Single-molecule Real-time Sequencing Research Articles

The genome of the rice planthopper egg parasitoid wasps Anagrus nilaparvatae casts light on the chemo- and mechanosensation in parasitism

BackgroundMymaridae is an ancient insect group and is a basal lineage of the superfamily Chalcidoidea. Species of Mymaridae have great potential for biological control. Anagrus nilaparvatae, a representative species of Mymaridae, is ideal for controlling rice planthopper due to its high rate of parasitism and ability to find hosts efficiently in paddy ridges and fields.ResultsUsing both PacBio single-molecule real-time and Illumina sequencing, we sequenced and assembled the whole genome of A. nilaparvatae, a first for the family Mymaridae. The assembly consists of 394 scaffolds, totaling 488.8 Mb. The assembly is of high continuity and completeness, indicated by the N50 value of 25.4 Mb and 98.2% mapping rate of Benchmarking Universal Single-Copy Orthologs. In total, 16,894 protein-coding genes in the genome were annotated. A phylogenomic tree constructed for A. nilaparvatae and other 12 species of Hymenoptera confirmed that the family Mymaridae is sister to all remaining chalcidoids. The divergence time between A. nilaparvatae and the other seven Chalcidoidea species was dated at ~ 126.9 Mya. Chemoreceptor and mechanoreceptor genes are important in explaining parasitic behavior. We identified 17 odorant binding proteins, 11 chemosensory proteins, four Niemann-Pick type C2 proteins, 88 olfactory receptors, 12 gustatory receptors, 22 ionotropic receptors and 13 sensory neuron membrane proteins in the genome of A. nilaparvatae, which are associated with the chemosensory functions. Strikingly, there is only one pickpocket receptors and nine transient receptor potential genes in the genome that have a mechanosensory function.ConclusionsWe obtained a high-quality genome assembly for A. nilaparvatae using PacBio single-molecule real-time sequencing, which provides phylogenomic insights for its evolutionary history. The small numbers of chemo- and mechanosensory genes in A. nilaparvatae indicate the species-specific host detection and oviposition behavior of A. nilaparvatae might be regulated by relatively simple molecular pathways.

Full-length transcriptome sequencing analysis and characterization, development and validation of microsatellite markers in Kengyilia melanthera.

As a typical psammophyte of the Triticeae, Kengyilia melanthera possesses high feeding potential and great utilization values in desertification control in the Qinghai-Tibet Plateau. However, few gene function and genetic studies have been performed in K. melanthera. In this study, single-molecule real-time sequencing technology was used to obtain the full-length transcriptome sequence of K. melanthera, following the functional annotation of transcripts and prediction of coding sequences (CDSs), transcription factors (TFs), and long noncoding RNA (lncRNA) sequences. Meanwhile, a total of 42,433 SSR loci were detected, with 5′-UTRs having the most SSR loci and trinucleotide being the most abundant type. In total, 108,399 SSR markers were designed, and 300 SSR markers were randomly selected for diversity verification of K. melanthera. A total of 49 polymorphic SSR markers were used to construct the genetic relationships of 56 K. melanthera accessions, among which 21 SSR markers showed good cross-species transferability among the related species. In conclusion, the full-length transcriptome sequence of the K. melanthera will assist gene prediction and promote molecular biology and genomics research, and the polymorphic SSR markers will promote molecular-assisted breeding and related research of K. melanthera and its relatives.

Adaptation to a new environment with pre-adaptive genomic features - Evidence from woody plants colonizing the land-sea interface.

Adaptation to new environments is a key evolutionary process which presumably involves complex genomic changes. Mangroves, a collection of approximately 80 woody plants that have independently invaded intertidal zones >20 times, are ideal for studying this process. We assembled near-chromosome-scale genomes of three Xylocarpus species as well as an outgroup species using single-molecule real-time sequencing. Phylogenomic analysis reveals two separate lineages, one with the mangrove Xylocarpus granatum and the other comprising a mangrove Xylocarpus moluccensis and a terrestrial Xylocarpus rumphii. In conjunction with previous studies, we identified several genomic features associated with mangroves: (i) signals of positive selection in genes related to salt tolerance and root development; (ii) genome-wide elevated ratios of non-synonymous to synonymous substitution relative to terrestrial relatives; and (iii) active elimination of long terminal repeats. These features are found in the terrestrial X. rumphii in addition to the two mangroves. These genomic features, not being strictly mangrove-specific, are hence considered pre-adaptive. We infer that the coastal but non-intertidal habitat of X. rumphii may have predisposed the common ancestor to invasion of true mangrove habitats. Other features including the preferential retention of duplicated genes and intolerance to pseudogenization are not found in X. rumphii and are likely true adaptive features in mangroves. In conclusion, by studying adaptive shift and partial shifts among closely related species, we set up a framework to study genomic features that are acquired at different stages of the pre-adaptation and adaptation to new environments.

Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Integrated Analysis of Single-Molecule Real-Time Sequencing and Next-Generation Sequencing Eveals Insights into Drought Tolerance Mechanism of Lolium multiflorum.

Lolium multiflorum is widely planted in temperate and subtropical regions globally, and it has high economic value owing to its use as forage grass for a wide variety of livestock and poultry. However, drought seriously restricts its yield and quality. At present, owing to the lack of available genomic resources, many types of basic research cannot be conducted, which severely limits the in-depth functional analysis of genes in L. multiflorum. Therefore, we used single-molecule real-time (SMRT) and next-generation sequencing (NGS) to sequence the complex transcriptome of L. multiflorum under drought. We identified 41,141 DEGs in leaves, 35,559 DEGs in roots, respectively. Moreover, we identified 1243 alternative splicing events under drought. LmPIP5K9 produced two different transcripts with opposite expression patterns, possibly through the phospholipid signaling pathway or the negatively regulated sugar-mediated root growth response to drought stress, respectively. Additionally, 13,079 transcription factors in 90 families were obtained. An in-depth analysis of R2R3-MYB gene family members was performed to preliminarily demonstrate their functions by utilizing subcellular localization and overexpression in yeast. Our data make a significant contribution to the genetics of L. multiflorum, offering a current understanding of plant adaptation to drought stress.

Whole-genome long-read TAPS deciphers DNA methylation patterns at base resolution using PacBio SMRT sequencing technology.

Long-read sequencing provides valuable information on difficult-to-map genomic regions, which can complement short-read sequencing to improve genome assembly, yet limited methods are available to accurately detect DNA methylation over long distances at a whole-genome scale. By combining our recently developed TET-assisted pyridine borane sequencing (TAPS) method, which enables direct detection of 5-methylcytosine and 5-hydroxymethylcytosine, with PacBio single-molecule real-time sequencing, we present here whole-genome long-read TAPS (wglrTAPS). To evaluate the performance of wglrTAPS, we applied it to mouse embryonic stem cells as a proof of concept, and an N50 read length of 3.5 kb is achieved. By sequencing wglrTAPS to 8.2× depth, we discovered a significant proportion of CpG sites that were not covered in previous 27.5× short-read TAPS. Our results demonstrate that wglrTAPS facilitates methylation profiling on problematic genomic regions with repetitive elements or structural variations, and also in an allelic manner, all of which are extremely difficult for short-read sequencing methods to resolve. This method therefore enhances applications of third-generation sequencing technologies for DNA epigenetics.

Integration analysis of PacBio SMRT- and Illumina RNA-seq reveals P450 genes involved in thiamethoxam detoxification in Bradysia odoriphaga

The sciarid fly Bradysia odoriphaga is a serious pest of Chinese chive (Liliaceae). Neonicotinoid insecticides including thiamethoxam have been used for B. odoriphaga control. However, thiamethoxam resistance in B. odoriphaga has developed in recent years. To identify potential genes involved in detoxification metabolism of thiamethoxam in B. odoriphaga, a PacBio single-molecule real-time (SMRT) transcriptome sequencing and Illumina RNA-seq analysis on thiamethoxam treated B. odoriphaga were performed to explore differentially expressed genes in B. odoriphaga. After SMRT sequencing, analysis of Illumina RNA-Seq data showed a total of 172 differentially expressed genes (DEGs) after thiamethoxam treatment, among which eight upregulated DEGs were P450 genes that may be related to thiamethoxam metabolism. The qRT-PCR results of the eight up-regulated P450 unigenes after thiamethoxam treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of the eight upregulated P450 transcripts followed by insecticide bioassay was conducted, and three P450 unigenes were verified to be related to thiamethoxam detoxification in B. odoriphaga. This study provides new information about the P450 genes involved in thiamethoxam detoxification in B. odoriphaga.

