Genome Mining of Antimicrobial Gene Clusters from Phyllospheric Bacteria Isolated from Solanum Lycopersicum and Lactuca Sativa to Identify Biocontrol Properties

Abstract BackgroundBiocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from the tomato and lettuce phyllosphere that can promote plant growth and antagonize mammalian pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), several of which are good candidates to produce protectants against microbial plant and mammalian pathogens.Results The antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and mammalian pathogens was determined with plates assays. Three strains with the highest antimicrobial activity against the relevant pathogens were selected and characterized (Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillus sp. PL91). All three strains showed a plant growth promotion effect by the production of volatile compounds (VOCs) on tomato and lettuce. In addition, genome mining of these isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 39 BGCs were identified, of which several are already known, such as bacilysin, bacillibactin, surfactin, subtilomycin, etc., but also several novel ones. Further analysis revealed that among the novel BGCs, one NRPS and two bacteriocins are encoded which were analyzed in more depth.Conclusions Several antimicrobial BGCs were found in the selected strains, including the rediscovery of known ones, but also the discovery of novel ones. Our study serves as support for subsequent examination and characterization of novel antimicrobial metabolites, and the possibility of developing biocontrol agents.

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A Community Effort: Combining Functional Amplicon Sequencing and Metagenomics Reveals Potential Biosynthetic Gene Clusters Associated with Protective Phenotypes in Rhizosphere Microbiomes

mSystems ◽

10.1128/msystems.00587-21 ◽

2021 ◽

Author(s):

Jaclyn M. Winter

Keyword(s):

Plant Growth ◽

Plant Pathogens ◽

Gene Clusters ◽

Amplicon Sequencing ◽

Plant Roots ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

First Line ◽

Community Effort ◽

Promote Plant Growth

Soil-dwelling microorganisms associated with plant roots carry out essential processes that promote plant growth and productivity. In addition to these beneficial functions, the rhizosphere microbiome also serves as the first line of defense against many plant pathogens.

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Genetic Potential of the Biocontrol Agent Pseudomonas brassicacearum (Formerly P. trivialis) 3Re2-7 Unraveled by Genome Sequencing and Mining, Comparative Genomics and Transcriptomics

Genes ◽

10.3390/genes10080601 ◽

2019 ◽

Vol 10 (8) ◽

pp. 601 ◽

Cited By ~ 4

Author(s):

Johanna Nelkner ◽

Gonzalo Torres Tejerizo ◽

Julia Hassa ◽

Timo Wentong Lin ◽

Julian Witte ◽

...

Keyword(s):

Comparative Genomics ◽

Plant Growth ◽

Plant Growth Promotion ◽

Growth Promotion ◽

Genome Mining ◽

Gene Clusters ◽

Disease Suppression ◽

Biocontrol Agents ◽

Circular Chromosome ◽

Pseudomonas Brassicacearum

The genus Pseudomonas comprises many known plant-associated microbes with plant growth promotion and disease suppression properties. Genome-based studies allow the prediction of the underlying mechanisms using genome mining tools and the analysis of the genes unique for a strain by implementing comparative genomics. Here, we provide the genome sequence of the strain Pseudomonas brassicacearum 3Re2-7, formerly known as P. trivialis and P. reactans, elucidate its revised taxonomic classification, experimentally verify the gene predictions by transcriptome sequencing, describe its genetic biocontrol potential and contextualize it to other known Pseudomonas biocontrol agents. The P. brassicacearum 3Re2-7 genome comprises a circular chromosome with a size of 6,738,544 bp and a GC-content of 60.83%. 6267 genes were annotated, of which 6113 were shown to be transcribed in rich medium and/or in the presence of Rhizoctonia solani. Genome mining identified genes related to biocontrol traits such as secondary metabolite and siderophore biosynthesis, plant growth promotion, inorganic phosphate solubilization, biosynthesis of lipo- and exopolysaccharides, exoproteases, volatiles and detoxification. Core genome analysis revealed, that the 3Re2-7 genome exhibits a high collinearity with the representative genome for the species, P. brassicacearum subsp. brassicacearum NFM421. Comparative genomics allowed the identification of 105 specific genes and revealed gene clusters that might encode specialized biocontrol mechanisms of strain 3Re2-7. Moreover, we captured the transcriptome of P. brassicacearum 3Re2-7, confirming the transcription of the predicted biocontrol-related genes. The gene clusters coding for 2,4-diacetylphloroglucinol (phlABCDEFGH) and hydrogen cyanide (hcnABC) were shown to be highly transcribed. Further genes predicted to encode putative alginate production enzymes, a pyrroloquinoline quinone precursor peptide PqqA and a matrixin family metalloprotease were also found to be highly transcribed. With this study, we provide a basis to further characterize the mechanisms for biocontrol in Pseudomonas species, towards a sustainable and safe application of P. brassicacearum biocontrol agents.

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Expanding the Natural Products Heterologous Expression Repertoire in the Model Cyanobacterium Anabaena sp. Strain PCC 7120: Production of Pendolmycin and Teleocidin B-4

10.26434/chemrxiv.11316098.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Patrick Videau ◽

Kaitlyn Wells ◽

Arun Singh ◽

Jessie Eiting ◽

Philip Proteau ◽

...

