scholarly journals Integrated Genomic and Metabolomic Approach to the Discovery of Potential Anti-Quorum Sensing Natural Products from Microbes Associated with Marine Samples from Singapore

Marine Drugs ◽  
2019 ◽  
Vol 17 (1) ◽  
pp. 72 ◽  
Author(s):  
Ji Ong ◽  
Hui Goh ◽  
Swee Lim ◽  
Li Pang ◽  
Joyce Chin ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Quorum Sensing ◽  
Large Scale ◽  
Marine Ecosystem ◽  
Gene Clusters ◽  
Integrated Approach ◽  
Rrna Gene ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Reporter System

With 70% of the Earth’s surface covered in water, the marine ecosystem offers immense opportunities for drug discovery and development. Due to the decreasing rate of novel natural product discovery from terrestrial sources in recent years, many researchers are beginning to look seaward for breakthroughs in new therapeutic agents. As part of an ongoing marine drug discovery programme in Singapore, an integrated approach of combining metabolomic and genomic techniques were initiated for uncovering novel anti-quorum sensing molecules from bacteria associated with subtidal samples collected in the Singapore Strait. Based on the culture-dependent method, a total of 102 marine bacteria strains were isolated and the identities of selected strains were established based on their 16S rRNA gene sequences. About 5% of the marine bacterial organic extracts showed quorum sensing inhibitory (QSI) activity in a dose-dependent manner based on the Pseudomonas aeruginosa QS reporter system. In addition, the extracts were subjected to mass spectrometry-based molecular networking and the genome of selected strains were analysed for known as well as new biosynthetic gene clusters. This study revealed that using integrated techniques, coupled with biological assays, can provide an effective and rapid prioritization of marine bacterial strains for downstream large-scale culturing for the purpose of isolation and structural elucidation of novel bioactive compounds.

scholarly journals Large-scale identification of viral quorum sensing systems reveal convergent evolution of density-dependent sporulation-hijacking in bacteriophages

2021 ◽  
Author(s):  
Charles Bernard ◽  
Yanyan Li ◽  
Philippe Lopez ◽  
Eric Bapteste
Keyword(s):  
Quorum Sensing ◽  
Large Scale ◽  
Supporting Cell ◽  
Computational Method ◽  
Dependent Manner ◽  
Genomic Context ◽  
Density Dependent ◽  
Sensing Systems ◽  
Public Data ◽  
Genetic Systems

Quorum sensing systems (QSSs) are genetic systems supporting cell-cell or bacteriophage-bacteriophage communication. By regulating behavioral switches as a function of the encoding population density, QSSs shape the social dynamics of microbial communities. However, their diversity is tremendously overlooked in bacteriophages, which implies that many density-dependent behaviors likely remains to be discovered in these viruses. Here, we developed a signature-based computational method to identify novel peptide-based RRNPP QSSs in gram-positive bacteria (e.g. Firmicutes) and their mobile genetic elements. The large-scale application of this method against available genomes of Firmicutes and bacteriophages revealed 2708 candidate RRNPP-type QSSs, including 382 found in (pro)phages. These 382 viral candidate QSSs are classified into 25 different groups of homologs, of which 22 were never described before in bacteriophages. Remarkably, genomic context analyses suggest that candidate viral QSSs from 6 different families dynamically manipulate the host biology. Specifically, many viral candidate QSSs are predicted to regulate, in a density-dependent manner, adjacent (pro)phage-encoded regulator genes whose bacterial homologs are key regulators of the sporulation initiation pathway (either Rap, Spo0E, or AbrB). Consistently, we found evidence from public data that certain of our candidate (pro)phage-encoded QSSs dynamically manipulate the timing of sporulation of the bacterial host. These findings challenge the current paradigm assuming that bacteria decide to sporulate in adverse situation. Indeed, our survey highlights that bacteriophages have evolved, multiple times, genetic systems that dynamically influence this decision to their advantage, making sporulation a survival mechanism of last resort for phage-host collectives.

scholarly journals Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

2004 ◽  
Vol 70 (4) ◽  
pp. 2452-2463 ◽  
Author(s):  
Asuncion Martinez ◽  
Steven J. Kolvek ◽  
Choi Lai Tiong Yip ◽  
Joern Hopke ◽  
Kara A. Brown ◽  
...  
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Bacterial Artificial Chromosome ◽  
Environmental Dna ◽  
Gene Clusters ◽  
Artificial Chromosome ◽  
Bacterial Strains ◽  
Soil Dna ◽  
Shuttle Vectors ◽  
E Coli

