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September 22, 2019

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown.In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes.Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.


September 22, 2019

Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis isolates.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glässer’s disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.


September 22, 2019

Characterization of Streptococcus pluranimalium from a cattle with mastitis by whole genome sequencing and functional validation.

Streptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism.The genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G?+?C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a ?11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit – CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).We reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model.


September 22, 2019

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.


September 22, 2019

Unraveling microbial communities associated with methylmercury production in paddy soils.

Rice consumption is now recognized as an important pathway of human exposure to the neurotoxin methylmercury (MeHg), particularly in countries where rice is a staple food. Although the discovery of a two-gene cluster hgcAB has linked Hg methylation to several phylogenetically diverse groups of anaerobic microorganisms converting inorganic mercury (Hg) to MeHg, the prevalence and diversity of Hg methylators in microbial communities of rice paddy soils remain unclear. We characterized the abundance and distribution of hgcAB genes using third-generation PacBio long-read sequencing and Illumina short-read metagenomic sequencing, in combination with quantitative PCR analyses in several mine-impacted paddy soils from southwest China. Both Illumina and PacBio sequencing analyses revealed that Hg methylating communities were dominated by iron-reducing bacteria (i.e., Geobacter) and methanogens, with a relatively low abundance of hgcA + sulfate-reducing bacteria in the soil. A positive correlation was observed between the MeHg content in soil and the relative abundance of Geobacter carrying the hgcA gene. Phylogenetic analysis also uncovered some hgcAB sequences closely related to three novel Hg methylators, Geobacter anodireducens, Desulfuromonas sp. DDH964, and Desulfovibrio sp. J2, among which G. anodireducens was validated for its ability to methylate Hg. These findings shed new light on microbial community composition and major clades likely driving Hg methylation in rice paddy soils.


September 22, 2019

A strain of an emerging Indian Xanthomonas oryzae pv. oryzae pathotype defeats the rice bacterial blight resistance gene xa13 without inducing a clade III SWEET gene and is nearly identical to a recent Thai isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host “susceptibility” (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.


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.


September 22, 2019

Antibiotic-resistant indicator bacteria in irrigation water: High prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

Irrigation water is a major source of fresh produce contamination with undesired microorganisms including antibiotic-resistant bacteria (ARB), and contaminated fresh produce can transfer ARB to the consumer especially when consumed raw. Nevertheless, no legal guidelines exist so far regulating quality of irrigation water with respect to ARB. We therefore examined irrigation water from major vegetable growing areas for occurrence of antibiotic-resistant indicator bacteria Escherichia coli and Enterococcus spp., including extended-spectrum ß-lactamase (ESBL)-producing E. coli and vancomycin-resistant Enterococcus spp. Occurrence of ARB strains was compared to total numbers of the respective species. We categorized water samples according to total numbers and found that categories with higher total E. coli or Enterococcus spp. numbers generally had an increased proportion of respective ARB-positive samples. We further detected high prevalence of ESBL-producing E. coli with eight positive samples of thirty-six (22%), while two presumptive vancomycin-resistant Enterococcus spp. were vancomycin-susceptible in confirmatory tests. In disk diffusion assays all ESBL-producing E. coli were multidrug-resistant (n = 21) and whole-genome sequencing of selected strains revealed a multitude of transmissible resistance genes (ARG), with blaCTX-M-1 (4 of 11) and blaCTX-M-15 (3 of 11) as the most frequent ESBL genes. Overall, the increased occurrence of indicator ARB with increased total indicator bacteria suggests that the latter might be a suitable estimate for presence of respective ARB strains. Finally, the high prevalence of ESBL-producing E. coli with transmissible ARG emphasizes the need to establish legal critical values and monitoring guidelines for ARB in irrigation water.


September 22, 2019

An improved genome assembly for Larimichthys crocea reveals hepcidin gene expansion with diversified regulation and function.

Larimichthys crocea (large yellow croaker) is a type of perciform fish well known for its peculiar physiological properties and economic value. Here, we constructed an improved version of the L. crocea genome assembly, which contained 26,100 protein-coding genes. Twenty-four pseudo-chromosomes of L. crocea were also reconstructed, comprising 90% of the genome assembly. This improved assembly revealed several expansions in gene families associated with olfactory detection, detoxification, and innate immunity. Specifically, six hepcidin genes (LcHamps) were identified in L. crocea, possibly resulting from lineage-specific gene duplication. All LcHamps possessed similar genomic structures and functional domains, but varied substantially with respect to expression pattern, transcriptional regulation, and biological function. LcHamp1 was associated specifically with iron metabolism, while LcHamp2s were functionally diverse, involving in antibacterial activity, antiviral activity, and regulation of intracellular iron metabolism. This functional diversity among gene copies may have allowed L. crocea to adapt to diverse environmental conditions.


