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September 22, 2019  |  

Genomic approaches for studying crop evolution.

Understanding how crop plants evolved from their wild relatives and spread around the world can inform about the origins of agriculture. Here, we review how the rapid development of genomic resources and tools has made it possible to conduct genetic mapping and population genetic studies to unravel the molecular underpinnings of domestication and crop evolution in diverse crop species. We propose three future avenues for the study of crop evolution: establishment of high-quality reference genomes for crops and their wild relatives; genomic characterization of germplasm collections; and the adoption of novel methodologies such as archaeogenetics, epigenomics, and genome editing.

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September 22, 2019  |  

Complete genome sequence of a blaKPC-2-positive Klebsiella pneumoniae strain isolated from the effluent of an urban sewage treatment plant in Japan.

Antimicrobial resistance genes (ARGs) and the bacteria that harbor them are widely distributed in the environment, especially in surface water, sewage treatment plant effluent, soil, and animal waste. In this study, we isolated a KPC-2-producing Klebsiella pneumoniae strain (GSU10-3) from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP) and determined its complete genome sequence. Strain GSU10-3 is resistant to most ß-lactam antibiotics and other antimicrobial agents (quinolones and aminoglycosides). This strain is classified as sequence type 11 (ST11), and a core genome phylogenetic analysis indicated that strain GSU10-3 is closely related to KPC-2-positive Chinese clinical isolates from 2011 to 2017 and is clearly distinct from strains isolated from the European Union (EU), United States, and other Asian countries. Strain GSU10-3 harbors four plasmids, including a blaKPC-2-positive plasmid, pGSU10-3-3 (66.2?kb), which is smaller than other blaKPC-2-positive plasmids and notably carries dual replicons (IncFII [pHN7A8] and IncN). Such downsizing and the presence of dual replicons may promote its maintenance and stable replication, contributing to its broad host range with low fitness costs. A second plasmid, pGSU10-3-1 (159.0?kb), an IncA/C2 replicon, carries a class 1 integron (containing intI1, dfrA12, aadA2, qacE?1, and sul1) with a high degree of similarity to a broad-host-range plasmid present in the family Enterobacteriaceae The plasmid pGSU10-3-2 (134.8?kb), an IncFII(K) replicon, carries the IS26-mediated ARGs [aac(6′)Ib-cr,blaOXA-1, catB4 (truncated), and aac(3)-IId], tet(A), and a copper/arsenate resistance locus. GSU10-3 is the first nonclinical KPC-2-producing environmental Enterobacteriaceae isolate from Japan for which the whole genome has been sequenced.IMPORTANCE We isolated and determined the complete genome sequence of a KPC-2-producing K. pneumoniae strain from a sampling site in Tokyo Bay, Japan, near a wastewater treatment plant (WWTP). In Japan, the KPC type has been very rarely detected, while IMP is the most predominant type of carbapenemase in clinical carbapenemase-producing Enterobacteriaceae (CPE) isolates. Although laboratory testing thus far suggested that Japan may be virtually free of KPC-producing Enterobacteriaceae, we have detected it from effluent from a WWTP. Antimicrobial resistance (AMR) monitoring of WWTP effluent may contribute to the early detection of future AMR bacterial dissemination in clinical settings and communities; indeed, it will help illuminate the whole picture in which environmental contamination through WWTP effluent plays a part. Copyright © 2018 Sekizuka et al.

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September 22, 2019  |  

Characterisation of pathogen-specific regions and novel effector candidates in Fusarium oxysporum f. sp. cepae.

A reference-quality assembly of Fusarium oxysporum f. sp. cepae (Foc), the causative agent of onion basal rot has been generated along with genomes of additional pathogenic and non-pathogenic isolates of onion. Phylogenetic analysis confirmed a single origin of the Foc pathogenic lineage. Genome alignments with other F. oxysporum ff. spp. and non pathogens revealed high levels of syntenic conservation of core chromosomes but little synteny between lineage specific (LS) chromosomes. Four LS contigs in Foc totaling 3.9?Mb were designated as pathogen-specific (PS). A two-fold increase in segmental duplication events was observed between LS regions of the genome compared to within core regions or from LS regions to the core. RNA-seq expression studies identified candidate effectors expressed in planta, consisting of both known effector homologs and novel candidates. FTF1 and a subset of other transcription factors implicated in regulation of effector expression were found to be expressed in planta.

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September 22, 2019  |  

Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt.

