scholarly journals Combination of Pseudo-LC-NMR and HRMS/MS-Based Molecular Networking for the Rapid Identification of Antimicrobial Metabolites From Fusarium petroliphilum

2021 ◽  
Vol 8 ◽  
Author(s):  
Abdulelah Alfattani ◽  
Laurence Marcourt ◽  
Valérie Hofstetter ◽  
Emerson Ferreira Queiroz ◽  
Sara Leoni ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Phylogenetic Analyses ◽  
Posidonia Oceanica ◽  
Antimicrobial Activities ◽  
Rapid Identification ◽  
Single Step ◽  
Preparative Hplc ◽  
Molecular Networking ◽  
Innovative Strategy ◽  
Fusarium Petroliphilum

An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea (Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call “pseudo-LC-NMR,” and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.

New Potential Biologically Active Compounds: Synthesis and Characterization of Urea and Thiourea Derivativpes Bearing 1,2,4-oxadiazole Ring

2020 ◽  
Vol 17 (7) ◽  
pp. 525-534 ◽  
Author(s):  
Nevin Arıkan Ölmez ◽  
Faryal Waseer
Keyword(s):  
Microwave Irradiation ◽  
Room Temperature ◽  
Antimicrobial Activities ◽  
Phenyl Isocyanate ◽  
Single Step ◽  
Biologically Active Compounds ◽  
Biologically Active ◽  
Active Compounds ◽  
Thiourea Derivatives ◽  
Acyl Chlorides

Background: Urea, thiourea, and 1,2,4-oxadiazole compounds are of great interest due to their different activities such as anti-inflammatory, antiviral, analgesic, fungicidal, herbicidal, diuretic, antihelminthic and antitumor along with antimicrobial activities. Objective: In this work, we provide a new series of potential biologically active compounds containing both 1,2,4-oxadiazole and urea/thiouprea moiety. Materials and Methods: Firstly, 5-chloromethyl-3-aryl-1,2,4-oxadiazoles (3a-j) were synthesized from the reaction of different substituted amidoximes (2a-j) and chloroacetyl chloride in the presence of pyridine by conventional and microwave-assisted methods. In the conventional method, 1,2,4-oxadiazoles were obtained in two steps. O-acylamidoximes obtained in the first step at room temperature were heated in toluene for an average of one hour to obtain 1,2,4-oxadiazoles. The yields varied from 70 to 96 %. 1,2,4-oxadiazoles were obtained under microwave irradiation in a single step in a 90-98 % yield at 160 °C in five minutes. 5-aminomethyl-3-aryl-1,2,4- oxadiazoles (5a-j) were obtained by Gabriel amine synthesis in two steps from corresponding 5-chloromethyl-3- aryl-1,2,4-oxadiazoles. Finally, twenty new urea (6a-j) and thiourea (7a-j) compounds bearing oxadiazole ring were synthesized by reacting 5-aminomethyl-3-aryl-1,2,4-oxadiazoles with phenyl isocyanate and isothiocyanate in tetrahydrofuran (THF) at room temperature with average yields (40-70%). Results and Discussions: An efficient and rapid method for the synthesis of 1,2,4-oxadiazoles from the reaction of amidoximes and acyl halides without using any coupling reagent under microwave irradiation has been developed, and twenty new urea/thiourea compounds bearing 1,2,4-oxadiazole ring have been synthesized and characterized. Conclusion: We have synthesized a new series of urea/thiourea derivatives bearing 1,2,4-oxadiazole ring. Also facile synthesis of 3,5-disubstituted 1,2,4-oxadiazoles from amidoximes and acyl chlorides under microwave irradiation was reported. The compounds were characterized using FTIR, 1H NMR, 13C NMR, and elemental analysis techniques.

scholarly journals Tools for Rapid Genetic Engineering of Vibrio fischeri

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Karen L. Visick ◽  
Kelsey M. Hodge-Hanson ◽  
Alice H. Tischler ◽  
Allison K. Bennett ◽  
Vincent Mastrodomenico
Keyword(s):  
Antibiotic Resistance ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Vibrio Fischeri ◽  
Single Step ◽  
Universal Primers ◽  
Resistance Marker ◽  
Content Type ◽  
Flp Recombinase ◽  
Overlap Extension