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.


September 22, 2019

N6-methyladenine DNA modification in Xanthomonas oryzae pv. oryzicola genome.

DNA N6-methyladenine (6mA) modifications expand the information capacity of DNA and have long been known to exist in bacterial genomes. Xanthomonas oryzae pv. Oryzicola (Xoc) is the causative agent of bacterial leaf streak, an emerging and destructive disease in rice worldwide. However, the genome-wide distribution patterns and potential functions of 6mA in Xoc are largely unknown. In this study, we analyzed the levels and global distribution patterns of 6mA modification in genomic DNA of seven Xoc strains (BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8 and RS105). The 6mA modification was found to be widely distributed across the seven Xoc genomes, accounting for percent of 3.80, 3.10, 3.70, 4.20, 3.40, 2.10, and 3.10 of the total adenines in BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8, and RS105, respectively. Notably, more than 82% of 6mA sites were located within gene bodies in all seven strains. Two specific motifs for 6?mA modification, ARGT and AVCG, were prevalent in all seven strains. Comparison of putative DNA methylation motifs from the seven strains reveals that Xoc have a specific DNA methylation system. Furthermore, the 6?mA modification of rpfC dramatically decreased during Xoc infection indicates the important role for Xoc adaption to environment.


September 22, 2019

Complete genome sequence of Bacillus velezensis ZY-1-1 reveals the genetic basis for its hemicellulosic/cellulosic substrate-inducible xylanase and cellulase activities.

Bacillus velezensis ZY-1-1 was isolated from the larval gut of the lignocellulose-rich diet-fed scarab beetle, Holotrichia parallela, and confirmed to possess extremely high xylanase (48153.8?±?412.1 U/L) and relatively moderate cellulase activity (610.1?±?8.2 U/L). Notably, these xylanase and cellulase activities were enhanced by xylan (1.4 and 5.8-fold, respectively) and cellulose (1.1 and 3.5-fold, respectively), which indicated the hemicellulosic/cellulosic substrate-inducible lignocellulolytic activities of this strain. The complete genome of B. velezensis ZY-1-1 comprises of 3,899,251 bp in a circular chromosome with a G?+?C content of 46.6%. Among the predicted 3688 protein-coding genes, 24 genes are involved in the degradation of lignocellulose and other polysaccharides, including 8, 7 and 2 critical genes for the degradation of xylan, cellulose and lignin, respectively. This genome-based analysis will facilitate our understanding of the mechanism underlying the biodegradation of lignocellulose and the biotechnological application of this novel lignocellulolytic bacteria or related enzymes.


September 22, 2019

Complete genome sequence of Burkholderia sp. JP2-270, a rhizosphere isolate of rice with antifungal activity against Rhizoctonia solani.

Burkholderia sp. JP2-270, a bacterium with a strong ability to inhibit the growth of Rhizoctonia solani, was isolated from the rhizosphere of rice. The phylogenetic analysis based on 16S rRNA gene revealed that JP2-270 belonged to Burkholderia cepacia complex. Here, we present the complete genome sequence of Burkholderia sp. JP2-270, which consists of three circular chromosomes (Chr1 3,723,585 bp, Chr2 3,274,969 bp, Chr3 1,483,367 bp) and two plasmids (Plas1 15,126 bp, Plas2 428,263 bp). A total of 8193 protein coding genes were predicted in the genome, including 67 tRNA genes, 18 rRNA genes and 4 ncRNA genes. In addition, mutation analysis of Burkholderia sp. JP2-270 revealed that the gene bysR (DM992_17470), encoding a lysR-type transcriptional regulator, was essential for the antagonistic activity of Burkholderia sp. JP2-270 against R. solani GD118 in vitro and in vivo. Identification of regulatory gene associated with antagonistic activity will contribute to understand the antagonistic mechanism of Burkholderia sp. JP2-270. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019

Complete genome sequence of the cyprodinil-degrading bacterium Acinetobacter johnsonii LXL_C1.

Acinetobacter johnsonii LXL_C1, a cyprodinil degrader, was isolated and purified from cyprodinil-contaminated agricultural soil. Here, we report the complete genome sequence of LXL_C1. The genome comprises one 3,398,706 bp circular chromosome with 41.2% G + C content and one 44,866 bp plasmid. Annotation based on COG and KEGG database analyses revealed genes encoding a cytochrome P450 monooxygenase and hydrolase, which can effectively degrade cyprodinil. The complete genome sequence of LXL_C1 can facilitate genetic engineering of a recombinant cyprodinil degrader. Copyright © 2018 Elsevier Ltd. All rights reserved.


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