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

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September 22, 2019  |  

Complete genome sequence and characterization of a protein-glutaminase producing strain, Chryseobacterium proteolyticum QSH1265.

Recently, an enzyme named protein-glutaminase (PG) has been identified as a new type of enzyme with significant potential for deamidation of food proteins. The enzyme is shown to be expressed as a pre-pro-protein with a putative signal peptide of 21 amino acids, a pro-sequence of 114 amino acids, and a mature PG of 185 amino acids. The microbial enzyme PG specifically catalyzes deamidation of proteins without protein hydrolysis pretreatment and only reacts with glutamine residues in the side-chains of proteins or long peptides. All these attributes suggest that it has a great potential for food industrial applications. However, until recently, there have been relatively few studies of the PG-producing strains. A strain named Chryseobacterium proteolyticum QSH1265 which can produce PG was isolated from a soil sample collected in Songjiang, Shanghai, China. Its enzyme activity was about 0.34 ± 0.01 U/mL when using carboxybenzoxy-Gln-Gly as a substrate. The strain can produce acid from D-glucose, maltose, L-arabinose sucrose, glycerol, and mannitol but not fructose, and it is also positive for indole production and urease. Here we describe the complete genome sequence of this strain via PacBio RSII sequencing. The C. proteolyticum QSH1265 genome consists of a circular chromosome with total length of 4,849,803 bp without any plasmids. All of 4563 genes were predicted including 4459 genes for protein-coding and 104 RNA-relative genes with an average G+C content of 36.16%. The KEGG and COG annotation provide information for the specific function of proteins encoded in the genome, such as proteases, chromoproteins, stress proteins, antiporters, etc. A highly conserved hypothetical protein shares a promoter with the gene encoding the protein-glutaminase enzyme. The genome sequence and preliminary annotation provide valuable genetic information for further study of C. proteolyticum.

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September 22, 2019  |  

Screening and whole-genome sequencing of two Streptomyces species from the rhizosphere soil of peony reveal their characteristics as plant growth-promoting Rhizobacteria.

Two bacteria, Streptomyces albireticuli MDJK11 and S. alboflavus MDJK44, which are potential plant growth-promoting rhizobacteria against pathogenic fungi were isolated from the rhizosphere soil of peony in Shandong, China. Their biological characteristics and complete genome sequences were reported in this study. The total genome size of MDJK11 was only 8.14?Mb with 6,550 protein-coding genes and a high GC content of 72.8?mol%. The MDJK44 genome comprises a 9.62 Mb chromosome with 72.1?mol% GC content, 7,285 protein-coding genes, and two plasmids. Some gene sequences in these two genomes were analyzed to be heterologously obtained by horizontal transfer. Gene or gene cluster candidates responding to secondary metabolites production, antimicrobial activities, and plant growth-promoting capacities were also analyzed in this paper. The genomic information and biological characteristics will facilitate the understanding and application of S. albireticuli and S. alboflavus species as biocontrol agents in future agriculture.

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September 22, 2019  |  

Complete genome sequencing and analysis of endophytic Sphingomonas sp. LK11 and its potential in plant growth.

Our study aimed to elucidate the plant growth-promoting characteristics and the structure and composition of Sphingomonas sp. LK11 genome using the single molecule real-time (SMRT) sequencing technology of Pacific Biosciences. The results revealed that LK11 produces different types of gibberellins (GAs) in pure culture and significantly improves soybean plant growth by influencing endogenous GAs compared with non-inoculated control plants. Detailed genomic analyses revealed that the Sphingomonas sp. LK11 genome consists of a circular chromosome (3.78 Mbp; 66.2% G+C content) and two circular plasmids (122,975 bps and 34,160 bps; 63 and 65% G+C content, respectively). Annotation showed that the LK11 genome consists of 3656 protein-coding genes, 59 tRNAs, and 4 complete rRNA operons. Functional analyses predicted that LK11 encodes genes for phosphate solubilization and nitrate/nitrite ammonification, which are beneficial for promoting plant growth. Genes for production of catalases, superoxide dismutase, and peroxidases that confer resistance to oxidative stress in plants were also identified in LK11. Moreover, genes for trehalose and glycine betaine biosynthesis were also found in LK11 genome. Similarly, Sphingomonas spp. analysis revealed an open pan-genome and a total of 8507 genes were identified in the Sphingomonas spp. pan-genome and about 1356 orthologous genes were found to comprise the core genome. However, the number of genomes analyzed was not enough to describe complete gene sets. Our findings indicated that the genetic makeup of Sphingomonas sp. LK11 can be utilized as an eco-friendly bioresource for cleaning contaminated sites and promoting growth of plants confronted with environmental perturbations.

