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September 22, 2019

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.

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September 22, 2019

Whole-genome resequencing and pan-transcriptome reconstruction highlight the impact of genomic structural Variation on secondary metabolite gene clusters in the grapevine Esca pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

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September 22, 2019

Phenotypic and genomic properties of Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov.

Two strains, VM2412T and VR2415T, were isolated from the feces of an Andean condor (Vultur gryphus) living in Seoul Grand Park, Gyeonggi-do, South Korea. Cells of both strains were observed to be Gram-stain positive, non-motile, aerobic, catalase positive and oxidase negative. Growth was found to occur at 10-30°C, showing optimum growth at 30°C. The strains could tolerate up to 15% (w/v) NaCl concentration and grow at pH 6-9. The strains shared 99.3% 16S rRNA gene sequence similarity to each other but were identified as two distinct species based on 89.0-89.2% ANIb, 90.3% ANIm, 89.7% OrthoANI and 38.0% dDDH values calculated using whole genome sequences. Among species with validly published names, Brachybacterium ginsengisoli DCY80T shared high 16S rRNA gene sequence similarities with strains VM2412T (98.7%) and VR2415T (98.4%) and close genetic relatedness with strains VM2412T (83.3-83.5% ANIb, 87.0% ANIm, 84.3% OrthoANI and 27.8% dDDH) and VR2415T (82.8-83.2% ANIb, 86.7% ANIm, 83.9% OrthoANI and 27.2% dDDH). The major fatty acid of the two strains was identified as anteiso-C15:0 and the polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, presumptively phosphatidylethanolamine and three unidentified glycolipids. Strain VR2415T also produced an unidentified phospholipid. The cell walls of the two strains contained meso-diaminopimelic acid as diagnostic diamino acid and the whole cell sugars were ribose, glucose, and galactose. The strains contained MK-7 as their predominant menaquinone. The genomes of strains VM2412T, VR2415T, and B. ginsengisoli DCY80T were sequenced in this study. The genomic G+C contents of strains VM2412T and VR2415T were determined to be 70.8 and 70.4 mol%, respectively. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that the strains formed a clade with members of the genus Brachybacterium, supporting their taxonomic classification into the genus Brachybacterium. Based on phenotypic and genotypic analyses in this study, strains VM2412T and VR2415T are considered to represent two novel species of the genus Brachybacterium and the names Brachybacterium vulturis sp. nov. and Brachybacterium avium sp. nov. are proposed for strains VM2412T (=KCTC 39996T = JCM 32142T) and VR2415T (=KCTC 39997T = JCM 32143T), respectively.

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September 22, 2019

Complete genome of streamlined marine actinobacterium Pontimonas salivibrio strain CL-TW6T adapted to coastal planktonic lifestyle.

Pontimonas salivibrio strain CL-TW6T (=KCCM 90105?=?JCM18206) was characterized as the type strain of a new genus within the Actinobacterial family Microbacteriaceae. It was isolated from a coastal marine environment in which members of Microbactericeae have not been previously characterized.The genome of P. salivibrio CL-TW6T was a single chromosome of 1,760,810 bp. Genomes of this small size are typically found in bacteria growing slowly in oligotrophic zones and said to be streamlined. Phylogenetic analysis showed it to represent a lineage originating in the Microbacteriaceae radiation occurring before the snowball Earth glaciations, and to have a closer relationship with some streamlined bacteria known through metagenomic data. Several genomic characteristics typical of streamlined bacteria are found: %G?+?C is lower than non-streamlined members of the phylum; there are a minimal number of rRNA and tRNA genes, fewer paralogs in most gene families, and only two sigma factors; there is a noticeable absence of some nonessential metabolic pathways, including polyketide synthesis and catabolism of some amino acids. There was no indication of any phage genes or plasmids, however, a system of active insertion elements was present. P. salivibrio appears to be unusual in having polyrhamnose-based cell wall oligosaccharides instead of mycolic acid or teichoic acid-based oligosaccharides. Oddly, it conducts sulfate assimilation apparently for sulfating cell wall components, but not for synthesizing amino acids. One gene family it has more of, rather than fewer of, are toxin/antitoxin systems, which are thought to down-regulate growth during nutrient deprivation or other stressful conditions.Because of the relatively small number of paralogs and its relationship to the heavily characterized Mycobacterium tuberculosis, we were able to heavily annotate the genome of P. salivibrio CL-TW6T. Its streamlined status and relationship to streamlined metagenomic constructs makes it an important reference genome for study of the streamlining concept. The final evolutionary trajectory of CL-TW6 T was to adapt to growth in a non-oligotrophic coastal zone. To understand that adaptive process, we give a thorough accounting of gene content, contrasting with both oligotrophic streamlined bacteria and large genome bacteria, and distinguishing between genes derived by vertical and horizontal descent.

