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September 22, 2019

Intraspecific comparative genomics of isolates of the Norway spruce pathogen (Heterobasidion parviporum) and identification of its potential virulence factors.

Heterobasidion parviporum is an economically most important fungal forest pathogen in northern Europe, causing root and butt rot disease of Norway spruce (Picea abies (L.) Karst.). The mechanisms underlying the pathogenesis and virulence of this species remain elusive. No reference genome to facilitate functional analysis is available for this species.To better understand the virulence factor at both phenotypic and genomic level, we characterized 15 H. parviporum isolates originating from different locations across Finland for virulence, vegetative growth, sporulation and saprotrophic wood decay. Wood decay capability and latitude of fungal origins exerted interactive effects on their virulence and appeared important for H. parviporum virulence. We sequenced the most virulent isolate, the first full genome sequences of H. parviporum as a reference genome, and re-sequenced the remaining 14 H. parviporum isolates. Genome-wide alignments and intrinsic polymorphism analysis showed that these isolates exhibited overall high genomic similarity with an average of at least 96% nucleotide identity when compared to the reference, yet had remarkable intra-specific level of polymorphism with a bias for CpG to TpG mutations. Reads mapping coverage analysis enabled the classification of all predicted genes into five groups and uncovered two genomic regions exclusively present in the reference with putative contribution to its higher virulence. Genes enriched for copy number variations (deletions and duplications) and nucleotide polymorphism were involved in oxidation-reduction processes and encoding domains relevant to transcription factors. Some secreted protein coding genes based on the genome-wide selection pressure, or the presence of variants were proposed as potential virulence candidates.Our study reported on the first reference genome sequence for this Norway spruce pathogen (H. parviporum). Comparative genomics analysis gave insight into the overall genomic variation among this fungal species and also facilitated the identification of several secreted protein coding genes as putative virulence factors for the further functional analysis. We also analyzed and identified phenotypic traits potentially linked to its virulence.

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September 22, 2019

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae.

Vibrio cholerae, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other Vibrio species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering crtS site recently discovered in V. cholerae were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.

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September 22, 2019

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

The sequence of the salamander.

The genome of the aquatic axolotl salamander, a native of Mexico’s lakes, has yielded some surprises, and the technique used could point the way to analysis of other organisms that have complex genomes with large numbers of sequence repeats, such as the lungfish and many species of plants.

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September 22, 2019

Genome sequence, assembly and characterization of two Metschnikowia fructicola strains used as biocontrol agents of postharvest diseases.

The yeast Metschnikowia fructicola was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product “Shemer.” Several mechanisms of action by which M. fructicola inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of M. fructicola using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of M. fructicola proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two M. fructicola strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among M. fructicola genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.

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September 22, 2019

Complete genome analysis of Gluconacetobacter xylinus CGMCC 2955 for elucidating bacterial cellulose biosynthesis and metabolic regulation.

Complete genome sequence of Gluconacetobacter xylinus CGMCC 2955 for fine control of bacterial cellulose (BC) synthesis is presented here. The genome, at 3,563,314?bp, was found to contain 3,193 predicted genes without gaps. There are four BC synthase operons (bcs), among which only bcsI is structurally complete, comprising bcsA, bcsB, bcsC, and bcsD. Genes encoding key enzymes in glycolytic, pentose phosphate, and BC biosynthetic pathways and in the tricarboxylic acid cycle were identified. G. xylinus CGMCC 2955 has a complete glycolytic pathway because sequence data analysis revealed that this strain possesses a phosphofructokinase (pfk)-encoding gene, which is absent in most BC-producing strains. Furthermore, combined with our previous results, the data on metabolism of various carbon sources (monosaccharide, ethanol, and acetate) and their regulatory mechanism of action on BC production were explained. Regulation of BC synthase (Bcs) is another effective method for precise control of BC biosynthesis, and cyclic diguanylate (c-di-GMP) is the key activator of BcsA-BcsB subunit of Bcs. The quorum sensing (QS) system was found to positively regulate phosphodiesterase, which decomposed c-di-GMP. Thus, in this study, we demonstrated the presence of QS in G. xylinus CGMCC 2955 and proposed a possible regulatory mechanism of QS action on BC production.

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September 22, 2019

Comparative genomics of bdelloid rotifers: Insights from desiccating and nondesiccating species.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer.

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September 22, 2019

Genomic architecture of haddock (Melanogrammus aeglefinus) shows expansions of innate immune genes and short tandem repeats.

Increased availability of genome assemblies for non-model organisms has resulted in invaluable biological and genomic insight into numerous vertebrates, including teleosts. Sequencing of the Atlantic cod (Gadus morhua) genome and the genomes of many of its relatives (Gadiformes) demonstrated a shared loss of the major histocompatibility complex (MHC) II genes 100 million years ago. An improved version of the Atlantic cod genome assembly shows an extreme density of tandem repeats compared to other vertebrate genome assemblies. Highly contiguous assemblies are therefore needed to further investigate the unusual immune system of the Gadiformes, and whether the high density of tandem repeats found in Atlantic cod is a shared trait in this group.Here, we have sequenced and assembled the genome of haddock (Melanogrammus aeglefinus) – a relative of Atlantic cod – using a combination of PacBio and Illumina reads. Comparative analyses reveal that the haddock genome contains an even higher density of tandem repeats outside and within protein coding sequences than Atlantic cod. Further, both species show an elevated number of tandem repeats in genes mainly involved in signal transduction compared to other teleosts. A characterization of the immune gene repertoire demonstrates a substantial expansion of MCHI in Atlantic cod compared to haddock. In contrast, the Toll-like receptors show a similar pattern of gene losses and expansions. For the NOD-like receptors (NLRs), another gene family associated with the innate immune system, we find a large expansion common to all teleosts, with possible lineage-specific expansions in zebrafish, stickleback and the codfishes.The generation of a highly contiguous genome assembly of haddock revealed that the high density of short tandem repeats as well as expanded immune gene families is not unique to Atlantic cod – but possibly a feature common to all, or most, codfishes. A shared expansion of NLR genes in teleosts suggests that the NLRs have a more substantial role in the innate immunity of teleosts than other vertebrates. Moreover, we find that high copy number genes combined with variable genome assembly qualities may impede complete characterization of these genes, i.e. the number of NLRs in different teleost species might be underestimates.