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September 22, 2019  |  

Comparison of the mitochondrial genome sequences of six Annulohypoxylon stygium isolates suggests short fragment insertions as a potential factor leading to larger genomic size.

Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium.

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September 22, 2019  |  

Ma orthologous genes in Prunus spp. shed light on a noteworthy NBS-LRR cluster conferring differential resistance to root-knot nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

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September 22, 2019  |  

Comparative genomics of degradative Novosphingobium strains with special reference to the microcystin-degrading Novosphingobium sp. THN1

Bacteria in genus Novosphingobium associated with biodegradation of substrates are prevalent in environments such as lakes, soil, sea, wood and sediments. To better understand the characteristics linked to their wide distribution and metabolic versatility, we report the whole genome sequence of Novosphingobium sp. THN1, a microcystin-degrading strain previously isolated by Jiang et al. (2011) from cyanobacteria-blooming water samples from Lake Taihu, China. We performed a genomic comparison analysis of Novosphingobium sp. THN1 with 21 other degradative Novosphingobium strains downloaded from GenBank. Phylogenetic trees were constructed using 16S rRNA genes, core genes, protein-coding sequences, and average nucleotide identity of whole genomes. Orthologous protein analysis showed that the 22 genomes contained 674 core genes and each strain contained a high proportion of distributed genes that are shared by a subset of strains. Inspection of their genomic plasticity revealed a high number of insertion sequence elements and genomic islands that were distributed on both chromosomes and plasmids. We also compared the predicted functional profiles of the Novosphingobium protein-coding genes. The flexible genes and all protein-coding genes produced the same heatmap clusters. The COG annotations were used to generate a dendrogram correlated with the compounds degraded. Furthermore, the metabolic profiles predicted from KEGG pathways showed that the majority of genes involved in central carbon metabolism, nitrogen, phosphate, sulfate metabolism, energy metabolism and cell mobility (above 62.5%) are located on chromosomes. Whereas, a great many of genes involved in degradation pathways (21–50%) are located on plasmids. The abundance and distribution of aromatics-degradative mono- and dioxygenases varied among 22 Novosphingoibum strains. Comparative analysis of the microcystin-degrading mlr gene cluster provided evidence for horizontal acquisition of this cluster. The Novosphingobium sp. THN1 genome sequence contained all the functional genes crucial for microcystin degradation and the mlr gene cluster shared high sequence similarity (=85%) with the sequences of other microcystin-degrading genera isolated from cyanobacteria-blooming water. Our results indicate that Novosphingobium species have high genomic and functional plasticity, rearranging their genomes according to environment variations and shaping their metabolic profiles by the substrates they are exposed to, to better adapt to their environments.

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September 22, 2019  |  

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019  |  

Draft genome sequence and transcriptional analysis of Rosellinia necatrix infected with a virulent mycovirus.

Understanding the molecular mechanisms of pathogenesis is useful in developing effective control methods for fungal diseases. The white root rot fungus Rosellinia necatrix is a soilborne pathogen that causes serious economic losses in various crops, including fruit trees, worldwide. Here, using next-generation sequencing techniques, we first produced a 44-Mb draft genome sequence of R. necatrix strain W97, an isolate from Japan, in which 12,444 protein-coding genes were predicted. To survey differentially expressed genes (DEGs) associated with the pathogenesis of the fungus, the hypovirulent W97 strain infected with Rosellinia necatrix megabirnavirus 1 (RnMBV1) was used for a comprehensive transcriptome analysis. In total, 545 and 615 genes are up- and down-regulated, respectively, in R. necatrix infected with RnMBV1. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the DEGs suggested that primary and secondary metabolism would be greatly disturbed in R. necatrix infected with RnMBV1. The genes encoding transcriptional regulators, plant cell wall-degrading enzymes, and toxin production, such as cytochalasin E, were also found in the DEGs. The genetic resources provided in this study will accelerate the discovery of genes associated with pathogenesis and other biological characteristics of R. necatrix, thus contributing to disease control.

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September 22, 2019  |  

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

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September 22, 2019  |  

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

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September 22, 2019  |  

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores. A dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein is at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicate that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.© 2018 John Wiley & Sons Ltd.

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