scholarly journals Pseudomonas Species Isolated via High-throughput Screening Significantly Protect Cotton Plants Against Verticillium Wilt

2020 ◽  
Author(s):  
Xiaoyuan Tao ◽  
Hailin Zhang ◽  
Mengtao Gao ◽  
Menglin Li ◽  
Ting Zhao ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Gene Clusters ◽  
Comparative Genomic ◽  
Wild Type ◽  
New Agents ◽  
Genomic Analyses ◽  
Cotton Plants ◽  
Pseudomonas Species ◽  
Significant Efficacy

Abstract Verticillium wilt (VW) caused by Verticillium dahliae is a devastating soil-borne disease that causes severe yield losses in cotton and other major crops worldwide. Here we conducted a high-throughput screening of isolates recovered from 886 plant rhizosphere samples taken from the three main cotton-producing areas of China. Fifteen isolates distributed in different genera of bacteria that showed inhibitory activity against V. dahliae were screened out. Of these, two Pseudomonas strains, P. protegens XY2F4 and P. donghuensis 22G5, showed significant inhibitory action against V. dahliae. Additional comparative genomic analyses and phenotypical assays confirmed that P. protegens XY2F4 and P. donghuensis 22G5 were the strains most efficient at protecting cotton plants against VW due to specific biological control products they produced. Importantly, we identified a significant efficacy of the natural tropolone compound 7-hydroxytropolone (7-HT) against VW. By phenotypical assay using the wild-type 22G5 and its mutant strain in 7-HT production, we revealed that the 7-HT produced by P. donghuensis is the major substance protecting cotton against VW. This study reveals that Pseudomonas specifically has gene clusters that allow the production of effective antipathogenic metabolites that can now be used as new agents in the biocontrol of VW.

scholarly journals Pseudomonas species isolated via high-throughput screening significantly protect cotton plants against verticillium wilt

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xiaoyuan Tao ◽  
Hailin Zhang ◽  
Mengtao Gao ◽  
Menglin Li ◽  
Ting Zhao ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Gene Clusters ◽  
Comparative Genomic ◽  
Wild Type ◽  
New Agents ◽  
Genomic Analyses ◽  
Cotton Plants ◽  
Pseudomonas Species ◽  
Significant Efficacy

Abstract Verticillium wilt (VW) caused by Verticillium dahliae is a devastating soil-borne disease that causes severe yield losses in cotton and other major crops worldwide. Here we conducted a high-throughput screening of isolates recovered from 886 plant rhizosphere samples taken from the three main cotton-producing areas of China. Fifteen isolates distributed in different genera of bacteria that showed inhibitory activity against V. dahliae were screened out. Of these, two Pseudomonas strains, P. protegens XY2F4 and P. donghuensis 22G5, showed significant inhibitory action against V. dahliae. Additional comparative genomic analyses and phenotypical assays confirmed that P. protegens XY2F4 and P. donghuensis 22G5 were the strains most efficient at protecting cotton plants against VW due to specific biological control products they produced. Importantly, we identified a significant efficacy of the natural tropolone compound 7-hydroxytropolone (7-HT) against VW. By phenotypical assay using the wild-type 22G5 and its mutant strain in 7-HT production, we revealed that the 7-HT produced by P. donghuensis is the major substance protecting cotton against VW. This study reveals that Pseudomonas specifically has gene clusters that allow the production of effective antipathogenic metabolites that can now be used as new agents in the biocontrol of VW.

scholarly journals High-throughput, image-based screening of genetic variant libraries

2017 ◽  
Author(s):  
George Emanuel ◽  
Jeffrey R. Moffitt ◽  
Xiaowei Zhuang
Keyword(s):  
High Throughput ◽  
Genetic Variants ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Genetic Variant ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Wild Type ◽  
Genetic Perturbations