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September 22, 2019

A gene-rich fraction analysis of the Passiflora edulis genome reveals highly conserved microsyntenic regions with two related Malpighiales species.

Passiflora edulis is the most widely cultivated species of passionflowers, cropped mainly for industrialized juice production and fresh fruit consumption. Despite its commercial importance, little is known about the genome structure of P. edulis. To fill in this gap in our knowledge, a genomic library was built, and now completely sequenced over 100 large-inserts. Sequencing data were assembled from long sequence reads, and structural sequence annotation resulted in the prediction of about 1,900 genes, providing data for subsequent functional analysis. The richness of repetitive elements was also evaluated. Microsyntenic regions of P. edulis common to Populus trichocarpa and Manihot esculenta, two related Malpighiales species with available fully sequenced genomes were examined. Overall, gene order was well conserved, with some disruptions of collinearity identified as rearrangements, such as inversion and translocation events. The microsynteny level observed between the P. edulis sequences and the compared genomes is surprising, given the long divergence time that separates them from the common ancestor. P. edulis gene-rich segments are more compact than those of the other two species, even though its genome is much larger. This study provides a first accurate gene set for P. edulis, opening the way for new studies on the evolutionary issues in Malpighiales genomes.

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September 22, 2019

Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs.

Classical swine fever virus (CSFV) infection causes most variable clinical syndromes from chronic or latent infection to acute death, and it is generally acknowledged that the course of disease is affected by both virus and host factors. To compare host immune responses to differentially virulent CSFV strains in pigs, fifteen 8-week-old specific-pathogen-free pigs were randomly divided into four groups and inoculated with the CSFV Shimen strain (a highly virulent strain), the HLJZZ2014 strain (a moderately virulent strains), C-strain (an avirulent strain), and DMEM (mock control), respectively. Infection with the Shimen or HLJZZ2014 strain resulted in fever, clinical signs and histopathological lesions, which were not observed in the C-strain-inoculated pigs, though low viral genome copies were detected in the peripheral blood and tissue samples. The data showed that the virulence of the strains affected the outcome of duration and intensity of the disease rather than the tissue tropism of the virus. Furthermore, leukopenia, lymphocytopenia, differentiation of T-cells, and the secretion of cytokines associated with inflammation or apoptosis such as interferon alpha (IFN-a), tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), IL-4, IL-6, and IL-10 were induced by the virulent CSFV infection, the differences reflected in onset and extent of the regulation. Taken together, our results revealed that the major differences among the three strains resided in the kinetics of host response to the infection: severe and immediate with the highly virulent strain, while progressive and delayed with the moderately virulent one. This comparative study will help to dissect the pathogenesis of CSFV. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Opposite polarity monospore genome de novo sequencing and comparative analysis reveal the possible heterothallic life cycle of Morchella importuna.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

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September 22, 2019

Co-location of the blaKPC-2, blaCTX-M-65, rmtB and virulence relevant factors in an IncFII plasmid from a hypermucoviscous Klebsiella pneumoniae isolate.

Hypervirulent variants of klebsiella pneumoniae (hvKP), which cause serious infections not only healthy individuals, but also the immunocompromised patients, have been increasingly reported recently. One conjugation of a hypermucoviscous strian SWU01 co-carried the resistance gene blaKPC-2 and virulence gene iroN by the PCR detection from three carbapenem-resistance hvKP. To know the genetic context of this plasmid. The whole genome of this strain was sequenced. We got a 162,552-bp plasmid (pSWU01) which co-carried the resistance gene blaKPC-2 and virulence gene iroN. It is composed of a typical IncFII-type backbone, five resistance genes including blaCTX-M-65, blaKPC-2, blaSHV-12, blaTEM-1 and rmtB, and several virulence relevant factors including iroN, traT and toxin-antitoxin systems. The plasmid pSWU01 co-carrying the multidrug resistance determinants and virulence relevant factors from the hypermucoviscous K. pneumoniae represents a novel therapeutic challenge. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Characterization of Haemophilus parasuis serovar 2 CL120103, a moderately virulent strain in China

Haemophilus parasuis is an important bacterium affecting pigs, causing Glässer’s disease. To further characterize this species, we determined the complete genomic sequence of H. parasuis CL120103, which was isolated from diseased pigs. The strain H. parasuis CL120103 was identified as serovar 2. The size of the largest scaffold is 2,326,318 bp and contains 145 large contigs, with the N50 contig being 20,573 bp in length. The complete genome of H. parasuis CL120103 is 2,305,354 bp in length with 39.97% GC content and contains 2227 protein-coding genes, 19 ribosomal rRNA operons and 60 tRNA genes. Sequence similarity of the genome of H. parasuis CL120103 to the previously sequenced genome of H. parasuis was up to 96% and query cover to 86%. Annotation of the genome of H. parasuis CL120103 identified a number of genes encoding potential virulence factors. These virulence factors are involved in metabolism, adhesion, secretion and LPS biosynthesis. These related genes pave the way to better understand mechanisms underlying metabolic capabilities. The comprehensive genetic and phylogenetic analysis shows that H. parasuis is closely related to Actinobacillus pleuropneumoniae and provides a foundation for future experimental confirmation of the virulence and pathogen-host interactions in H. parasuis.

