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September 22, 2019

The pathogenic mechanisms of Tilletia horrida as revealed by comparative and functional genomics.

Tilletia horrida is a soil-borne, mononucleate basidiomycete fungus with a biotrophic lifestyle that causes rice kernel smut, a disease that is distributed throughout hybrid rice growing areas worldwide. Here we report on the high-quality genome sequence of T. horrida; it is composed of 23.2?Mb that encode 7,729 predicted genes and 6,973 genes supported by RNA-seq. The genome contains few repetitive elements that account for 8.45% of the total. Evolutionarily, T. horrida lies close to the Ustilago fungi, suggesting grass species as potential hosts, but co-linearity was not observed between T. horrida and the barley smut Ustilago hordei. Genes and functions relevant to pathogenicity were presumed. T. horrida possesses a smaller set of carbohydrate-active enzymes and secondary metabolites, which probably reflect the specific characteristics of its infection and biotrophic lifestyle. Genes that encode secreted proteins and enzymes of secondary metabolism, and genes that are represented in the pathogen-host interaction gene database genes, are highly expressed during early infection; this is consistent with their potential roles in pathogenicity. Furthermore, among the 131 candidate pathogen effectors identified according to their expression patterns and functionality, we validated two that trigger leaf cell death in Nicotiana benthamiana. In summary, we have revealed new molecular mechanisms involved in the evolution, biotrophy, and pathogenesis of T. horrida.

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September 22, 2019

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

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September 22, 2019

Coculture of marine Streptomyces sp. with Bacillus sp. produces a newpiperazic acid-bearing cyclic peptide.

Microbial culture conditions in the laboratory, which conventionally involve the cultivation of one strain in one culture vessel, are vastly different from natural microbial environments. Even though perfectly mimicking natural microbial interactions is virtually impossible, the cocultivation of multiple microbial strains is a reasonable strategy to induce the production of secondary metabolites, which enables the discovery of new bioactive natural products. Our coculture of marine Streptomyces and Bacillus strains isolated together from an intertidal mudflat led to discover a new metabolite, dentigerumycin E (1). Dentigerumycin E was determined to be a new cyclic hexapeptide incorporating three piperazic acids, N-OH-Thr, N-OH-Gly, ß-OH-Leu, and a pyran-bearing polyketide acyl chain mainly by analysis of its NMR and MS spectroscopic data. The putative PKS-NRPS biosynthetic gene cluster for dentigerumycin E was found in the Streptomyces strain, providing clear evidence that this cyclic peptide is produced by the Streptomyces strain. The absolute configuration of dentigerumycin E was established based on the advanced Marfey’s method, ROESY NMR correlations, and analysis of the amino acid sequence of the ketoreductase domain in the biosynthetic gene cluster. In biological evaluation of dentigerumycin E (1) and its chemical derivatives [2-N,16-N-deoxydenteigerumycin E (2) and dentigerumycin methyl ester (3)], only dentigerumycin E exhibited antiproliferative and antimetastatic activities against human cancer cells, indicating that N-OH and carboxylic acid functional groups are essential for the biological activity.

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September 22, 2019

The landscape of repetitive elements in the refined genome of chilli anthracnose fungus Colletotrichum truncatum.

The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and “two-speed” genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

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September 22, 2019

Comparative genomic analysis of Pseudomonas amygdali pv. lachrymans NM002: Insights into its potential virulence genes and putative invasion determinants.

Pseudomonas amygdali pv. lachrymans is currently of important plant pathogenic bacteria that causes cucumber angular leaf spot worldwide. The pathogen has been studied for its roles in pathogenicity and plant inheritance resistance. To further delineate traits critical to virulence, invasion and survival in the phyllosphere, we reported the first complete genome of P. amygdali pv. lachrymans NM002. Analysis of the whole genome in comparison with three closely-related representative pathovars of P. syringae identified the conservation of virulence genes, including flagella and chemotaxis, quorum-sensing systems, two-component systems, and lipopolysaccharide and antiphagocytosis. It also revealed differences of invasion determinants, such as type III effectors, phytotoxin (coronatine, syringomycin and phaseolotoxin) and cell wall-degrading enzyme, which may contribute to infectivity. The aim of this study was to derive genomic information that would reveal the probable molecular mechanisms underlying the virulence, infectivity and provide a better understanding of the pathogenesis of the P. syringae pathovars. Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

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September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.

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September 22, 2019

A complete Cannabis chromosome assembly and adaptive admixture for elevated cannabidiol (CBD) content

Cannabis has been cultivated for millennia with distinct cultivars providing either fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol/cannabidiol segregating population and a complete chromosome assembly from a high-cannabidiol cultivar together resolve the linkage of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated with transposable elements. High-cannabidiol cultivars appear to have been generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests that overall drug potency, however, is associated with other genomic regions needing additional study.

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September 22, 2019

Phenazines in plant-beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics.