AbstractImage-based, high-throughput, high-content screening of pooled libraries of genetic perturbations will greatly advance our understanding biological systems and facilitate many biotechnology applications. Here we introduce a high-throughput screening method that allows highly diverse genotypes and the corresponding phenotypes to be imaged in numerous individual cells. To facilitate genotyping by imaging, barcoded genetic variants are introduced into the cells, each cell carrying a single genetic variant connected to a unique, nucleic-acid barcode. To identify the genotype-phenotype correspondence, we perform live-cell imaging to determine the phenotype of each cell, and massively multiplexed FISH imaging to measure the barcode expressed in the same cell. We demonstrated the utility of this approach by screening for brighter and more photostable variants of the fluorescent protein YFAST. We imaged 20 million cells expressing ~60,000 YFAST mutants and identified novel YFAST variants that are substantially brighter and/or more photostable than the wild-type protein.

scholarly journals High Throughput Screening of the NatureBank ‘Marine Collection’ in a Haemonchus Bioassay Identifies Anthelmintic Activity in Extracts from a Range of Sponges from Australian Waters

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5846
Author(s):  
Aya C. Taki ◽  
Joseph J. Byrne ◽  
Abdul Jabbar ◽  
Kah Yean Lum ◽  
Sasha Hayes ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Marine Invertebrates ◽  
Chemical Space ◽  
Anthelmintic Activity ◽  
Distinct Species ◽  
Parasitic Nematodes ◽  
Wild Type ◽  
Control Programs

Widespread resistance in parasitic nematodes to most classes of anthelmintic drugs demands the discovery and development of novel compounds with distinct mechanisms of action to complement strategic or integrated parasite control programs. Products from nature—which assume a diverse ‘chemical space’—have significant potential as a source of anthelmintic compounds. In the present study, we screened a collection of extracts (n = 7616) derived from marine invertebrates sampled from Australian waters in a high throughput bioassay for in vitro anti-parasitic activity against the barber’s pole worm (Haemonchus contortus)—an economically important parasitic nematode of livestock animals. In this high throughput screen (HTS), we identified 58 active extracts that reduced larval motility by ≥70% (at 90 h), equating to an overall ‘hit rate’ of ~0.8%. Of these 58 extracts, 16 also inhibited larval development by ≥80% (at 168 h) and/or induced ‘non-wild-type’ (abnormal) larval phenotypes with reference to ‘wild-type’ (normal) larvae not exposed to extract (negative controls). Most active extracts (54 of 58) originated from sponges, three from chordates (tunicates) and one from a coral; these extracts represented 37 distinct species/taxa of 23 families. An analysis of samples by 1H NMR fingerprinting was utilised to dereplicate hits and to prioritise a set of 29 sponge samples for future chemical investigation. Overall, these results indicate that a range of sponge species from Australian waters represents a rich source of natural compounds with nematocidal or nematostatic properties. Our plan now is to focus on in-depth chemical investigations of the sample set prioritised herein.

scholarly journals Rapid, noninvasive, and unsupervised detection of sleep/wake using piezoelectric monitoring for pharmacological studies in narcoleptic mice

2017 ◽  
Author(s):  
Sarah Wurts Black ◽  
Jessica D. Sun ◽  
Alex Laihsu ◽  
Nikki Kimura ◽  
Pamela Santiago ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Body Movement ◽  
Rapid Screening ◽  
Wild Type ◽  
Preclinical Development ◽  
Pharmacological Studies ◽  
Dose Response Study

