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April 21, 2020

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.


April 21, 2020

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.


April 21, 2020

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.


April 21, 2020

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (>?5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.


April 21, 2020

Metabolite-based mutualism enhances hydrogen production in a two-species microbial consortium

Sustainable hydrogen production from renewable and low-cost substrates is very important to mitigate environmental and energy-related issues. Microbial consortia are promising for diverse bioenergy and environmental applications, yet microbial interactions are not fully understood. Here, we present comprehensive investigation on how two species in an artificial microbial consortium, consisting of Bacillus cereus A1 and Brevundimonas naejangsanensis B1, mutually cooperate to achieve an overall enhancement in hydrogen production and starch utilization. In this consortium, strains A1 and B1 secrete a-amylase and glucoamylase that are functionally complementary in starch hydrolysis. Moreover, strain A1 converts starch into lactate as a carbon source and electron donor, supporting the cell growth and hydrogen generation of strain B1. In return, strain B1 produces formate as an electron shuttle to strain A1 to enhance hydrogen production. The co-culture re-directs the overall metabolic flux, facilitates the cell growth, and up-regulates the key genes of hydrogen production and starch hydrolysis.


April 21, 2020

Genomic characterisation of the new Dickeya fangzhongdai species regrouping plant pathogens and environmental isolates.

The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity.The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100-150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways.This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn’t find any relationship between gene content and phenotypic differences or sharing of environmental habitats.


April 21, 2020

Resource Concentration Modulates the Fate of Dissimilated Nitrogen in a Dual-Pathway Actinobacterium.

Respiratory ammonification and denitrification are two evolutionarily unrelated dissimilatory nitrogen (N) processes central to the global N cycle, the activity of which is thought to be controlled by carbon (C) to nitrate (NO3-) ratio. Here we find that Intrasporangium calvum C5, a novel dual-pathway denitrifier/respiratory ammonifier, disproportionately utilizes ammonification rather than denitrification when grown under low C concentrations, even at low C:NO3- ratios. This finding is in conflict with the paradigm that high C:NO3- ratios promote ammonification and low C:NO3- ratios promote denitrification. We find that the protein atomic composition for denitrification modules (NirK) are significantly cost minimized for C and N compared to ammonification modules (NrfA), indicating that limitation for C and N is a major evolutionary selective pressure imprinted in the architecture of these proteins. The evolutionary precedent for these findings suggests ecological importance for microbial activity as evidenced by higher growth rates when I. calvum grows predominantly using its ammonification pathway and by assimilating its end-product (ammonium) for growth under ammonium-free conditions. Genomic analysis of I. calvum further reveals a versatile ecophysiology to cope with nutrient stress and redox conditions. Metabolite and transcriptional profiles during growth indicate that enzyme modules, NrfAH and NirK, are not constitutively expressed but rather induced by nitrite production via NarG. Mechanistically, our results suggest that pathway selection is driven by intracellular redox potential (redox poise), which may be lowered when resource concentrations are low, thereby decreasing catalytic activity of upstream electron transport steps (i.e., the bc1 complex) needed for denitrification enzymes. Our work advances our understanding of the biogeochemical flexibility of N-cycling organisms, pathway evolution, and ecological food-webs.


October 23, 2019

Gene editing and genetic engineering approaches for advanced probiotics: A review.

The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher’s attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance. The genetic manipulation of probiotic microorganisms is crucial for defining their role in intestinal microbiota and exploring their beneficial properties. This review describes an overview of gene editing and systems biology approaches, highlighting the advent of omics methods which allows the study of new routes for studying probiotic bacteria. We have also summarized gene editing tools like TALEN, ZFNs and CRISPR-Cas that edits or cleave the specific target DNA. Furthermore, in this review an overview of proposed design of advanced customized probiotic is also hypothesized to improvise the probiotics.