ABSTRACT The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

Marine Microbial Metabolites: A new wave of drugs for Combating Antimicrobial Resistance

2021 ◽  
pp. 2348-2352
Author(s):  
Venkat Abhiram Earny ◽  
Venkatesh Kamath ◽  
Anuraag Muralidharan ◽  
Vandana K E ◽  
Kanav Khera
Keyword(s):  
Drug Discovery ◽  
Antimicrobial Resistance ◽  
Health Care Professionals ◽  
Marine Ecosystem ◽  
Integrated Approach ◽  
Multidrug Resistant ◽  
Bioactive Metabolites ◽  
Resistant Bacteria ◽  
Steady Increase ◽  
Terrestrial Source

The steady increase in the emergence of multidrug-resistant bacteria amongst medical centers, environment, animals, and food is of major concern for health care professionals. Most of the currently used mainline antibacterial drugs were discovered during the golden era of antibiotic discovery (1950-60). During this period, many natural, semi-synthetic, and synthetic molecules were screened for their antimicrobial potential against a spectrum of clinical pathogens. Nevertheless, there was a gap of forty long years until the release of a newer class of antibiotics in the market. It is very vital to develop an integrated approach to combat antimicrobial resistance. There has been a paradigm shift in the field of marine drug discovery in the last two decades. Bioactive metabolites derived from the marine ecosystem are known to exhibit a wide array of pharmacological activity than the terrestrial source. Among all marine organisms, secondary metabolites derived from microbes are the most underexplored natural source. Screening of marine microbes for various antimicrobial molecules has become a noteworthy trend in marine drug discovery and provides a ray of hope for combating antimicrobial resistance.

scholarly journals Gut bacterial aggregates as living gels

2021 ◽  
Author(s):  
Brandon H Schlomann ◽  
Raghuveer Parthasarathy
Keyword(s):  
Large Scale ◽  
Spatial Organization ◽  
Three Dimensional ◽  
Intestinal Transport ◽  
Power Laws ◽  
Soft Materials ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Host Microbe Interactions ◽  
Bacterial Aggregates

The spatial organization of gut microbiota influences both microbial abundances and host-microbe interactions, but the underlying rules relating bacterial dynamics to large-scale structure remain unclear. To this end we studied experimentally and theoretically the formation of three-dimensional bacterial clusters, a key parameter controlling susceptibility to intestinal transport and access to the epithelium. Inspired by models of structure formation in soft materials, we sought to understand how the distribution of gut bacterial cluster sizes emerges from bacterial-scale kinetics. Analyzing imaging-derived data on cluster sizes for eight different bacterial strains in the larval zebrafish gut, we find a common family of size distributions that decay approximately as power laws with exponents close to -2, becoming shallower for large clusters in a strain-dependent manner. We show that this type of distribution arises naturally from a Yule-Simons-type process in which bacteria grow within clusters and can escape from them, coupled to an aggregation process that tends to condense the system toward a single massive cluster, reminiscent of gel formation. Together, these results point to the existence of general, biophysical principles governing the spatial organization of the gut microbiome that may be useful for inferring fast-timescale dynamics that are experimentally inaccessible.

scholarly journals Roadmap for the use of base editors to decipher drug mechanism of action

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257537
Author(s):  
Estel Aparicio-Prat ◽  
Dong Yan ◽  
Marco Mariotti ◽  
Michael Bassik ◽  
Gaelen Hess ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Reporter System ◽  
Experimental Conditions ◽  
Drug Mechanism ◽  
Β2 Adrenergic Receptor ◽  
Set Up ◽  
G Protein Coupled ◽  
Early Drug

CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context focusing on G-protein coupled receptors. A pooled mutagenesis screening framework was set up based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. The β2 Adrenergic Receptor (B2AR) was targeted in this way employing a 6xCRE-mCherry reporter system to monitor its response to isoproterenol. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology.

scholarly journals Degradation of 2,3,7,8-TCDD by a consortium of bacterial strains isolated from heavil herbicide/dioxin contaminated soil in Bienhoa airbase

2020 ◽  
Vol 16 (4) ◽  
pp. 777-784
Author(s):  
Pham Quang Huy ◽  
Nguyen Kim Thoa ◽  
Dang Thi Cam Ha
Keyword(s):  
Contaminated Soil ◽  
Large Scale ◽  
Morphological Characteristics ◽  
Bacterial Consortium ◽  
Soil Extract ◽  
High Ratio ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Agrobacterium Strain ◽  
Microbial Strains

