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April 21, 2020

A prophage and two ICESa2603-family integrative and conjugative elements (ICEs) carrying optrA in Streptococcus suis.

To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene optrA and identify the genetic elements involved in the horizontal transfer of the optrA gene in Streptococcus suis.A total of 237 S. suis isolates were screened for the presence of the optrA gene by PCR. Whole-genome DNA of three optrA-positive strains was completely sequenced using the Illumina MiSeq and Pacbio RSII platforms. MICs were determined by broth microdilution. Transferability of the optrA gene in S. suis was investigated by conjugation. The presence of circular intermediates was examined by inverse PCR.The optrA gene was present in 11.8% (28/237) of the S. suis strains. In three strains, the optrA gene was flanked by two copies of IS1216 elements in the same orientation, located either on a prophage or on ICESa2603-family integrative and conjugative elements (ICEs), including one tandem ICE. In one isolate, the optrA-carrying ICE transferred with a frequency of 2.1?×?10-8. After the transfer, the transconjugant displayed elevated MICs of the respective antimicrobial agents. Inverse PCRs revealed that circular intermediates of different sizes were formed in the three optrA-carrying strains, containing one copy of the IS1216E element and the optrA gene alone or in combination with other resistance genes.A prophage and two ICESa2603-family ICEs (including one tandem ICE) associated with the optrA gene were identified in S. suis. The association of the optrA gene with the IS1216E elements and its location on either a prophage or ICEs will aid its horizontal transfer. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.

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April 21, 2020

Highly flexible infection programs in a specialized wheat pathogen.

Many filamentous plant pathogens exhibit high levels of genomic variability, yet the impact of this variation on host-pathogen interactions is largely unknown. We have addressed host specialization in the wheat pathogen Zymoseptoria tritici. Our study builds on comparative analyses of infection and gene expression phenotypes of three isolates and reveals the extent to which genomic variation translates into phenotypic variation. The isolates exhibit genetic and genomic variation but are similarly virulent. By combining confocal microscopy, disease monitoring, staining of ROS, and comparative transcriptome analyses, we conducted a detailed comparison of the infection processes of these isolates in a susceptible wheat cultivar. We characterized four core infection stages: establishment, biotrophic growth, lifestyle transition, and necrotrophic growth and asexual reproduction that are shared by the three isolates. However, we demonstrate differentiated temporal and spatial infection development and significant differences in the expression profiles of the three isolates during the infection stages. More than 20% of the genes were differentially expressed and these genes were located significantly closer to transposable elements, suggesting an impact of epigenetic regulation. Further, differentially expressed genes were enriched in effector candidates suggesting that isolate-specific strategies for manipulating host defenses are present in Z. tritici. We demonstrate that individuals of a host-specialized pathogen have highly differentiated infection programs characterized by flexible infection development and functional redundancy. This illustrates how high genetic diversity in pathogen populations results in highly differentiated infection phenotypes, which fact needs to be acknowledged to understand host-pathogen interactions and pathogen evolution.

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April 21, 2020

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.

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April 21, 2020

Multidrug-Resistant Bovine Salmonellosis Predisposing for Severe Human Clostridial Myonecrosis.

BACKGROUND The overuse of antibiotics in animals promotes the development of multidrug-resistance predisposing for severe polymicrobial human infections. CASE REPORT We describe a case of spontaneous clostridial myonecrosis due to ulcerative colonic infection with multidrug-resistant Salmonella enterica subsp. enterica, serotype 4,[5],12: i: -. Serotyping of the colonic Salmonella isolate in the index case and the bovine farm outbreak isolates from where the patient worked indicated they were both serotype I 4,[5],12: i: -, which is linked with a multitude of large reported disease outbreaks. Further analysis revealed that they are highly genetically related and antibiotic susceptibility testing indicated that they are phenotypically identical. CONCLUSIONS Enteritis due to human acquisition of multidrug-resistant Salmonella from cattle led to the invasion and dissemination of Clostridium septicum resulting in devastating myonecrotic disease. This highlights the ramifications of co-existence and evolution of pathogenic bacteria in animals and humans and lends support to reducing the use of antibiotics in animals.

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April 21, 2020

Complete chloroplast genome sequence of Carthamus tinctorius L. from PacBio Sequel Platform

Carthamus tinctorius L, also known as safflower, is an important oil crop planted worldwide. The com- plete chloroplast (cp) genome was reported in this study using the PacBio Sequel Platform. The cp genome with a total size of 152,963bp consisted of two inverted repeats (25,128bp) separated by a large single-copy region (84,124bp) and a small single-copy region (18,583bp). Further annotation revealed the cp genome contains 112 genes, including 79 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. The information of the cp genome will be useful for investigation of evolution and molecular breeding of safflower in the future.

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April 21, 2020

Carbohydrate catabolic capability of a Flavobacteriia bacterium isolated from hadal water.