AbstractBackgroundAssessment of sleep/wake by electroencephalography (EEG) and electromyography (EMG) is invasive, resource intensive, and not amenable to rapid screening at scale for drug discovery. In the preclinical development of therapeutics for narcolepsy, efficacy tests are hindered by the lack of a non-EEG/EMG based translational test of symptom severity. The current methods study offers proof-of-principle that PiezoSleep (noninvasive, unsupervised piezoelectric monitoring of gross body movement, together with respiration patterns during behavioral quiescence), can be used to determine sleep/wake as applicable to the development of wake-promoting therapeutics. First, the translational wake-maintenance score (WMS, the ratio of time during the first half of the dark period spent in long wake bouts to short sleep bouts) of the PiezoSleep narcolepsy screen was introduced as a means by which to rank narcoleptic orexin/ataxin-3 mice and wild type mice by sleep/wake fragmentation severity. Accuracy of the WMS to detect narcoleptic phenotypes were determined in genotype-confirmed orexin/ataxin-3 mice and wild type colony mates. The WMS was used to identify the most highly symptomatic mice for resource-intensive EEG/EMG studies for further analysis of specific arousal states. Second, PiezoSleep was demonstrated for use in high-throughput screening of wake-promoting compounds using modafinil in orexin/ataxin-3 and wild type mice.ResultsThe WMS detected a narcoleptic phenotype with 89% sensitivity, 92% specificity and 98% positive predictive value. A 15-fold difference in WMS differentiated wild type littermates from the most severely affected orexin/ataxin-3 mice. Follow-up EEG/EMG study indicated 82% of the orexin/ataxin-3 mice with the lowest wake-maintenance scores met or exceeded the cataplexy-occurrence threshold (≥ 3 bouts) for inclusion in therapeutic efficacy studies. In the PiezoSleep dose-response study, the ED50 for wake-promotion by modafinil was approximately 50 mg/kg in both genotypes. Using unsupervised piezoelectric monitoring, the efficacy of wake-promoting compounds can be determined in a 5-arm study with 60 mice in less than one week—a fraction of the time compared to EEG/EMG studies.ConclusionsThe WMS on the PiezoSleep narcolepsy screen quantifies the inability to sustain wakefulness and provides an accurate measure of the narcoleptic phenotype in mice. PiezoSleep offers rapid, scalable assessment of sleep/wake for high-throughput screening in drug discovery.

Application of directed evolution in the development of enantioselective enzymes

2000 ◽  
Vol 72 (9) ◽  
pp. 1615-1622 ◽  
Author(s):  
Manfred T. Reetz
Keyword(s):  
Organic Chemistry ◽  
Gene Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Resolution ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Hydrolytic Kinetic Resolution ◽  
Novel Approach

A novel approach to developing enantioselective enzymes for use in organic chemistry has been devised which is independent of structural or mechanistic aspects. The underlying idea is to combine appropriate methods of random mutagenesis, gene expression, and high-throughput screening for enantioselectivity. If these actions are performed in repetitive cycles, an evolutionary pressure is created that leads to sequential improvements of the enantioselectivity of a given enzyme-catalyzed reaction. The concept is illustrated by an example involving the lipase-catalyzed hydrolytic kinetic resolution of an α-chiral ester, the enantio-selectivity increasing from ee = 2% (E =1.1) for a wild-type enzyme to ee = 90-93% (E = 25) for the best mutants.

scholarly journals At the Gate of Mutualism: Identification of Genomic Traits Predisposing to Insect-Bacterial Symbiosis in Pathogenic Strains of the Aphid Symbiont Serratia symbiotica

2021 ◽  
Vol 11 ◽  
Author(s):  
François Renoz ◽  
Vincent Foray ◽  
Jérôme Ambroise ◽  
Patrice Baa-Puyoulet ◽  
Bertrand Bearzatto ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Comparative Genomic ◽  
Intermediate Hosts ◽  
Bacterial Symbiosis ◽  
Early Stages ◽  
Genomic Analyses ◽  
Significant Enrichment ◽  
New Hosts ◽  
Serratia Symbiotica ◽  
Metabolism Gene

Mutualistic associations between insects and heritable bacterial symbionts are ubiquitous in nature. The aphid symbiont Serratia symbiotica is a valuable candidate for studying the evolution of bacterial symbiosis in insects because it includes a wide diversity of strains that reflect the diverse relationships in which bacteria can be engaged with insects, from pathogenic interactions to obligate intracellular mutualism. The recent discovery of culturable strains, which are hypothesized to resemble the ancestors of intracellular strains, provide an opportunity to study the mechanisms underlying bacterial symbiosis in its early stages. In this study, we analyzed the genomes of three of these culturable strains that are pathogenic to aphid hosts, and performed comparative genomic analyses including mutualistic host-dependent strains. All three genomes are larger than those of the host-restricted S. symbiotica strains described so far, and show significant enrichment in pseudogenes and mobile elements, suggesting that these three pathogenic strains are in the early stages of the adaptation to their host. Compared to their intracellular mutualistic relatives, the three strains harbor a greater diversity of genes coding for virulence factors and metabolic pathways, suggesting that they are likely adapted to infect new hosts and are a potential source of metabolic innovation for insects. The presence in their genomes of secondary metabolism gene clusters associated with the production of antimicrobial compounds and phytotoxins supports the hypothesis that S. symbiotia symbionts evolved from plant-associated strains and that plants may serve as intermediate hosts. Mutualistic associations between insects and bacteria are the result of independent transitions to endosymbiosis initiated by the acquisition of environmental progenitors. In this context, the genomes of free-living S. symbiotica strains provide a rare opportunity to study the inventory of genes held by bacterial associates of insects that are at the gateway to a host-dependent lifestyle.