From two different soil sources in Bienhoa airbase (heavy herbicide/dioxin contaminated West-South region and bioremediated cell), five microbial strains were isolated and their 2,3,7,8-TCDD biodegrability in consortium was investigated. Based on the colony and cell morphological characteristics as well as 16S rRNA gene sequences, these strains were classified into 5 genera, including Methylobacterium (strain BHBi1), Hydrocarboniphaga (strain BHBi4), Agrobacterium (strain BHBi5), Bosea (strain BHBi7) and Microbacterium (strain BH09). Two strains BHBi7 and BHBi4 were the first representatives of the genera Bosea and Hydrocarboniphaga that were isolated from heavyly herbicide/dioxin contaminated soil. All five strains were able to grow well in mineral salt medium (MSM) supplemented with soil extract (SE) containing 2,3,7,8-TCDD (this congener is the main soil total compound toxicity) and other congeners, including PCDDs, PCDFs, 2,4,5-T, 2,4-D, PAHs and their intermediates. This microbial consortium degraded 2,537.34 ngTEQ/kg of 2,3,7,8-TCDD congener in soil, equivalent to 59.1% lost of total toxicity in comparison to the control without bacterial seeding (4,294.12 ng TEQ/kg). Such a high ratio of dioxin degradation by a bacterial consortium was reported here for the first time, contributing more evidences for convincing the successful dioxin bioremediation of “Active Landfill” technology at large scale in Z1 area at Bienhoa airbase, Dongnai, Vietnam.

scholarly journals Phytochemical Screening, Antibacterial, Antifungal, Antiviral, Cytotoxic, and Anti-Quorum-Sensing Properties of Teucrium polium L. Aerial Parts Methanolic Extract

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1418
Author(s):  
Mousa Alreshidi ◽  
Emira Noumi ◽  
Lamjed Bouslama ◽  
Ozgur Ceylan ◽  
Vajid N. Veettil ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Methanolic Extract ◽  
High Resolution Mass Spectrometry ◽  
Antimicrobial Activities ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Concentration Dependent Manner ◽  
African Green Monkey Kidney ◽  
Teucrium Polium ◽  
Teucrium Polium L

The chemical profile of Teucrium polium L. (T. polium) methanolic extract was tested using liquid chromatography coupled with high resolution mass spectrometry (HR-LCMS). Disc diffusion and microdilution assays were used for the antimicrobial activities. Coxsackievirus B-3 (CVB3) and Herpes simplex virus type 2 (HSV-2) were used for the antiviral activities. Chromobacterium violaceum (ATCC 12472 and CV026) and Pseudomonas aeruginosa PAO1 were used as starter strains for the anti-quorum sensing tests. Isoprenoids are the main class of compounds identified, and 13R-hydroxy-9E,11Z-octadecadienoic acid, valtratum, rhoifolin, sericetin diacetate, and dihydrosamidin were the dominant phytoconstituents. The highest mean diameter of growth inhibition zone was recorded for Acinetobacter baumannii (19.33 ± 1.15 mm). The minimal inhibitory concentrations were ranging from 6.25 to 25 mg/mL for bacterial strains, and from 6.25 to 25 mg/mL for Candida species. The 50% cytotoxic concentration on VERO (African Green Monkey Kidney) cell lines was estimated at 209 µg/mL. No antiviral activity was recorded. Additionally, T. polium extract was able to inhibit P. aeruginosa PAO1 motility in a concentration-dependent manner. However, the tested extract was able to inhibit 23.66% of the swarming and 35.25% of swimming capacities of PAO1 at 100 µg/mL. These results highlighted the role of germander as a potent antimicrobial agent that can interfere with the virulence factors controlled by the quorum-sensing systems.

scholarly journals Isolation and Screening of Pectinolytic Bacterial Strains using Rotten Apples from Lahore, Pakistan

2019 ◽  
Vol 01 (03) ◽  
pp. 23-36
Author(s):  
Amna Yaqoob ◽  
Fatima Amanat ◽  
Asif Ali ◽  
Muhammad Sajjad
Keyword(s):  
Large Scale ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Scale Production ◽  
Bacterial Strains ◽  
Large Scale Production ◽  
Pectinolytic Activity ◽  
Juice Extraction ◽  
Rrna Gene Sequencing ◽  
Pectinase Production