ABSTRACT Vibrio fischeri is used as a model for a number of processes, including symbiosis, quorum sensing, bioluminescence, and biofilm formation. Many of these studies depend on generating deletion mutants and complementing them. Engineering such strains, however, is a time-consuming, multistep process that relies on cloning and subcloning. Here, we describe a set of tools that can be used to rapidly engineer deletions and insertions in the V. fischeri chromosome without cloning. We developed a uniform approach for generating deletions using PCR splicing by overlap extension (SOEing) with antibiotic cassettes flanked by standardized linker sequences. PCR SOEing of the cassettes to sequences up- and downstream of the target gene generates a DNA product that can be directly introduced by natural transformation. Selection for the introduced antibiotic resistance marker yields the deletion of interest in a single step. Because these cassettes also contain FRT (FLP recognition target) sequences flanking the resistance marker, Flp recombinase can be used to generate an unmarked, in-frame deletion. We developed a similar methodology and tools for the rapid insertion of specific genes at a benign site in the chromosome for purposes such as complementation. Finally, we generated derivatives of these tools to facilitate different applications, such as inducible gene expression and assessing protein production. We demonstrated the utility of these tools by deleting and inserting genes known or predicted to be involved in motility. While developed for V. fischeri strain ES114, we anticipate that these tools can be adapted for use in other V. fischeri strains and, potentially, other microbes. IMPORTANCE Vibrio fischeri is a model organism for studying a variety of important processes, including symbiosis, biofilm formation, and quorum sensing. To facilitate investigation of these biological mechanisms, we developed approaches for rapidly generating deletions and insertions and demonstrated their utility using two genes of interest. The ease, consistency, and speed of the engineering is facilitated by a set of antibiotic resistance cassettes with common linker sequences that can be amplified by PCR with universal primers and fused to adjacent sequences using splicing by overlap extension and then introduced directly into V. fischeri , eliminating the need for cloning and plasmid conjugation. The antibiotic cassettes are flanked by FRT sequences, permitting their removal using Flp recombinase. We augmented these basic tools with a family of constructs for different applications. We anticipate that these tools will greatly accelerate mechanistic studies of biological processes in V. fischeri and potentially other Vibrio species.

scholarly journals Sequence-Based Analysis of Secondary-Metabolite Biosynthesis in Marine Actinobacteria

2010 ◽  
Vol 76 (8) ◽  
pp. 2487-2499 ◽  
Author(s):  
Erin A. Gontang ◽  
Susana P. Gaud�ncio ◽  
William Fenical ◽  
Paul R. Jensen
Keyword(s):  
Secondary Metabolite ◽  
Phylogenetic Analyses ◽  
Rapid Identification ◽  
Secondary Metabolite Production ◽  
Type I ◽  
Marine Actinobacteria ◽  
Metabolite Production ◽  
A Genome ◽  
Secondary Metabolite Biosynthesis ◽  
Primer Sets

ABSTRACT A diverse collection of 60 marine-sediment-derived Actinobacteria representing 52 operational taxonomic units was screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were employed to specifically target adenylation domains associated with nonribosomal peptide synthetases (NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne polyketide synthases (PKSs). In total, two-thirds of the strains yielded a sequence-verified PCR product for at least one of these biosynthetic types. Genes associated with enediyne biosynthesis were detected in only two genera, while 88% of the ketosynthase sequences shared greatest homology with modular PKSs. Positive strains included representatives of families not traditionally associated with secondary-metabolite production, including the Corynebacteriaceae, Gordoniaceae, Intrasporangiaceae, and Micrococcaceae. In four of five cases where phylogenetic analyses of KS sequences revealed close evolutionary relationships to genes associated with experimentally characterized biosynthetic pathways, secondary-metabolite production was accurately predicted. Sequence clustering patterns were used to provide an estimate of PKS pathway diversity and to assess the biosynthetic richness of individual strains. The detection of highly similar KS sequences in distantly related strains provided evidence of horizontal gene transfer, while control experiments designed to amplify KS sequences from Salinispora arenicola strain CNS-205, for which a genome sequence is available, led to the detection of 70% of the targeted PKS pathways. The results provide a bioinformatic assessment of secondary-metabolite biosynthetic potential that can be applied in the absence of fully assembled pathways or genome sequences. The rapid identification of strains that possess the greatest potential to produce new secondary metabolites along with those that produce known compounds can be used to improve the process of natural-product discovery by providing a method to prioritize strains for fermentation studies and chemical analysis.

