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September 22, 2019  |  

Rapid allopolyploid radiation of moonwort ferns (Botrychium; Ophioglossaceae) revealed by PacBio sequencing of homologous and homeologous nuclear regions.

Polyploidy is a major speciation process in vascular plants, and is postulated to be particularly important in shaping the diversity of extant ferns. However, limitations in the availability of bi-parental markers for ferns have greatly limited phylogenetic investigation of polyploidy in this group. With a large number of allopolyploid species, the genus Botrychium is a classic example in ferns where recurrent polyploidy is postulated to have driven frequent speciation events. Here, we use PacBio sequencing and the PURC bioinformatics pipeline to capture all homeologous or allelic copies of four long (~1?kb) low-copy nuclear regions from a sample of 45 specimens (25 diploids and 20 polyploids) representing 37 Botrychium taxa, and three outgroups. This sample includes most currently recognized Botrychium species in Europe and North America, and the majority of our specimens were genotyped with co-dominant nuclear allozymes to ensure species identification. We analyzed the sequence data using maximum likelihood (ML) and Bayesian inference (BI) concatenated-data (“gene tree”) approaches to explore the relationships among Botrychium species. Finally, we estimated divergence times among Botrychium lineages and inferred the multi-labeled polyploid species tree showing the origins of the polyploid taxa, and their relationships to each other and to their diploid progenitors. We found strong support for the monophyly of the major lineages within Botrychium and identified most of the parental donors of the polyploids; these results largely corroborate earlier morphological and allozyme-based investigations. Each polyploid had at least two distinct homeologs, indicating that all sampled polyploids are likely allopolyploids (rather than autopolyploids). Our divergence-time analyses revealed that these allopolyploid lineages originated recently-within the last two million years-and thus that the genus has undergone a recent radiation, correlated with multiple independent allopolyploidizations across the phylogeny. Also, we found strong parental biases in the formation of allopolyploids, with individual diploid species participating multiple times as either the maternal or paternal donor (but not both). Finally, we discuss the role of polyploidy in the evolutionary history of Botrychium and the interspecific reproductive barriers possibly involved in these parental biases. Copyright © 2017 Elsevier Inc. All rights reserved.


September 22, 2019  |  

Comparative genomics of the Baltic Sea toxic cyanobacteria Nodularia spumigena UHCC 0039 and its response to varying salinity.

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.


September 22, 2019  |  

Comparative genomic insights into endofungal lifestyles of two bacterial endosymbionts, Mycoavidus cysteinexigens and Burkholderia rhizoxinica.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.


September 22, 2019  |  

Capnocytophaga endodontalis sp. nov., isolated from a human refractory periapical abscess.

A novel Gram-negative, capnophilic, fusiform bacterium, designated strain ChDC OS43T, was isolated from a human refractory periapical abscess in the left mandibular second molar and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene sequence revealed that the strain belongs to the genus Capnocytophaga, as it showed sequence similarities to Capnocytophaga ochracea ATCC 27872T(96.30%) and C. sputigena ATCC 33612T(96.16%). The prevalent fatty acids of strain ChDC OS43Twere isoC15:0(57.54%), C16:0(5.93%), C16:03OH (5.72%), and C18:1cis 9 (4.41%). The complete genome of strain ChDC OS43Twas 3,412,686 bp, and the G+C content was 38.2 mol%. The average nucleotide identity (ANI) value between strain ChDC OS43Tand C. ochracea ATCC 27872Tor C. sputigena ATCC 33612Twas >92.01%. The genome-to-genome distance (GGD) value between strain ChDC OS43Tand C. ochracea ATCC 27872Tor C. sputigena ATCC 33612Twas 32.0 and 45.7%, respectively. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC OS43T(=?KCOM 1579T?=?KCTC 5562T?=?KCCM 42841T?=?JCM 32133T) should be classified as the type strain of a novel species of genus Capnocytophaga, for which the name Capnocytophaga endodontalis sp. nov. is proposed.


September 22, 2019  |  

Transcriptional profiling, molecular cloning, and functional analysis of C1 inhibitor, the main regulator of the complement system in black rockfish, Sebastes schlegelii.