Genome Sequence Resource of Bacillus velezensis Strain HC-8, a Native Bacterial Endophyte with Biocontrol Potential Against the Honeysuckle Powdery Mildew Causative Pathogen Erysiphe lonicerae var. lonicerae.

Genome Sequence Resource of Bacillus velezensis Strain HC-8, a Native Bacterial Endophyte with Biocontrol Potential Against the Honeysuckle Powdery Mildew Causative Pathogen Erysiphe lonicerae var. lonicerae.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Long-read PacBio genome sequencing of four environmental saprophytic Sporothrix species spanning the pathogenic clade

BackgroundThe genus Sporothrix belongs to the order Ophiostomatales and contains mainly saprobic soil and plant fungi, although pathogenic species capable of causing human infections are also present. The whole-genomes of disease-causing species have already been sequenced and annotated but no comprehensive genomic resources for environmental Sporothrix species are available, thus limiting our understanding of the evolutionary origin of virulence-related genes and pathogenicity.ResultThe genome assembly of four environmental Sporothrix species resulted in genome size of ~ 30.9 Mbp in Sporothrix phasma, ~ 35 Mbp in S. curviconia, ~ 38.7 Mbp in S. protearum, and ~ 39 Mbp in S. variecibatus, with a variable gene content, ranging from 8142 (S. phasma) to 9502 (S. variecibatus). The analysis of mobile genetic elements showed significant differences in the content of transposable elements within the sequenced genomes, with the genome of S. phasma lacking several class I and class II transposons, compared to the other Sporothrix genomes investigated. Moreover, the comparative analysis of orthologous genes shared by clinical and environmental Sporothrix genomes revealed the presence of 3622 orthogroups shared by all species, whereas over 4200 genes were species-specific single-copy gene products. Carbohydrate-active enzyme analysis revealed a total of 2608 protein-coding genes containing single and/or multiple CAZy domains, resulting in no statistically significant differences among pathogenic and environmental species. Nevertheless, some families were not found in clinical species. Furthermore, for each sequenced Sporothrix species, the mitochondrial genomes was assembled in a single circular DNA molecule, ranging from 25,765 bp (S. variecibatus) to 58,395 bp (S. phasma).ConclusionIn this study, we present four annotated genome assemblies generated using PacBio SMRT sequencing data from four environmental species: S. curviconia, S. phasma, S. protearum and S. variecibatus with the aim to provide a starting point for future comparative genome evolution studies addressing species diversification, ecological/host adaptation and origin of pathogenic lineages within the genus Sporothrix.

Full-Length Transcriptome Reconstruction Reveals the Genetic Mechanisms of Eyestalk Displacement and Its Potential Implications on the Interspecific Hybrid Crab (Scylla serrata ♀ × S. paramamosain ♂).

Simple SummaryThe eyestalk is a key organ in crustaceans that produces neurohormones and regulates a range of physiological functions. Eyestalk displacement was discovered in some first-generation (F1) offspring of the novel interspecific hybrid crab (Scylla serrata ♀ × S. paramamosain ♂). To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, high-quality transcriptome was reconstructed using single-molecule real-time (SMRT) sequencing. A total of 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in hybrid crabs with displaced eyestalks (DH). The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant gene ontology (GO) terms were related to the cuticle or chitin. Overall, this study highlights the underlying genetic mechanisms of eyestalk displacement and provide useful knowledge for mud crab (Scylla spp.) crossbreeding.The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring’s eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding.

Distribution Pattern of N6-Methyladenine DNA Modification in the Seashore Paspalum (Paspalum vaginatum) Genome.

N6-methyladenine (6mA) DNA modification has been detected in several eukaryotic organisms, in some of them, it plays important role in the regulation process of stress-resistance response. However, the genome-wide distribution patterns and potential functions of 6mA DNA modification in halophyte Seashore paspalum (Paspalum vaginatum) remain largely unknown. Here, we examined the 6mA landscape in the P. vaginatum genome by adopting single molecule real-time sequencing technology and found that 6mA modification sites were broadly distributed across the P. vaginatum genome. We demonstrated distinct 6mA methylation levels and 6mA distribution patterns in different types of transcription genes, which hinted at different epigenetic rules. Furthermore, the moderate 6mA density genes in P. vaginatum functionally correlated with stress resistance, which also maintained a higher transcriptional level. On the other hand, a specific 6mA distribution pattern in the gene body and near TSS was observed in gene groups with higher RNA expression, which maybe implied some kind of regularity between 6mA site distribution and the protein coding genes transcription was possible. Our study provides new insights into the association between 6mA methylation and gene expression, which may also contribute to key agronomic traits in P. vaginatum.