scholarly journals Phytophthora Database 2.0: Update and Future Direction

2013 ◽  
Vol 103 (12) ◽  
pp. 1204-1208 ◽  
Author(s):  
Bongsoo Park ◽  
Frank Martin ◽  
David M. Geiser ◽  
Hye-Seon Kim ◽  
Michele A. Mansfield ◽  
...  
Keyword(s):  
Diagnostic Tool ◽  
Online Community ◽  
Phylogenetic Analyses ◽  
Global Scale ◽  
Rapid Identification ◽  
Tool Development ◽  
Sequence Alignments ◽  
Geographic Origins ◽  
Community Forum ◽  
Future Direction

The online community resource Phytophthora database (PD) was developed to support accurate and rapid identification of Phytophthora and to help characterize and catalog the diversity and evolutionary relationships within the genus. Since its release in 2008, the sequence database has grown to cover 1 to 12 loci for ≈2,600 isolates (representing 138 described and provisional species). Sequences of multiple mitochondrial loci were added to complement nuclear loci-based phylogenetic analyses and diagnostic tool development. Key characteristics of most newly described and provisional species have been summarized. Other additions to improve the PD functionality include: (i) geographic information system tools that enable users to visualize the geographic origins of chosen isolates on a global-scale map, (ii) a tool for comparing genetic similarity between isolates via microsatellite markers to support population genetic studies, (iii) a comprehensive review of molecular diagnostics tools and relevant references, (iv) sequence alignments used to develop polymerase chain reaction-based diagnostics tools to support their utilization and new diagnostic tool development, and (v) an online community forum for sharing and preserving experience and knowledge accumulated in the global Phytophthora community. Here we present how these improvements can support users and discuss the PD's future direction.

scholarly journals Metagenomic Insights Into the Microbial Assemblage Capable of Quorum Sensing and Quorum Quenching in Particulate Organic Matter in the Yellow Sea

2021 ◽  
Vol 11 ◽  
Author(s):  
Ying Su ◽  
Yuanzhi Yang ◽  
Xiao-Yu Zhu ◽  
Xiao-Hua Zhang ◽  
Min Yu
Keyword(s):  
Organic Matter ◽  
Quorum Sensing ◽  
Particulate Organic Matter ◽  
Yellow Sea ◽  
Current Knowledge ◽  
Phylogenetic Analyses ◽  
Quorum Quenching ◽  
The Yellow Sea ◽  
Rrna Gene ◽  
Wide Range

Quorum sensing (QS) is a density-dependent communicating mechanism that allows bacteria to regulate a wide range of biogeochemical important processes and could be inhibited by quorum quenching (QQ). Increasing researches have demonstrated that QS can affect the degradation of particulate organic matter (POM) in the photic zone. However, knowledge of the diversity and variation of microbial QS and QQ systems in sinking POM is scarce. Here, POM samples were collected from surface seawater (SW), bottom seawater (BW), and surficial sediment (SS) in the Yellow Sea of China. 16S rRNA gene amplicon and metagenome sequencing were performed to analyze the community structure of particle-associated microorganisms and distribution of QS genes [acylated homoserine lactone (AHL) synthesizing gene luxI and AHL sensing gene luxR] and QQ genes (genes encoding for AHL lactonase and acylase) in POM. Shifting community structures were observed at different sampling depths, with an increase of microbial abundance and diversity from SW to BW. Along with the variation of microbial communities, the abundances of luxI and luxR decreased slightly but were restored or even exceeded when POM arrived at SS. Comparatively, abundances of AHL lactonase and acylase remained constant during the transportation process from SW to BW but increased dramatically in SS. Correlation tests indicated that abundances of luxI and luxR were positively correlated with temperature, while those of AHL acylase were positively correlated with depth, SiO42–, PO43–, and NO3–, but negatively correlated with temperature and pH. According to phylogenetic analyses, the retrieved QS and QQ genes are more diverse and distinctive than ever experimentally identified. Besides, the vertical transmission of QS and QQ genes along with POM sinking was observed, which could be one of the key factors leading to the prevalence of QS and QQ genes in marine ecosystems. Overall, our results increase the current knowledge of QS and QQ metabolic pathways in marine environment and shed light on the intertwined interspecies relationships to better investigate their dynamics and ecological roles in POM cycling.