C1-inhibitor (C1inh) plays a crucial role in assuring homeostasis and is the central regulator of the complement activation involved in immunity and inflammation. A C1-inhibitor gene from Sebastes schlegelii was identified and designated as SsC1inh. The identified genomic DNA and cDNA sequences were 6837 bp and 2161 bp, respectively. The genomic DNA possessed 11 exons, interrupted by 10 introns. The amino acid sequence possessed two immunoglobulin-like domains and a serpin domain. Multiple sequence alignment revealed that the serpin domain of SsC1inh was highly conserved among analyzed species where the two immunoglobulin-like domains showed divergence. The distinctiveness of teleost C1inh from other homologs was indicated by the phylogenetic analysis, genomic DNA organization, and their extended N-terminal amino acid sequences. Under normal physiological conditions, SsC1inh mRNA was most expressed in the liver, followed by the gills. The involvement of SsC1inh in homeostasis was demonstrated by modulated transcription profiles in the liver and spleen upon pathogenic stress by different immune stimulants. The protease inhibitory potential of recombinant SsC1inh (rSsC1inh) and the potentiation effect of heparin on rSsC1inh was demonstrated against C1esterase and thrombin. For the first time, the anti-protease activity of the teleost C1inh against its natural substrates C1r and C1s was proved in this study. The protease assay conducted with recombinant black rockfish C1r and C1s proteins in the presence or absence of rSsC1inh showed that the activities of both proteases were significantly diminished by rSsC1inh. Taken together, results from the present study indicate that SsC1inh actively plays a significant role in maintaining homeostasis in the immune system of black rock fish. Copyright © 2018. Published by Elsevier Ltd.


September 22, 2019  |  

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models.Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.


September 22, 2019  |  

Virgibacillus phasianinus sp. nov., a halophilic bacterium isolated from faeces of a Swinhoe’s pheasant, Lophura swinhoii.

A rod-shaped, Gram-stain-positive, motile and aerobic bacterium, designated LM2416T, was isolated from faeces of Lophuras winhoii living in Seoul Grand Park, Gyeonggi-do, Republic of Korea. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain LM2416T belonged to the genus Virgibacillus, sharing high 16S rRNA gene sequence similarities to Virgibacillus necropolis LMG 19488T (99.0?%), Virgibacillus carmonensis LMG 20964T (98.4?%), Virgibacillus arcticus Hal 1T (98.3?%) and Virgibacillus flavescens S1-20T (97.9?%). The isolate grew at 10-30?°C, pH 6-7 and 0-20?% (w/v) NaCl. Optimal growth was observed at 30?°C, pH 6-7 and 10?% (w/v) NaCl. The major fatty acid was anteiso-C15?:?0. Polar lipids were composed of phosphatidylglycerol, diphosphatidylglycerol, three unknown phospholipids and two unknown aminophospholipids. The main menaquinone was MK-7. Strain LM2416T had alanine, lysine, glutamic acid, glycine and aspartic acid as cell-wall amino acids and ribose as a cell-wall sugar. The whole genome sequences of strain LM2416T and V. necropolis KCTC 3820T were sequenced by PacBio RS II sequencing. The genome sequence-based G+C?content of strain LM2416T was 39.5?mol%. The orthologous average nucleotide identity value, showing genetic relatedness between strain LM2416T and V. necropolis KCTC 3820T, was 78.3?%. Based on the phylogenetic, biochemical, chemotaxonomic and genotypic data presented in this study, strain LM2416T is considered to represent a novel species of the genus Virgibacillus, for which the name Virgibacillus phasianinus is proposed. The type strain is LM2416T (=KCTC 33927T=JCM 32144T).


September 22, 2019  |  

Characterization of two novel bacteriophages infecting multidrug-resistant (MDR) Acinetobacter baumannii and evaluation of their therapeutic efficacy in vivo.