September 22, 2019

Cow, yak, and camel milk diets differentially modulated the systemic immunity and fecal microbiota of rats

Cow milk is most widely consumed; however, non-cattle milk has gained increasing interest because of added nutritive values. We compared the health effects of yak, cow, and camel milk in rats. By measuring several plasma immune factors, significantly more interferon-? was detected in the camel than the yak (P=0.0020) or cow (P=0.0062) milk group. Significantly more IgM was detected in the yak milk than the control group (P=0.0071). The control group had significantly less interleukin 6 than the yak (P=0.0499) and cow (P=0.0248) milk groups. The fecal microbiota of the 144 samples comprised mainly of the Firmicutes (76.70±11.03%), Bacteroidetes (15.27±7.79%), Proteobacteria (3.61±4.34%), and Tenericutes (2.61±2.53%) phyla. Multivariate analyses revealed a mild shift in the fecal microbiota along the milk treatment. We further identified the differential microbes across the four groups. At day 14, 22 and 28 differential genera and species were identified (P=0.0000–0.0462), while 8 and 11 differential genera and species (P=0.0000–0.0013) were found at day 28. Some short-chain fatty acid and succinate producers increased, while certain health-concerned bacteria (Prevotella copri, Phascolarctobacterium faecium, and Bacteroides uniformis) decreased after 14days of yak or camel milk treatment. We demonstrated that different animal milk could confer distinctive nutritive value to the host.


September 22, 2019

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such ‘talented’ producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus ‘Entotheonella’ with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. ‘Entotheonella’ spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum ‘Tectomicrobia’. The pronounced bioactivities and chemical uniqueness of ‘Entotheonella’ compounds provide significant opportunities for ecological studies and drug discovery.


September 22, 2019

Novel syntrophic populations dominate an ammonia-tolerant methanogenic microbiome.

Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H2 and CO2, followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1’s metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or “biogas” is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.


September 22, 2019

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, ?-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage.


September 22, 2019

A novel lactobacilli-based teat disinfectant for improving bacterial communities in the milks of cow teats with subclinical mastitis.

Teat disinfection pre- and post-milking is important for the overall health and hygiene of dairy cows. The objective of this study was to evaluate the efficacy of a novel probiotic lactobacilli-based teat disinfectant based on changes in somatic cell count (SCC) and profiling of the bacterial community. A total of 69 raw milk samples were obtained from eleven Holstein-Friesian dairy cows over 12 days of teat dipping in China. Single molecule, real-time sequencing technology (SMRT) was employed to profile changes in the bacterial community during the cleaning protocol and to compare the efficacy of probiotic lactic acid bacteria (LAB) and commercial teat disinfectants. The SCC gradually decreased following the cleaning protocol and the SCC of the LAB group was slightly lower than that of the commercial disinfectant (CD) group. Our SMRT sequencing results indicate that raw milk from both the LAB and CD groups contained diverse microbial populations that changed over the course of the cleaning protocol. The relative abundances of some species were significantly changed during the cleaning process, which may explain the observed bacterial community differences. Collectively, these results suggest that the LAB disinfectant could reduce mastitis-associated bacteria and improve the microbial environment of the cow teat. It could be used as an alternative to chemical pre- and post-milking teat disinfectants to maintain healthy teats and udders. In addition, the Pacific Biosciences SMRT sequencing with the full-length 16S ribosomal RNA gene was shown to be a powerful tool for monitoring changes in the bacterial population during the cleaning protocol.


September 22, 2019

Global dissection of alternative splicing uncovers transcriptional diversity in tissues and associates with the flavonoid pathway in tea plant (Camellia sinensis).

Alternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea.In our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~?20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc. Thirteen co-expressed modules of AS transcripts were identified, which revealed a similar pattern between the bud and young leaves as well as a distinct pattern between seasons. AS events of structural genes including anthocyanidin reductase and MYB transcription factors were involved in biosynthesis of flavonoid, especially in vegetative tissues. The AS isoforms rather than the full-length ones were the major transcripts involved in flavonoid synthesis pathway, and is positively correlated with the catechins content conferring the tea taste. We propose that the AS is an important functional mechanism in regulating flavonoid metabolites.Our study provides the insight into the AS events underlying tea plant’s uniquely different developmental process and highlights the important contribution and efficacy of alternative splicing regulatory function to biosynthesis of flavonoids.


September 22, 2019

Researches on transcriptome sequencing in the study of traditional Chinese medicine

Due to its incomparable advantages, the application of transcriptome sequencing in the study of traditional Chinese medicine attracts more and more attention of researchers, which greatly promote the development of traditional Chinese medicine. In this paper, the applications of transcriptome sequencing in traditional Chinese medicine were summarized by reviewing recent related papers.


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