A Novel 5 kb Deletion in the β-Globin Gene Cluster Identified in a Chinese Patient

β-Thalassemia (β-thal), a highly prevalent disease in tropical and subtropical regions of Southern China, is caused mainly by point mutations in the β-globin gene cluster. However, large deletions have also been found to contribute to some types of β-thal. We identified a novel 5 kb deletion in the β-globin cluster in a Chinese patient using multiplex ligation-dependent probe amplification (MLPA), and characterized it with single molecule real-time (SMRT) sequencing, gap-polymerase chain reaction (gap-PCR) and Sanger sequencing. The deletion was located between positions 5226189 and 5231091 on chromosome 11 (GRCh38), extending from 4 kb upstream of the 5' untranslated region (5'UTR) to the second intron of the β-globin gene. The patient with this deletion presented with microcytosis and hypochromic red cells, as well as relatively high Hb F and Hb A2 levels. Our research indicated that SMRT sequencing is a useful tool for accurate detection of large deletions. Our study broadens the spectrum of deletional β-thalassemias and provides a perspective for further study of the function of the β-globin cluster.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50 =9.7Mbp/32.4Mbp, L50 =13/11) and complete (BUSCO complete =99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

The complete and closed genome of the facultative generalist Candidatus Endoriftia persephone from deep-sea hydrothermal vents.

The mutualistic interactions between Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone (short Endoriftia) have been extensively researched. However, the closed Endoriftia genome is still lacking. Here, by employing single-molecule real-time sequencing we present the closed chromosomal sequence of Endoriftia. In contrast to theoretical predictions of enlarged and mobile genetic element-rich genomes related to facultative endosymbionts, the closed Endoriftia genome is streamlined with fewer than expected coding sequence regions, insertion-, prophage-sequences and transposase-coding sequences. Automated and manually curated functional analyses indicated that Endoriftia is more versatile regarding sulphur metabolism than previously reported. We identified the presence of two identical rRNA operons and two long CRISPR regions in the closed genome. Additionally, pangenome analyses revealed the presence of three types of secretion systems (II, IV and VI) in the different Endoriftia populations indicating lineage-specific adaptations. The in depth mobilome characterization identified the presence of shared genomic islands in the different Endoriftia drafts and in the closed genome, suggesting that the acquisition of foreign DNA predates the geographical dispersal of the different endosymbiont populations. Finally, we found no evidence of epigenetic regulation in Endoriftia, as revealed by gene screenings and absence of methylated modified base motifs in the genome. As a matter of fact, the restriction-modification system seems to be dysfunctional in Endoriftia, pointing to a higher importance of molecular memory-based immunity against phages via spacer incorporation into CRISPR system. The Endoriftia genome is the first closed tubeworm endosymbiont to date and will be valuable for future gene oriented and evolutionary comparative studies.

Long-read-based draft genome sequence of Indian black gram IPU-94-1 'Uttara': Insights into disease resistance and seed storage protein genes.

Black gram [Vigna mungo (L.) Hepper var. mungo] is a warm-season legume highly prized for its protein content along with significant folate and iron proportions. To expedite the genetic enhancement of black gram, a high-quality draft genome from the center of origin of the crop is indispensable. Here, we established a draft genome sequence of an Indian black gram cultivar, 'Uttara' (IPU 94-1), known for its high resistance to mungbean yellow mosaic virus. Pacific Biosciences of California, Inc. (PacBio) single-molecule real-time (SMRT) and Illumina sequencing assembled a draft reference-guided assembly with a cumulative size of ∼454.4 Mb, of which, 444.4 Mb was anchored on 11 pseudomolecules corresponding to 11 chromosomes. Uttara assembly denotes features of a high-quality draft genome illustrated through high N50 value (42.88 Mb), gene completeness (benchmarking universal single-copy ortholog [BUSCO] score 94.17%) and low levels of ambiguous nucleotides (N) percent (0.0005%). Gene discovery using transcript evidence predicted 28,881 protein-coding genes, from which, ∼95% were functionally annotated. A global survey of genes associated with disease resistance revealed 119 nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, while 23 genes encoding seed storage proteins (SSPs) were discovered in black gram. A large set of microsatellite loci were discovered for marker development in the crop. Our draft genome of an Indian black gram provides the foundational genomic resources for the improvement of important agronomic traits and ultimately will help in accelerating black gram breeding programs.