Acinetobacter baumannii is emerging as a challenging nosocomial pathogen due to its rapid evolution of antibiotic resistance. We report characterization of two novel bacteriophages, PBAB08 and PBAB25, infecting clinically isolated, multidrug-resistant (MDR) A. baumannii strains. Both phages belonged to Myoviridae of Caudovirales as their morphology observed under an electron microscope. Their genomes were double stranded linear DNAs of 42,312 base pairs and 40,260 base pairs, respectively. The two phages were distinct from known Acinetobacter phages when whole genome sequences were compared. PBAB08 showed a 99% similarity with 57% sequence coverage to phage AB1 and PBAB25 showed a 97% similarity with 78% sequence coverage to phage IME_AB3. BLASTN significant alignment coverage of all other known phages were <30%. Seventy six and seventy genes encoding putative phage proteins were found in the genomes of PBAB08 and PBAB25, respectively. Their genomic organizations and sequence similarities were consistent with the modular theory of phage evolution. Therapeutic efficacy of a phage cocktail containing the two and other phages were evaluated in a mice model with nasal infection of MDR A. baumannii. Mice treated with the phage cocktail showed a 2.3-fold higher survival rate than those untreated in 7 days post infection. In addition, 1/100 reduction of the number of A. baumannii in the lung of the mice treated with the phage cocktail was observed. Also, inflammatory responses of mice which were injected with the phage cocktail by intraperitoneal, intranasal, or oral route was investigated. Increase in serum cytokine was minimal regardless of the injection route. A 20% increase in IgE production was seen in intraperitoneal injection route, but not in other routes. Thus, the cocktail containing the two newly isolated phages could serve as a potential candidate for therapeutic interventions to treat A. baummannii infections.


September 22, 2019  |  

Antioxidative properties and structural features of atypical 2-Cys peroxiredoxin from Sebastes schlegelii.

Atypical 2-Cys peroxiredoxin (Prx5) is an antioxidant protein that exerts its antioxidant function by detoxifying different reactive oxygen species (ROS). Here, we identified mitochondrial Prx5 from rockfish (SsPrx5) and described its specific structural and functional characteristics. The open reading frame (ORF) of SsPrx5 (570 bp) was translated into a 190-amino acid polypeptide that contained a mitochondrial targeting sequence (MTS), thioredoxin 2 domain, two Prx-specific signature motifs, and three conserved cysteine residues. Sequence comparison indicated that the SsPrx5 protein sequence shared greatest identity with teleost orthologs, where the phylogenetic results showed an evolutionary position within the fish Prx5. The coding sequence of SsPrx5 was scattered in six exons as found in other vertebrates. Additionally, the potent antioxidant functions of recombinantly expressed SsPrx5 protein was demonstrated by insulin reduction and extracellular H2O2 scavenging both in vitro and in vivo. Quantitative real time PCR (qPCR) detected ubiquitous mRNA expression of SsPrx5 in healthy rockfish tissues, with remarkable expression observed in gill, liver, and reproductive tissues. Prompt transcription of SsPrx5 was shown in the immune-stimulated gill and liver tissues against Streptococcus iniae and lipopolysaccharide injection. Taken together, present results suggest the indispensable role of SsPrx5 in the rockfish antioxidant defense system against oxidative stresses and its role in maintaining redox balance upon pathogen invasion. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Isolation, functional characterization and transmissibility of p3PS10, a multidrug resistance plasmid of the fish pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128-256 µg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.


September 22, 2019  |  

Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism.

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.


September 22, 2019  |  

First report of the occurrence and whole-genome characterization of Edwardsiella tarda in the false killer whale (Pseudorca crassidens).

Although several Edwardsiella tarda infections have been reported, its pathogenic role in marine mammals has not been investigated at the genome level. We investigated the genome of E. tarda strain KC-Pc-HB1, isolated from the false killer whale (Pseudorca crassidens) found bycaught in South Korea. The obtained genome was similar to that of human pathogenic E. tarda strains, but distinct from other Edwardsiella species. Although type III and VI secretion systems, which are essential for the virulence of other Edwardsiella species, were absent, several virulence-related genes involved in the pathogenesis of E. tarda were found in the genome. These results provide important insights into the E. tarda infecting marine mammals and give valuable information on potential virulence factors in this pathogen.


September 22, 2019  |  

Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata.

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.


September 22, 2019  |  

Stendomycin and pantomycin are identical natural products: Preparation of a functionalized bioactive analogue.

The natural products pantomycin and stendomycin were both reported as antimicrobial agents. We demonstrate by gene cluster analysis, LC-MS analysis, and isolation that these polypeptides are identical, and we identify previously unknown congeners. We show that stendomycin can be chemically modified at its electrophilic dehydrobutyrine moiety yielding the first bioactive analogue of this natural product which can undergo additional functionalization. This compound may be a valuable starting point for molecular probe development, and we invite its distribution to the scientific community.


September 22, 2019  |  

Protocol: a versatile, inexpensive, high-throughput plant genomic DNA extraction method suitable for genotyping-by-sequencing.

The recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or “orphan” crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.We modified a non-organic solvent protocol to extract microgram quantities (1-13 µg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.This versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.


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