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September 22, 2019

Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

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September 22, 2019

A transposable element annotation pipeline and expression analysis reveal potentially active elements in the microalga Tisochrysis lutea.

Transposable elements (TEs) are mobile DNA sequences known as drivers of genome evolution. Their impacts have been widely studied in animals, plants and insects, but little is known about them in microalgae. In a previous study, we compared the genetic polymorphisms between strains of the haptophyte microalga Tisochrysis lutea and suggested the involvement of active autonomous TEs in their genome evolution.To identify potentially autonomous TEs, we designed a pipeline named PiRATE (Pipeline to Retrieve and Annotate Transposable Elements, download: https://doi.org/10.17882/51795 ), and conducted an accurate TE annotation on a new genome assembly of T. lutea. PiRATE is composed of detection, classification and annotation steps. Its detection step combines multiple, existing analysis packages representing all major approaches for TE detection and its classification step was optimized for microalgal genomes. The efficiency of the detection and classification steps was evaluated with data on the model species Arabidopsis thaliana. PiRATE detected 81% of the TE families of A. thaliana and correctly classified 75% of them. We applied PiRATE to T. lutea genomic data and established that its genome contains 15.89% Class I and 4.95% Class II TEs. In these, 3.79 and 17.05% correspond to potentially autonomous and non-autonomous TEs, respectively. Annotation data was combined with transcriptomic and proteomic data to identify potentially active autonomous TEs. We identified 17 expressed TE families and, among these, a TIR/Mariner and a TIR/hAT family were able to synthesize their transposase. Both these TE families were among the three highest expressed genes in a previous transcriptomic study and are composed of highly similar copies throughout the genome of T. lutea. This sum of evidence reveals that both these TE families could be capable of transposing or triggering the transposition of potential related MITE elements.This manuscript provides an example of a de novo transposable element annotation of a non-model organism characterized by a fragmented genome assembly and belonging to a poorly studied phylum at genomic level. Integration of multi-omics data enabled the discovery of potential mobile TEs and opens the way for new discoveries on the role of these repeated elements in genomic evolution of microalgae.

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September 22, 2019

Signatures of host specialization and a recent transposable element burst in the dynamic one-speed genome of the fungal barley powdery mildew pathogen.

Powdery mildews are biotrophic pathogenic fungi infecting a number of economically important plants. The grass powdery mildew, Blumeria graminis, has become a model organism to study host specialization of obligate biotrophic fungal pathogens. We resolved the large-scale genomic architecture of B. graminis forma specialis hordei (Bgh) to explore the potential influence of its genome organization on the co-evolutionary process with its host plant, barley (Hordeum vulgare).The near-chromosome level assemblies of the Bgh reference isolate DH14 and one of the most diversified isolates, RACE1, enabled a comparative analysis of these haploid genomes, which are highly enriched with transposable elements (TEs). We found largely retained genome synteny and gene repertoires, yet detected copy number variation (CNV) of secretion signal peptide-containing protein-coding genes (SPs) and locally disrupted synteny blocks. Genes coding for sequence-related SPs are often locally clustered, but neither the SPs nor the TEs reside preferentially in genomic regions with unique features. Extended comparative analysis with different host-specific B. graminis formae speciales revealed the existence of a core suite of SPs, but also isolate-specific SP sets as well as congruence of SP CNV and phylogenetic relationship. We further detected evidence for a recent, lineage-specific expansion of TEs in the Bgh genome.The characteristics of the Bgh genome (largely retained synteny, CNV of SP genes, recently proliferated TEs and a lack of significant compartmentalization) are consistent with a “one-speed” genome that differs in its architecture and (co-)evolutionary pattern from the “two-speed” genomes reported for several other filamentous phytopathogens.

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September 22, 2019

The African Bullfrog (Pyxicephalus adspersus) genome unites the two ancestral ingredients for making vertebrate sex chromosomes

Heteromorphic sex chromosomes have evolved repeatedly among vertebrate lineages despite largely deleterious reductions in gene dose. Understanding how this gene dose problem is overcome is hampered by the lack of genomic information at the base of tetrapods and comparisons across the evolutionary history of vertebrates. To address this problem, we produced a chromosome-level genome assembly for the African Bullfrog (Pyxicephalus adspersus)–an amphibian with heteromorphic ZW sex chromosomes–and discovered that the Bullfrog Z is surprisingly homologous to substantial portions of the human X. Using this new reference genome, we identified ancestral synteny among the sex chromosomes of major vertebrate lineages, showing that non-mammalian sex chromosomes are strongly associated with a single vertebrate ancestral chromosome, while mammals are associated with another that displays increased haploinsufficiency. The sex chromosomes of the African Bullfrog however, share genomic blocks with both humans and non-mammalian vertebrates, connecting the two ancestral chromosome sequences that repeatedly characterize vertebrate sex chromosomes. Our results highlight the consistency of sex-linked sequences despite sex determination system lability and reveal the repeated use of two major genomic sequence blocks during vertebrate sex chromosome evolution.

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September 22, 2019

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

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September 22, 2019

Long-read sequencing data analysis for yeasts.

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ~41 h to generate a complete and well-annotated genome from ~100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

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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.

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