scholarly journals A novel and widespread class of ketosynthase is responsible for the head-to-head condensation of two acyl moieties in bacterial pyrone biosynthesis

2015 ◽  
Vol 11 ◽  
pp. 1412-1417 ◽  
Author(s):  
Darko Kresovic ◽  
Florence Schempp ◽  
Zakaria Cheikh-Ali ◽  
Helge B Bode
Keyword(s):  
Amino Acid ◽  
Quorum Sensing ◽  
Heterologous Expression ◽  
Phylogenetic Analyses ◽  
Bioinformatic Analysis ◽  
Evolutionary Origin ◽  
Biochemical Mechanism ◽  
Amino Acid Exchange ◽  
Quorum Sensing Signals ◽  
Acid Exchange

The biosynthesis of photopyrones, novel quorum sensing signals in Photorhabdus, has been studied by heterologous expression of the photopyrone synthase PpyS catalyzing the head-to-head condensation of two acyl moieties. The biochemical mechanism of pyrone formation has been investigated by amino acid exchange and bioinformatic analysis. Additionally, the evolutionary origin of PpyS has been studied by phylogenetic analyses also revealing homologous enzymes in Pseudomonas sp. GM30 responsible for the biosynthesis of pseudopyronines including a novel derivative. Moreover this novel class of ketosynthases is only distantly related to other pyrone-forming enzymes identified in the biosynthesis of the potent antibiotics myxopyronin and corallopyronin.

scholarly journals Construction of prcK and prcR Mutant Strains of Lactobacillus paracasei HD1.7 and the Impact on the Production of Paracin 1.7

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Jingping Ge ◽  
Xiaolei Ji ◽  
Tian You ◽  
Yanyang Sun ◽  
Wenxiang Ping
Keyword(s):  
Quorum Sensing ◽  
Antimicrobial Peptides ◽  
Parental Strain ◽  
Antimicrobial Activities ◽  
Qrt Pcr ◽  
Mutant Strains ◽  
Gene Knockouts ◽  
Pcr Assays ◽  
Chemical Preservatives ◽  
The Impact

Gene knockouts of prcK, prcR and both together were constructed in L. paracasei HD1.7. The antimicrobial activities of the prcK, prcR and prcKprcR mutant strains against B. subtilis were 23.6%, 21.9% and 36.6% lower than that of the parental strain, respectively, indicating that these genes affect production of bacteriocin antimicrobial peptides. qRT-PCR assays showed that the relative transcription levels of prcK and prcR mRNA in the DK and DR strains were 0.36 and 0.33 times of that in parental bacteria, respectively. Our data suggest that prcK and prcR are quorum sensing related genes that influence production of the bacteriocin Paracin 1.7. This research provides the basis for exploring the functions of these genes in the production of Paracin 1.7 and more generally for the exploration of the biological preservatives instead of chemical preservatives.

scholarly journals Rapid identification of compounds with enhanced antimicrobial activity by using conformationally defined combinatorial libraries

1996 ◽  
Vol 313 (1) ◽  
pp. 141-147 ◽  
Author(s):  
Sylvie E. BLONDELLE ◽  
Ema TAKAHASHI ◽  
Richard A. HOUGHTEN ◽  
Enrique PÉREZ-PAYÁ
Keyword(s):  
Combinatorial Library ◽  
Combinatorial Libraries ◽  
Antimicrobial Activities ◽  
Rapid Identification ◽  
Helical Conformation ◽  
Lipid Layer ◽  
Highly Active ◽  
Structure Activity ◽  
The Individual ◽  
The Relationship

We have combined the strength of our synthetic combinatorial library approach for the rapid identification of highly active compounds with prior knowledge of the relationship between the antimicrobial activities of individual peptides with specific induced conformations in order to identify new peptides with enhanced activity relative to a starting known antimicrobial sequence. In the current study, conformationally defined combinatorial libraries were generated based on an 18-mer antimicrobial peptide known to be induced into an α-helical conformation in a lipidic environment. Not only were novel sequences readily identified with 10-fold increases in activity, but detailed information about the structure-activity relationships of the peptides studied was also obtained during the deconvolution process. By using circular dichroism spectroscopy it was found that the individual 18-mer peptides could be induced into α-helical conformations on interaction with the cell lipid layer and/or sialic acids, which could result in bacterial cell lysis due to perturbation of the lipid packing of the cell wall.

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