negative community-acquired methicillin-resistant Staphylococcus aureus strain of sequence type 88 in China.

Sequence type 88 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain SR434, isolated from an outpatient with skin and soft tissue infection, was subjected to whole genome sequencing, antimicrobial susceptibility testing, mouse skin infection model and hemolysis analysis to identify its virulence and resistance determinants. MRSA strain SR434 is resistant to clindamycin, erythromycin and fosfomycin. Four plasmids with resistance genes were identified in this strain, including a 20,658bp blaZ-carrying plasmid, a 2473bp ermC-carrying plasmid, a 2622bp fosB7-carrying plasmid (86% identity with plasmid in a ST2590 MRSA strain) and a 4817bp lnuA-carrying plasmid (99% identity with pLNU4 from bovine coagulase-nagetive Staphylococci). This strain contains staphylococcal cassette chromosome mec type IV and does not contain arginine catabolic mobile element or Panton-Valentine-Leukocidin. SR434 harbors genomic islands ?Saa, ?Saß, ?Sa? and FSa3 and pathogenicity islands ?Sa2 that carries genes encoding toxic shock syndrome toxin 1, superantigen enterotoxin C and superantigen enterotoxin L. Mouse skin infection model results show that SR434 had similar virulence potential causing invasive skin infection as a PVL-negative epidemic Korea clone HL1 (ST72). CA-MRSA strain of ST88 lineage might be a great concern for its high virulence. PVL has limited contribution to virulence phenotype among this lineage. Copyright © 2017 Elsevier GmbH. All rights reserved.

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July 7, 2019

A review of methods used for studying the molecular epidemiology of Brachyspira hyodysenteriae.

Brachyspira (B.) spp. are intestinal spirochaetes isolated from pigs, other mammals, birds and humans. In pigs, seven Brachyspira spp. have been described, i.e. B. hyodysenteriae, B. pilosicoli, B. intermedia, B. murdochii, B. innocens, B. suanatina and B. hampsonii. Brachyspira hyodysenteriae is especially relevant in pigs as it causes swine dysentery and hence considerable economic losses to the pig industry. Furthermore, reduced susceptibility of B. hyodysenteriae to antimicrobials is of increasing concern. The epidemiology of B. hyodysenteriae infections is only partially understood, but different methods for detection, identification and typing have supported recent improvements in knowledge and understanding. In the last years, molecular methods have been increasingly used. Molecular epidemiology links molecular biology with epidemiology, offering unique opportunities to advance the study of diseases. This review is based on papers published in the field of epidemiology and molecular epidemiology of B. hyodysenteriae in pigs. Electronic databases were screened for potentially relevant papers using title and abstract and finally, Barcellos et al. papers were systemically selected and assessed. The review summarises briefly the current knowledge on B. hyodysenteriae epidemiology and elaborates on molecular typing techniques available. Results of the studies are compared and gaps in the knowledge are addressed. Finally, potential areas for future research are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Detection and genetic features of MCR-1-producing plasmid in human Escherichia coli infection in South Korea.

The plasmid-mediated colistin resistance gene, mcr-1, was identified for the first time from a hospitalized patient in South Korea. The mcr-1 gene was successfully transferred to E. coli J53 recipient and conferred resistance to colistin in the recipient. The mcr-1-harboring plasmid possessed a typical IncI2 group and did not have the mcr-1-associated ISApl1 element. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the a-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea’s defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

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July 7, 2019

Morphological and genetic analyses of the invasive forest pathogen Phytophthora austrocedri reveal two clonal lineages colonised Britain and Argentina from a common ancestral population.

Phytophthora austrocedri is causing widespread mortality of Austrocedrus chilensis in Argentina and Juniperus communis in Britain. The pathogen has also been isolated from J. horizontalis in Germany. Isolates from Britain, Argentina and Germany are homothallic with no clear differences in the dimensions of sporangia, oogonia or oospores. Argentinian and German isolates grew faster than British isolates across a range of media and had a higher temperature tolerance although most isolates regardless of origin grew best at 15°C and all isolates were killed at 25°C. Argentinian and British isolates caused lesions on both hosts when inoculated onto A. chilensis and J. communis; however the Argentinian isolate caused longer lesions on A. chilensis than on J. communis and vice versa for the British isolate. Genetic analyses of nuclear and mitochondrial loci showed that all British isolates are identical. Argentinian isolates and the German isolate are also identical but differ from the British isolates. Single nucleotide polymorphisms are shared between the British and Argentinian isolates. It is concluded that British isolates and Argentinian isolates conform to two distinct clonal lineages of P. austrocedri founded from the same as-yet unidentified source population. These lineages should be recognised and treated as separate risks by international plant health legislation.

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July 7, 2019

Comparative genomic analysis identifies a Campylobacter clade deficient in selenium metabolism.