Plant-beneficial phenazine-producing Pseudomonas spp. are proficient biocontrol agents of soil-dwelling plant pathogens. Phenazines are redox-active molecules that display broad-spectrum antibiotic activity toward many fungal, bacterial and oomycete plant pathogens. Phenazine compounds also play a role in the persistence and survival of Pseudomonas spp. in the rhizosphere. This mini-review focuses on plant-beneficial phenazine-producing Pseudomonas spp. from the P. fluorescens species complex, which includes numerous well-known phenazine-producing strains of biocontrol interest. In this review the current knowledge on phenazine biosynthesis and regulation, the role played by phenazines in biocontrol and rhizosphere colonization, as well as exciting new advances in the genomics of plant-beneficial phenazine-producing Pseudomonas spp. will be discussed.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Genome mining of Streptomyces xinghaiensis NRRL B-24674T for the discovery of the gene cluster involved in anticomplement activities and detection of novel xiamycin analogs.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.

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September 22, 2019

Genome sequence of the potato pathogenic fungus Alternaria solani HWC-168 reveals clues for its conidiation and virulence.

Alternaria solani is a known air-born deuteromycete fungus with a polycyclic life cycle and is the causal agent of early blight that causes significant yield losses of potato worldwide. However, the molecular mechanisms underlying the conidiation and pathogenicity remain largely unknown.We produced a high-quality genome assembly of A. solani HWC-168 that was isolated from a major potato-producing region of Northern China, which facilitated a comprehensive gene annotation, the accurate prediction of genes encoding secreted proteins and identification of conidiation-related genes. The assembled genome of A. solani HWC-168 has a genome size 32.8 Mb and encodes 10,358 predicted genes that are highly similar with related Alternaria species including Alternaria arborescens and Alternaria brassicicola. We identified conidiation-related genes in the genome of A. solani HWC-168 by searching for sporulation-related homologues identified from Aspergillus nidulans. A total of 975 secreted protein-encoding genes, which might act as virulence factors, were identified in the genome of A. solani HWC-168. The predicted secretome of A. solani HWC-168 possesses 261 carbohydrate-active enzymes (CAZy), 119 proteins containing RxLx[EDQ] motif and 27 secreted proteins unique to A. solani.Our findings will facilitate the identification of conidiation- and virulence-related genes in the genome of A. solani. This will permit new insights into understanding the molecular mechanisms underlying the A. solani-potato pathosystem and will add value to the global fungal genome database.

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September 22, 2019

Out in the cold: Identification of genomic regions associated with cold tolerance in the biocontrol fungus Clonostachys rosea through genome-wide association mapping.

There is an increasing importance for using biocontrol agents in combating plant diseases sustainably and in the long term. As large scale genomic sequencing becomes economically viable, the impact of single nucleotide polymorphisms (SNPs) on biocontrol-associated phenotypes can be easily studied across entire genomes of fungal populations. Here, we improved a previously reported genome assembly of the biocontrol fungus Clonostachys rosea strain IK726 using the PacBio sequencing platform, which resulted in a total genome size of 70.7 Mbp and 21,246 predicted genes. We further performed whole-genome re-sequencing of 52 additional C. rosea strains isolated globally using Illumina sequencing technology, in order to perform genome-wide association studies in conditions relevant for biocontrol activity. One such condition is the ability to grow at lower temperatures commonly encountered in cryic or frigid soils in temperate regions, as these will be prevalent for protecting growing crops in temperate climates. Growth rates at 10°C on potato dextrose agar of the 53 sequenced strains of C. rosea were measured and ranged between 0.066 and 0.413 mm/day. Performing a genome wide association study, a total of 1,478 SNP markers were significantly associated with the trait and located in 227 scaffolds, within or close to (< 1000 bp distance) 265 different genes. The predicted gene products included several chaperone proteins, membrane transporters, lipases, and proteins involved in chitin metabolism with possible roles in cold tolerance. The data reported in this study provides a foundation for future investigations into the genetic basis for cold tolerance in fungi, with important implications for biocontrol.

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September 22, 2019

Impacts of horizontal gene transfer on the compact genome of the clavulanic acid-producing Streptomyces strain F613-1.

Mobile genetic elements involved in mediating horizontal transfer events contribute to bacterial evolution, and bacterial genomic plasticity and instability result in variation in functional genetic information in Streptomyces secondary metabolism. In a previous study, we reported the complete genome sequence of the industrial Streptomyces strain F613-1, which produces high yields of clavulanic acid. In this study, we used comparative genomics and bioinformatics to investigate the unique genomic features of this strain. Taken together, comparative genomics were used to systematically investigate secondary metabolism capabilities and indicated that frequent exchange of genetic materials between Streptomyces replicons may shape the remarkable diversities in their secondary metabolite repertoires. Moreover, a 136.9-kb giant region of plasticity (RGP) was found in the F613-1 chromosome, and the chromosome and plasmid pSCL4 are densely packed with an exceptionally large variety of potential secondary metabolic gene clusters, involving several determinants putatively accounting for antibiotic production. In addition, the differences in the architecture and size of plasmid pSCL4 between F613-1 and ATCC 27064 suggest that the pSCL4 plasmid could evolve from pSCL4-like and pSCL2-like extrachromosomal replicons. Furthermore, the genomic analyses revealed that strain F613-1 has developed specific genomic architectures and genetic patterns that are well suited to meet the requirements of industrial innovation processes.

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September 22, 2019

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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