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September 22, 2019

Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association.

Salmonella enterica serovar Montevideo has been linked to recent foodborne illness outbreaks resulting from contamination of products such as fruits, vegetables, seeds and spices. Studies have shown that Montevideo also is frequently associated with healthy cattle and can be isolated from ground beef, yet human salmonellosis outbreaks of Montevideo associated with ground beef contamination are rare. This disparity fuelled our interest in characterizing the genomic differences between Montevideo strains isolated from healthy cattle and beef products, and those isolated from human patients and outbreak sources. To that end, we sequenced 13 Montevideo strains to completion, producing high-quality genome assemblies of isolates from human patients (n=8) or from healthy cattle at slaughter (n=5). Comparative analysis of sequence data from this study and publicly available sequences (n=72) shows that Montevideo falls into four previously established clades, differentially occupied by cattle and human strains. The results of these analyses reveal differences in metabolic islands, environmental adhesion determinants and virulence factors within each clade, and suggest explanations for the infrequent association between bovine isolates and human illnesses.

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September 22, 2019

Distinct genomic features characterize two clades of Corynebacterium diphtheriae: Proposal of Corynebacterium diphtheriae subsp. diphtheriae subsp. nov. and Corynebacterium diphtheriae subsp. lausannense subsp. nov.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

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September 22, 2019

Functional and genome sequence-driven characterization of tal effector gene repertoires reveals novel variants with altered specificities in closely related Malian Xanthomonas oryzae pv. oryzae strains.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

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September 22, 2019

The energy-coupling factor transporter module EcfAA’T, a novel candidate for the genetic basis of fatty acid-auxotrophic small-colony variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA’T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA’T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

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September 22, 2019

Improved Brassica rapa reference genome by single-molecule sequencing and chromosome conformation capture technologies.

Brassica rapa comprises several important cultivated vegetables and oil crops. Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous, thereby limiting extensive genetic and genomic analyses. Here, we report an improved assembly of the B. rapa genome (v3.0) using single-molecule sequencing, optical mapping, and chromosome conformation capture technologies (Hi-C). Relative to the previous reference genomes, our assembly features a contig N50 size of 1.45?Mb, representing a ~30-fold improvement. We also identified a new event that occurred in the B. rapa genome ~1.2 million years ago, when a long terminal repeat retrotransposon (LTR-RT) expanded. Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B. rapa genome. The B. rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B. rapa. This resource will facilitate breeding efforts in B. rapa, as well as comparative genomic analysis with other Brassica species.

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September 22, 2019

Comparative genomics reveal a flagellar system, a type VI secretion system and plant growth-promoting gene clusters unique to the endophytic bacterium Kosakonia radicincitans.

The recent worldwide discovery of plant growth-promoting (PGP) Kosakonia radicincitans in a large variety of crop plants suggests that this species confers significant influence on plants, both in terms of yield increase and product quality improvement. We provide a comparative genome analysis which helps to unravel the genetic basis for K. radicincitans’ motility, competitiveness and plant growth-promoting capacities. We discovered that K. radicincitans carries multiple copies of complex gene clusters, among them two flagellar systems and three type VI secretion systems (T6SSs). We speculate that host invasion may be facilitated by different flagella, and bacterial competitor suppression by effector proteins ejected via T6SSs. We found a large plasmid in K. radicincitans DSM 16656T, the species type strain, that confers the potential to exploit plant-derived carbon sources. We propose that multiple copies of complex gene clusters in K. radicincitans are metabolically expensive but provide competitive advantage over other bacterial strains in nutrient-rich environments. The comparison of the DSM 16656T genome to genomes of other genera of enteric plant growth-promoting bacteria (PGPB) exhibits traits unique to DSM 16656T and K. radicincitans, respectively, and traits shared between genera. We used the output of the in silico analysis for predicting the purpose of genomic features unique to K. radicincitans and performed microarray, PhyloChip, and microscopical analyses to gain deeper insight into the interaction of DSM 16656T, plants and associated microbiota. The comparative genome analysis will facilitate the future search for promising candidates of PGPB for sustainable crop production.

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