Flavobacteriia are abundant in many marine environments including hadal waters, as demonstrated recently. However, it is unclear how this flavobacterial population adapts to hadal conditions. In this study, extensive comparative genomic analyses were performed for the flavobacterial strain Euzebyella marina RN62 isolated from the Mariana Trench hadal water in low abundance. The complete genome of RN62 possessed a considerable number of carbohydrate-active enzymes with a different composition. There was a predominance of GH family 13 proteins compared to closely related relatives, suggesting that RN62 has preserved a certain capacity for carbohydrate utilization and that the hadal ocean may hold an organic matter reservoir distinct from the surface ocean. Additionally, RN62 possessed potential intracellular cycling of the glycogen/starch pathway, which may serve as a strategy for carbon storage and consumption in response to nutrient pulse and starvation. Moreover, the discovery of higher glycoside hydrolase dissimilarities among Flavobacteriia, compared to peptidases and transporters, suggested variation in polysaccharide utilization related traits as an important ecophysiological factor in response to environmental alterations, such as decreased labile organic carbon in hadal waters. The presence of abundant toxin exporting, transcription and signal transduction related genes in RN62 may further help to survive in hadal conditions, including high pressure/low temperature.Copyright © 2019 Elsevier GmbH. All rights reserved.

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April 21, 2020

Evolution of Goat’s Rue Rhizobia (Neorhizobium galegae): Analysis of Polymorphism of the Nitrogen Fixation and Nodule Formation Genes

The goat’s rue rhizobia (Neorhizobium galegae) represent a convenient model to study the evolution and speciation of symbiotic bacteria. This rhizobial species is composed of two biovars (bv. orientalis and bv. officinalis), which form N2-fixing nodules with certain species of goat’s rue (Galega orientalis and G. officinalis). The cross-inoculation between them results in the formation of nodules unable to fix nitrogen. On the basis of the data on the whole-genome sequencing, we studied the nucleotide polymorphism of 11 N. galegae strains isolated from the North Caucasus ecosystems, where G. orientalis has higher diversity than G. officinalis. The low level of differences in the polymorphism within the group of the sym genes in comparison with the nonsymbiotic genes can be associated with the active participation of host plants in the evolution of rhizobia. The intragenic polymorphism of bv. orientalis proved to be significantly higher than that of bv. officinalis. The level of polymorphism of nonsymbiotic genes was lower than that of the symbiotic genes, which are functionally more homogeneous. The divergence of the nitrogen fixation genes (nif/fix) is more pronounced than that of the nodule formation genes (nod) in the N. galegae biovars. These facts indicate the leading role of the host-specific nitrogen fixation in the evolution of the studied rizhobial species.

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April 21, 2020

Iron-associated protein interaction networks reveal the key functional modules related to survival and virulence of Pasteurella multocida.

Pasteurella multocida causes respiratory infectious diseases in a multitude of birds and mammals. A number of virulence-associated genes were reported across different strains of P. multocida, including those involved in the iron transport and metabolism. Comparative iron-associated genes of P. multocida among different animal hosts towards their interaction networks have not been fully revealed. Therefore, this study aimed to identify the iron-associated genes from core- and pan-genomes of fourteen P. multocida strains and to construct iron-associated protein interaction networks using genome-scale network analysis which might be associated with the virulence. Results showed that these fourteen strains had 1587 genes in the core-genome and 3400 genes constituting their pan-genome. Out of these, 2651 genes associated with iron transport and metabolism were selected to construct the protein interaction networks and 361 genes were incorporated into the iron-associated protein interaction network (iPIN) consisting of nine different iron-associated functional modules. After comparing with the virulence factor database (VFDB), 21 virulence-associated proteins were determined and 11 of these belonged to the heme biosynthesis module. From this study, the core heme biosynthesis module and the core outer membrane hemoglobin receptor HgbA were proposed as candidate targets to design novel antibiotics and vaccines for preventing pasteurellosis across the serotypes or animal hosts for enhanced precision agriculture to ensure sustainability in food security. Copyright © 2018. Published by Elsevier Ltd.

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April 21, 2020

Genome Organization and Adaptive Potential of Archetypal Organophosphate Degrading Sphingobium fuliginis ATCC 27551.

Sphingobium fuliginis ATCC 27551, previously classified as Flavobacterium sp. ATCC 27551, degrades neurotoxic organophosphate insecticides and nerve agents through the activity of a membrane-associated organophosphate hydrolase. This study was designed to determine the complete genome sequence of S. fuliginis ATCC 27551 to unravel its degradative potential and adaptability to harsh environments. The 5,414,624?bp genome with a GC content of 64.4% is distributed between two chromosomes and four plasmids and encodes 5,557 proteins. Of the four plasmids, designated as pSF1, pSF2, pSF3, and pSF4, only two (pSF1 and pSF2) are self-transmissible and contained the complete genetic repertoire for a T4SS. The other two plasmids (pSF3 and pSF4) are mobilizable and both showed the presence of an oriT and relaxase-encoding sequences. The sequence of plasmid pSF3 coincided with the previously determined sequence of pPDL2 and included an opd gene encoding organophosphate hydrolase as a part of the mobile element. About 15,455 orthologous clusters were identified from among the cumulatively annotated genes of 49 Sphingobium species. Phylogenetic analysis done using the core genome consisting of 802 orthologous clusters revealed a close relationship between S. fuliginis ATCC 27551 and bacteria capable of degradation of polyaromatic hydrocarbon compounds. Genes coding for transposases, efflux pumps conferring resistance to heavy metals, and TonR-type outer membrane receptors are selectively enriched in the genome of S. fuliginis ATCC 27551 and appear to contribute to the adaptive potential of the organism to challenging and harsh environments. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon.

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286-37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (~1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. © 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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April 21, 2020

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation.

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp. Copyright © 2017 Elsevier Inc. All rights reserved.

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April 21, 2020

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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