The nonthermotolerant Campylobacter species C. fetus, C. hyointestinalis, C. iguaniorum, and C. lanienae form a distinct phylogenetic cluster within the genus. These species are primarily isolated from foraging (swine) or grazing (e.g., cattle, sheep) animals and cause sporadic and infrequent human illness. Previous typing studies identified three putative novel C. lanienae-related taxa, based on either MLST or atpA sequence data. To further characterize these putative novel taxa and the C. fetus group as a whole, 76 genomes were sequenced, either to completion or to draft level. These genomes represent 26 C. lanienae strains and 50 strains of the three novel taxa. C. fetus, C. hyointestinalis and C. iguaniorum genomes were previously sequenced to completion; therefore, a comparative genomic analysis across the entire C. fetus group was conducted (including average nucleotide identity analysis) that supports the initial identification of these three novel Campylobacter species. Furthermore, C. lanienae and the three putative novel species form a discrete clade within the C. fetus group, which we have termed the C. lanienae clade. This clade is distinguished from other members of the C. fetus group by a reduced genome size and distinct CRISPR/Cas systems. Moreover, there are two signature characteristics of the C. lanienae clade. C. lanienae clade genomes carry four to ten unlinked and similar, but nonidentical, flagellin genes. Additionally, all 76 C. lanienae clade genomes sequenced demonstrate a complete absence of genes related to selenium metabolism, including genes encoding the selenocysteine insertion machinery, selenoproteins, and the selenocysteinyl tRNA. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.

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July 7, 2019

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

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July 7, 2019

Complete genome sequence of a livestock-associated methicillin-resistant Staphylococcus aureus sequence type 5 isolate from the United States.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) may be the largest MRSA reservoir outside the hospital setting. One concern with LA-MRSA is the acquisition of novel mobile genetic elements by these isolates. Here, we report the complete genome sequence of a swine LA-MRSA sequence type 5 isolate from the United States.

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July 7, 2019

Pearl millet genome sequence provides a resource to improve agronomic traits in arid environments.

Pearl millet [Cenchrus americanus (L.) Morrone] is a staple food for more than 90 million farmers in arid and semi-arid regions of sub-Saharan Africa, India and South Asia. We report the ~1.79 Gb draft whole genome sequence of reference genotype Tift 23D2B1-P1-P5, which contains an estimated 38,579 genes. We highlight the substantial enrichment for wax biosynthesis genes, which may contribute to heat and drought tolerance in this crop. We resequenced and analyzed 994 pearl millet lines, enabling insights into population structure, genetic diversity and domestication. We use these resequencing data to establish marker trait associations for genomic selection, to define heterotic pools, and to predict hybrid performance. We believe that these resources should empower researchers and breeders to improve this important staple crop.

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July 7, 2019

Complete genome sequence of livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 isolated from swine in the United States.

Methicillin-resistant Staphylococcus aureus (MRSA) colonizes and causes disease in many animal species. Livestock-associated MRSA (LA-MRSA) isolates are represented by isolates of the sequence type 398 (ST398). These isolates are considered to be livestock adapted. This report provides the complete genome sequence of one swine-associated LA-MRSA ST398 isolate from the United States.

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July 7, 2019

Genomic insights into the pathogenicity and environmental adaptability of Enterococcus hirae R17 isolated from pork offered for retail sale.

Genetic information about Enterococcus hirae is limited, a feature that has compromised our understanding of these clinically challenging bacteria. In this study, comparative analysis was performed of E. hirae R17, a daptomycin-resistant strain isolated from pork purchased from a retail market in Beijing, China, and three other enterococcal genomes (Enterococcus faecium DO, Enterococcus faecalis V583, and E. hirae ATCC™ 9790). Some 1,412 genes were identified that represented the core genome together with an additional 139 genes that were specific to E. hirae R17. The functions of these R17 strain-specific coding sequences relate to the COGs categories of carbohydrate transport and metabolism and transcription, a finding that suggests the carbohydrate utilization capacity of E. hirae R17 may be more extensive when compared with the other three bacterial species (spp.). Analysis of genomic islands and virulence genes highlighted the potential that horizontal gene transfer played as a contributor of variations in pathogenicity in this isolate. Drug-resistance gene prediction and antibiotic susceptibility testing indicated E. hirae R17 was resistant to several antimicrobial compounds, including bacitracin, ciprofloxacin, daptomycin, erythromycin, and tetracycline, thereby limiting chemotherapeutic treatment options. Further, tolerance to biocides and metals may confer a phenotype that facilitates the survival and adaptation of this isolate against food preservatives, disinfectants, and antibacterial coatings. The genomic plasticity, mediated by IS elements, transposases, and tandem repeats, identified in the E. hirae R17 genome may support adaptation to new environmental niches, such as those that are found in hospitalized patients. A predicted transmissible plasmid, pRZ1, was found to carry several antimicrobial determinants, along with some predicted pathogenic genes. These data supported the previously determined phenotype confirming that the foodborne E. hirae R17 is a multidrug-resistant pathogenic bacterium with evident genome plasticity and environmental adaptability.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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