Transcriptome Analysis of Fusarium-Tomato Interaction Based on an Updated Genome Annotation of Fusarium oxysporum f. sp. lycopersici Identifies Novel Effector Candidates That Suppress or Induce Cell Death in Nicotiana benthamiana.

Fusarium oxysporum f. sp. lycopersici (Fol) causes vascular wilt disease in tomato. Upon colonization of the host, Fol secretes many small effector proteins into the xylem sap to facilitate infection. Besides known SIX (secreted in xylem) proteins, the identity of additional effectors that contribute to Fol pathogenicity remains largely unexplored. We performed a deep RNA-sequencing analysis of Fol race 2-infected tomato, used the sequence data to annotate a published genome assembly generated via PacBio SMRT sequencing of the Fol race 2 reference strain Fol4287, and analysed the resulting transcriptome to identify Fol effector candidates among the newly annotated genes. We examined the Fol-infection expression profiles of all 13 SIX genes present in Fol race 2 and identified 27 new candidate effector genes that were likewise significantly upregulated upon Fol infection. Using Agrobacterium-mediated transformation, we tested the ability of 22 of the new candidate effector genes to suppress or induce cell death in leaves of Nicotiana benthamiana. One effector candidate designated Fol-EC19, encoding a secreted guanyl-specific ribonuclease, was found to trigger cell death and two effector candidates designated Fol-EC14 and Fol-EC20, encoding a glucanase and a secreted trypsin, respectively, were identified that can suppress Bax-mediated cell death. Remarkably, Fol-EC14 and Fol-EC20 were also found to suppress I-2/Avr2- and I/Avr1-mediated cell death. Using the yeast secretion trap screening system, we showed that these three biologically-active effector candidates each contain a functional signal peptide for protein secretion. Our findings provide a basis for further understanding the virulence functions of Fol effectors.

Identification of putative acyltransferase genes responsible for the biosynthesis of homogenous and heterogenous hydroxycinnamoyl-tartaric acid esters from Bidens pilosa

Bidens pilosa is an edible plant with highly sought-after nutraceutical properties. The purported bioactivities of this plant can be correlated to the high number of metabolites. Amongst these metabolites, different derivatives of hydroxy‑cinnamoyl esters have been shown to exist in high proportions. However, the enzymatic machinery, thus the biosynthetic pathways responsible for the accumulation of these compounds in the plant have not yet been identified. For the first time, we report the putative identification of two genes with sequence homology to hydroxycinnamoyl-CoA: tartaric acid hydroxycinnamoyl transferase (HTT) in B. pilosa. The full-length sequence of the two isoforms of the HTT gene was achieved using single-molecule real-time (SMRT) sequencing approach. Analyses of methanolic extracts of B. pilosa through Liquid-chromatography hyphenated with mass spectrometry (LC-MS) technique revealed the existence of heterogeneous hydroxycinnamoyl-tartaric acid esters consisting of different hydroxycinnamoyl derivatives. To the best of our knowledge, this is a first report on these molecules from B. pilosa. Taken altogether, this plant utilises hydroxycinnamoyl-CoA tartaric hydroxycinnamoyl transferase (HTT) genes to diversify its metabolite composition through esterification of tartaric acid acceptor molecule by acylating it with either homogeneous or heterogenous hydroxycinnamic acids (HCA) derivatives. Therefore, B. pilosa is a source of structurally diverse isomeric compounds with purported nutraceutical values. The enzyme products of the two identified HTT genes are therefore pointed out as possible catalysts which can be further exploited by incorporating them in other economically viable plants to enhance the nutraceutical values thereof.