scholarly journals Comparative Genomic Analyses of Seventeen Streptococcus pneumoniae Strains: Insights into the Pneumococcal Supragenome

2007 ◽  
Vol 189 (22) ◽  
pp. 8186-8195 ◽  
Author(s):  
N. Luisa Hiller ◽  
Benjamin Janto ◽  
Justin S. Hogg ◽  
Robert Boissy ◽  
Susan Yu ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Recombination ◽  
Orthologous Gene ◽  
Gene Clusters ◽  
Comparative Genomic ◽  
Host Interaction ◽  
Genomic Analyses ◽  
Strain Pair ◽  
Interaction Strategy ◽  
Genic Diversity

ABSTRACT The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strain's genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters (∼3,000) that are represented in the S. pneumoniae population at frequencies of ≥0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.

scholarly journals Analysis of the small RNA spf in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000

Microbiology ◽  
2014 ◽  
Vol 160 (5) ◽  
pp. 941-953 ◽  
Author(s):  
So Hae Park ◽  
Zhongmeng Bao ◽  
Bronwyn G. Butcher ◽  
Katherine D’Amico ◽  
Yun Xu ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Comparative Genomic ◽  
Wild Type ◽  
New Information ◽  
Genomic Analyses ◽  
Increased Sensitivity ◽  
Non Coding Rnas

Bacteria contain small non-coding RNAs (ncRNAs) that are typically responsible for altering transcription, translation or mRNA stability. ncRNAs are important because they often regulate virulence factors and susceptibility to various stresses. Here, the regulation of a recently described ncRNA of Pseudomonas syringae DC3000, spot 42 (now referred to as spf), was investigated. A putative RpoE binding site was identified upstream of spf in strain DC3000. RpoE is shown to regulate the expression of spf. Also, deletion of spf results in increased sensitivity to hydrogen peroxide compared with the wild-type strain, suggesting that spf plays a role in susceptibility to oxidative stress. Furthermore, expression of alg8 is shown to be influenced by spf, suggesting that this ncRNA plays a role in alginate biosynthesis. Structural and comparative genomic analyses show this ncRNA is well conserved among the pseudomonads. The findings provide new information on the regulation and role of this ncRNA in P. syringae.

scholarly journals High-Throughput Screening of Biodiversity for Antibiotic Discovery

Acta Naturae ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 23-29
Author(s):  
S. S. Terekhov ◽  
I. A. Osterman ◽  
I. V. Smirnov
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Genome Mining ◽  
Gene Clusters ◽  
Good Alternative ◽  
Antibiotic Activity ◽  
Antibiotic Resistant ◽  
Chemical Libraries ◽  
Antibiotic Discovery

The increasing number of infections caused by antibiotic-resistant strains of pathogens challenges modern technologies of drug discovery. Combinatorial chemistry approaches are based on chemical libraries. They enable the creation of high-affinity low-molecular-weight ligands of the therapeutically significant molecular targets of human cells, thus opening an avenue toward a directed design of highly effective therapeutic agents. Nevertheless, these approaches face insurmountable difficulties in antibiotic discovery. Natural compounds that have evolved for such important characteristics as broad specificity and efficiency are a good alternative to chemical libraries. However, unrestricted use of natural antibiotics and their analogues leads to avalanche-like spread of resistance among bacteria. The search for new natural antibiotics, in its turn, is extremely complicated nowadays by the problem of antibiotic rediscovery. This calls for the application of alternative high-throughput platforms for antibiotic activity screening, cultivation of unculturable microorganisms, exploration of novel antibiotic biosynthetic gene clusters, as well as their activation and heterologous expression. Microfluidic technologies for the screening of antibiotic activity at the level of single cells are, therefore, of great interest, since they enable the use of a single platform to combine the technology of ultrahigh-throughput screening, next-generation sequencing, and genome mining, thus opening up unique opportunities for antibiotic discovery.

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