Pectinases are pectin degrading enzymes predominantly used as biocatalysts in various industries such as wine extraction, fruit juice extraction, and making of paper pulp. Large scale production of pectinases using biological systems (bacteria, fungi, plants) is a common method used in the industry. In the current study, different bacterial isolates obtained from rotten apples were used for pectinase production and their pectinolytic activity was investigated. Five bacterial strains were isolated on the growth medium containing 0.3% KH2PO4, 0.6% Na2HPO4, 0.2% NH4Cl, 0.5% NaCl, 1% Pectin, 1.5% Agar, 1mM CaCl2, and 10mM MgSO4. The isolates of five samples A, B, C, D and E were then biochemically characterized as Serratia marcescens, Klebseilla pneumoniea, Pseudomonas aeruginosa and Escherichia coli, respectively. They were also identified at the molecular level through 16S rRNA gene sequencing.

scholarly journals Roadmap for the use of Base Editors to Decipher Drug Mechanism of Action

2020 ◽  
Author(s):  
Estel Aparicio Prat ◽  
Dong Yan ◽  
Marco Mariotti ◽  
Michael Bassik ◽  
Gaelen Hess ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Mechanism Of Action ◽  
Large Scale ◽  
Reporter System ◽  
Experimental Conditions ◽  
Base Editing ◽  
Drug Mechanism ◽  
Set Up ◽  
G Protein Coupled ◽  
Early Drug

Abstract Background: CRISPR base editors are powerful tools for large-scale mutagenesis studies. This kind of approach can elucidate the mechanism of action of compounds, a key process in drug discovery. Here, we explore the utility of base editors in an early drug discovery context, and we focus on G-protein coupled receptors.Results: We set up a pooled mutagenesis screening framework based on a modified version of the CRISPR-X base editor system. We determine optimized experimental conditions for mutagenesis where sgRNAs are delivered by cell transfection or viral infection over extended time periods (>14 days), resulting in high mutagenesis produced in a short region located at -4/+8 nucleotides with respect to the sgRNA match. We thus target the Beta 2 Adrenergic Receptor (B2AR) and employ a 6xCRE-mCherry reporter system to monitor its activity. The results of our screening indicate that residue 184 of B2AR is crucial for its activation. Based on our experience, we then outline the crucial points to consider when designing and performing CRISPR-based pooled mutagenesis screening, including the typical technical hurdles encountered when studying compound pharmacology. Conclusions: The base editing technology has a great potential to help deciphering the mechanism of action of drugs, and it is a very powerful tool in drug discovery. Here we show an application of pooled mutagenesis screening to study B2AR, and we provide a roadmap for successfully applying this approach to other target proteins.

scholarly journals Isolation of Polygalacturonase-Producing Bacterial Strain from Tomatoes (Lycopersicon esculentum Mill.)

2019 ◽  
Vol 2019 ◽  
pp. 1-9
Author(s):  
Yemisi Dorcas Obafemi ◽  
Adesola Adetutu Ajayi ◽  
Olugbenga Samson Taiwo ◽  
Shade John Olorunsola ◽  
Patrick Omoregie Isibor
Keyword(s):  
Lycopersicon Esculentum ◽  
Bacterial Strain ◽  
Large Scale ◽  
Maximum Yield ◽  
Precipitation Method ◽  
Molecular Characteristics ◽  
Rrna Gene ◽  
Scale Production ◽  
Optimum Conditions ◽  
Bacterial Strains

Background. Polygalacturonase (EC 3.2.1.15) enzyme aids in microbial spoilage of fruits and vegetables. It is very important to find economical ways to producing the enzyme so as to achieve maximum yield in industries due to its use at different areas of production process. Methods. Isolation of polygalacturonase-producing bacterial strain from tomatoes (Lycopersicon esculentum Mill.) was studied. Polygalacturonase-producing bacterial strains were isolated and screened from tomatoes stored at normal laboratory temperature (25 ± 2°C). They were identified based on their morphological, biochemical, and molecular characteristics. The enzyme produced was partially purified by the ammonium sulphate precipitation method. Molecular weights and optimum conditions for best enzyme activity were obtained by SDS PAGE technique. Results. Five bacterial isolates resulted after screening. Bacterial strain code B5 showed highest polygalacturonase activity. Optimum conditions for polygalacturonase PEC B5 were maintained at pH 4.5; temperature 35°C; substrate concentration 0.3 mg/ml, and best activity at less than 5 min of heating. The enzyme PEC B5 was found to weigh 65 kDa and 50 kDa for crude and partially purified aliquots, respectively. The result of 16S rRNA gene sequencing revealed bacterial strain code B5 as Enterobacter tabaci NR146667 having 79% similarity with the NCBI GenBank. Conclusion. Microorganisms should be developed for large-scale production of enzymes in developing countries.

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