Using PacBio SMRT Sequencing Technology and Metabolomics to Explore the Microbiota-Metabolome Interaction Related to Silage Fermentation of Woody Plant

Silage fermentation is a dynamic process involving the succession of microbial communities and changes in metabolites. Fresh branched and leaves of paper mulberry were used to prepared silage. Crop by-products including corn bran, rice bran, and wheat bran were used as exogenous additives. Pacific Biosciences single-molecule real-time (SMRT) sequencing technology and metabolomics are used to explore the interaction mechanism of microbial structure and metabolites during woody silage fermentation and to verify the principle that exogenous additives can modulate silage fermentation. Under the dual stress of anaerobic and acidic environment of silage fermentation, the microbial community changed from Gram-negative to Gram-positive bacteria, and a large amount of lactic acid and volatile fatty acid were produced, which lowered the pH value and caused the rapid death of aerobic bacteria with thin cell walls. The exogenous additives of corn bran and wheat bran accelerated the dynamic succession of lactic acid bacteria as the dominant microbial community in silage fermentation, increased the metabolic pathways of lactic acid, unsaturated fatty acids, citric acid, L-malic acid and other flavoring agents, and inhibited the growth of Clostridium and Enterobacter, thereby improving the flavor and quality of the silage. However, because rice bran contained butyric acid spore bacteria, it can multiply in an anaerobic environment, led to butyric acid fermentation, and promoted protein degradation and ammonia nitrogen production, thereby reduced the fermentation quality of woody silage. The results showed that during the silage fermentation process, the microbial community and the metabolome can interact, and exogenous additives can affect the fermentation quality of silage. SMRT sequencing technology and untargeted metabolomics revealed the microbiota-metabolome interaction during silage fermentation. Changes in the structure of the microbial community can affect the metabolic pathways, and the final metabolites can inhibit the growth of microorganisms that are not conducive to silage fermentation. Exogenous carbohydrate additives can change the fermentation substrate and affect microbial community structure, thus modulate the silage fermentation.

Genomic Analysis of KPC-2-Producing Klebsiella pneumoniae ST11 Isolates at the Respiratory Department of a Tertiary Care Hospital in Beijing, China.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is an important pathogen causing hospital-associated outbreaks worldwide. The spread of K. pneumoniae carbapenemase-2 (KPC-2)-producing CRKP is primarily associated with sequence type (ST) 11.MethodsA total of 152 KPC-2-producing K. pneumoniae ST11 isolates were collected from the respiratory department of a tertiary care hospital in Beijing, China between 2009 and 2018. The genome sequencing of these isolates was performed on the HiSeq X Ten sequencer. Multilocus sequence typing (MLST), capsular type, plasmid replicon types and resistance genes were identified. Fifteen isolates were selected for the subsequent single-molecule real-time (SMRT) sequencing on the PacBio RS II. Alignment of the complete sequences of the plasmids carrying blaKPC–2 and/or virulence genes was performed by using BRIG and Easyfig.ResultsFrom 2012 to 2018, the detection rate of the blaKPC–2-carrying CRKP rose rapidly from 3.3 to 28.1%. KPC-2-producing K. pneumoniae ST11 isolates were dominant in CRKP, which emerged in 2012 and caused several outbreaks. Most isolates exhibited multidrug-resistant to commonly used antibiotics, while all the isolates remained susceptible to tigecycline and polymyxin B. The single nucleotide polymorphism (SNP) analysis showed that all these 152 KPC-2-producing K. pneumoniae ST11 isolates could be divided into three genetically distinct clades (A, B, and C) and eleven subclades (A1–A9 and B1–B2). The majority belonged to clade A with KL47 serotype (n = 117, 77.0%), while KL64 and KL16 were identified in clades B and C, respectively. The blaKPC–2-carrying plasmids exhibited diverse types, namely, IncFII (pHN7A8)/IncR(6/15), IncFII (pHN7A8)/IncpA1763–KPC (5/15), IncFII (pHN7A8) (1/15), IncR (1/15), and IncpA1763–KPC (1/15). The genetic environment of blaKPC–2 showed nine IS26-based composite transposons, which had a basic core structure ISKpn27-blaKPC–2-ΔISKpn6. About 27.6% (42/152) isolates co-carried 2 to 4 virulence marker genes (namely, peg344, iucA, iroB, rmpA, and rmpA2) for hvKp strains. At least three isolates were identified to harbor virulence gene-carrying plasmids.ConclusionKPC-2-producing K. pneumoniae ST11 was highly heterogeneous in our hospital. Transmission of these strains was mainly mediated by twelve high-risk clones. The blaKPC–2-carrying plasmids and genetic environment of blaKPC–2 genes exhibited active evolution in K. pneumoniae ST11. More attention should be paid to the tendency of KPC-2-ST11 to acquire hypervirulent plasmids.