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July 7, 2019

Characterization of the emerging zoonotic pathogen Arcobacter thereius by whole genome sequencing and comparative genomics.

Four Arcobacter species have been associated with human disease, and based on current knowledge, these Gram negative bacteria are considered as potential food and waterborne zoonotic pathogens. At present, only the genome of the species Arcobacter butzleri has been analysed, and still little is known about their physiology and genetics. The species Arcobacter thereius has first been isolated from tissue of aborted piglets, duck and pig faeces, and recently from stool of human patients with enteritis. In the present study, the complete genome and analysis of the A. thereius type strain LMG24486T, as well as the comparative genome analysis with 8 other A. thereius strains are presented. Genome analysis revealed metabolic pathways for the utilization of amino acids, which represent the main source of energy, together with the presence of genes encoding for respiration-associated and chemotaxis proteins. Comparative genome analysis with the A. butzleri type strain RM4018 revealed a large correlation, though also unique features. Furthermore, in silico DDH and ANI based analysis of the nine A. thereius strains disclosed clustering into two closely related genotypes. No discriminatory differences in genome content nor phenotypic behaviour were detected, though recently the species Arcobacter porcinus was proposed to encompass part of the formerly identified Arcobacter thereius strains. The report of the presence of virulence associated genes in A. thereius, the presence of antibiotic resistance genes, verified by in vitro susceptibility testing, as well as other pathogenic related relevant features, support the classification of A. thereius as an emerging pathogen.


July 7, 2019

Recombination of virulence genes in divergent Acidovorax avenae strains that infect a common host.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.


July 7, 2019

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.


July 7, 2019

Whole-genome sequence and variant analysis of W303, a widely-used strain of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has emerged as a superior model organism. Selection of distinct laboratory strains of S. cerevisiae with unique phenotypic properties, such as superior mating or sporulation efficiencies, has facilitated advancements in research. W303 is one such laboratory strain that is closely related to the first completely sequenced yeast strain, S288C. In this work, we provide a high-quality, annotated genome sequence for W303 for utilization in comparative analyses and genome-wide studies. Approximately 9500 variations exist between S288C and W303, affecting the protein sequences of ~700 genes. A listing of the polymorphisms and divergent genes is provided for researchers interested in identifying the genetic basis for phenotypic differences between W303 and S288C. Several divergent functional gene families were identified, including flocculation and sporulation genes, likely representing selection for desirable laboratory phenotypes. Interestingly, remnants of ancestor wine strains were found on several chromosomes. Finally, as a test of the utility of the high-quality reference genome, variant mapping revealed more accurate identification of accumulated mutations in passaged mismatch repair-defective strains. Copyright © 2017 Matheson et al.


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.


July 7, 2019

Benchmark datasets for phylogenomic pipeline validation, applications for foodborne pathogen surveillance.

As next generation sequence technology has advanced, there have been parallel advances in genome-scale analysis programs for determining evolutionary relationships as proxies for epidemiological relationship in public health. Most new programs skip traditional steps of ortholog determination and multi-gene alignment, instead identifying variants across a set of genomes, then summarizing results in a matrix of single-nucleotide polymorphisms or alleles for standard phylogenetic analysis. However, public health authorities need to document the performance of these methods with appropriate and comprehensive datasets so they can be validated for specific purposes, e.g., outbreak surveillance. Here we propose a set of benchmark datasets to be used for comparison and validation of phylogenomic pipelines.We identified four well-documented foodborne pathogen events in which the epidemiology was concordant with routine phylogenomic analyses (reference-based SNP and wgMLST approaches). These are ideal benchmark datasets, as the trees, WGS data, and epidemiological data for each are all in agreement. We have placed these sequence data, sample metadata, and “known” phylogenetic trees in publicly-accessible databases and developed a standard descriptive spreadsheet format describing each dataset. To facilitate easy downloading of these benchmarks, we developed an automated script that uses the standard descriptive spreadsheet format.Our “outbreak” benchmark datasets represent the four major foodborne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) and one simulated dataset where the “known tree” can be accurately called the “true tree”. The downloading script and associated table files are available on GitHub: https://github.com/WGS-standards-and-analysis/datasets.These five benchmark datasets will help standardize comparison of current and future phylogenomic pipelines, and facilitate important cross-institutional collaborations. Our work is part of a global effort to provide collaborative infrastructure for sequence data and analytic tools-we welcome additional benchmark datasets in our recommended format, and, if relevant, we will add these on our GitHub site. Together, these datasets, dataset format, and the underlying GitHub infrastructure present a recommended path for worldwide standardization of phylogenomic pipelines.


July 7, 2019

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.


July 7, 2019

Dynamics and impact of homologous recombination on the evolution of Legionella pneumophila.

Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires’ disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic “hotspots” of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination.


July 7, 2019

Genome diversity and evolution in the budding yeasts (Saccharomycotina).

Considerable progress in our understanding of yeast genomes and their evolution has been made over the last decade with the sequencing, analysis, and comparisons of numerous species, strains, or isolates of diverse origins. The role played by yeasts in natural environments as well as in artificial manufactures, combined with the importance of some species as model experimental systems sustained this effort. At the same time, their enormous evolutionary diversity (there are yeast species in every subphylum of Dikarya) sparked curiosity but necessitated further efforts to obtain appropriate reference genomes. Today, yeast genomes have been very informative about basic mechanisms of evolution, speciation, hybridization, domestication, as well as about the molecular machineries underlying them. They are also irreplaceable to investigate in detail the complex relationship between genotypes and phenotypes with both theoretical and practical implications. This review examines these questions at two distinct levels offered by the broad evolutionary range of yeasts: inside the best-studied Saccharomyces species complex, and across the entire and diversified subphylum of Saccharomycotina. While obviously revealing evolutionary histories at different scales, data converge to a remarkably coherent picture in which one can estimate the relative importance of intrinsic genome dynamics, including gene birth and loss, vs. horizontal genetic accidents in the making of populations. The facility with which novel yeast genomes can now be studied, combined with the already numerous available reference genomes, offer privileged perspectives to further examine these fundamental biological questions using yeasts both as eukaryotic models and as fungi of practical importance. Copyright © 2017 by the Genetics Society of America.


July 7, 2019

Emergence and genomic diversification of a virulent serogroup W: ST-2881 (CC175) Neisseria meningitidis clone in the African meningitis belt

Countries of the African ‘meningitis belt’ are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the ‘cell envelope’ and the ‘transport/binding protein’ categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion.


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.


July 7, 2019

Neisseria lactamica Y92-1009 complete genome sequence.

We present the high quality, complete genome assembly of Neisseria lactamica Y92-1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.


July 7, 2019

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.


July 7, 2019

Novel urease-negative Helicobacter sp. ‘H. enhydrae sp. nov.’ isolated from inflamed gastric tissue of southern sea otters.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all ‘H. enhydrae’ isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named ‘H. enhydrae’, isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret.


July 7, 2019

Insights into Cedecea neteri strain M006 through complete genome sequence, a rare bacterium from aquatic environment.

Cedecea neteri M006 is a rare bacterium typically found as an environmental isolate from the tropical rainforest Sungai Tua waterfall (Gombak, Selangor, Malaysia). It is a Gram-reaction-negative, facultative anaerobic, bacillus. Here, we explore the features of Cedecea neteri M006, together with its genome sequence and annotation. The genome comprised 4,965,436 bp with 4447 protein-coding genes and 103 RNA genes.


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