Keyword(s):

Natural Products ◽

Genome Mining ◽

Gene Clusters ◽

Combinatorial Biosynthesis ◽

Test Case ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Cyanobacterium Anabaena ◽

Anabaena Sp ◽

Pcc 7120

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of <i>Anabaena </i>sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native <i>Anabaena</i>7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

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Perception of Biocontrol Potential of Bacillusinaquosorum KR2-7 against Tomato Fusarium Wilt through Merging Genome Mining with Chemical Analysis

Biology ◽

10.3390/biology11010137 ◽

2022 ◽

Vol 11 (1) ◽

pp. 137

Author(s):

Maedeh Kamali ◽

Dianjing Guo ◽

Shahram Naeimi ◽

Jafar Ahmadi

Keyword(s):

Chemical Analysis ◽

Plant Growth ◽

Fusarium Wilt ◽

Plant Growth Promotion ◽

Growth Promotion ◽

Genome Mining ◽

Gene Clusters ◽

Systemic Resistance ◽

Plant Colonization ◽

Biocontrol Potential

Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Fol), is a destructive disease that threatens the agricultural production of tomatoes. In the present study, the biocontrol potential of strain KR2-7 against Fol was investigated through integrated genome mining and chemical analysis. Strain KR2-7 was identified as B. inaquosorum based on phylogenetic analysis. Through the genome mining of strain KR2-7, we identified nine antifungal and antibacterial compound biosynthetic gene clusters (BGCs) including fengycin, surfactin and Bacillomycin F, bacillaene, macrolactin, sporulation killing factor (skf), subtilosin A, bacilysin, and bacillibactin. The corresponding compounds were confirmed through MALDI-TOF-MS chemical analysis. The gene/gene clusters involved in plant colonization, plant growth promotion, and induced systemic resistance were also identified in the KR2-7 genome, and their related secondary metabolites were detected. In light of these results, the biocontrol potential of strain KR2-7 against tomato Fusarium wilt was identified. This study highlights the potential to use strain KR2-7 as a plant-growth promotion agent.

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Discovery of Unusual Cyanobacterial Tryptophan-containing Anabaenopeptins by MS/MS Based Molecular Networking

10.20944/preprints202007.0562.v1 ◽

2020 ◽

Author(s):

Subhasish Saha ◽

Germana Esposito ◽

Petra Urajova ◽

Jan Mareš ◽

Daniela Ewe ◽

...

Keyword(s):

Multidisciplinary Approach ◽

Spectroscopic Analysis ◽

Chemical Space ◽

Genome Mining ◽

Gc Content ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Hela Cell Lines ◽

Bioactive Secondary Metabolites

Heterocytous cyanobacteria are among the most prolific source of bioactive secondary metabolites, including anabaenopeptins (APTs). A terrestrial filamentous Brasilonema sp. CT11 collected in Costa Rica bamboo forest, as black mat was studied using a multidisciplinary approach: genome mining and HPLC-HRMS/MS coupled with bionformatic analyses. Herein, we report the nearly complete genome consisting 8.79 Mbp with a GC content of 42.4%. Moreover, we report on three novel tryptophane-containing APTs; anabaenopeptin 788 (1), anabaenopeptin 802 (2) and anabaenopeptin 816 (3). Further, the structure of two homologues, i.e., anabaenopeptin 802 (2a) and anabaenopeptin 802 (2b) was determined by spectroscopic analysis (NMR and MS). Both compounds were shown to exert weak to moderate antiproliferative activity against HeLa cell lines. This study also provides the unique and diverse potential of biosynthetic gene clusters and an assessment of the predicted chemical space yet to be discovered from this genus.

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An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

Antibiotics ◽

10.3390/antibiotics9080494 ◽

2020 ◽

Vol 9 (8) ◽

pp. 494

Author(s):

Lena Mitousis ◽

Yvonne Thoma ◽

Ewa M. Musiol-Kroll

Keyword(s):

Genetic Engineering ◽

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Molecular Tools ◽

Biosynthetic Gene Clusters ◽

Streptomyces Antibioticus ◽

The Past ◽

Bioactive Agents ◽

Valuable Compounds

The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.

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A Comparative Analysis of Biosynthetic Gene Clusters in Lean and Obese Humans

BioMed Research International ◽

10.1155/2019/6361320 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Shengqin Wang ◽

Na Li ◽

Nan Li ◽

Huixi Zou ◽

Mingjiang Wu

Keyword(s):

Gut Microbiome ◽

Gene Clusters ◽

Human Adipose Tissue ◽

Taxonomic Diversity ◽

Metagenomic Data ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Intestinal Microbes ◽

Microbe Interactions ◽

Treatment Of Obesity

Obesity is intrinsically linked with the gut microbiome, and studies have identified several obesity-associated microbes. The microbe-microbe interactions can alter the composition of the microbial community and influence host health by producing secondary metabolites (SMs). However, the contribution of these SMs in the prevention and treatment of obesity has been largely ignored. We identified several SM-encoding biosynthetic gene clusters (BGCs) from the metagenomic data of lean and obese individuals and found significant association between some BGCs, including those that produce hitherto unknown SM, and obesity. In addition, the mean abundance of BGCs was positively correlated with obesity, consistent with the lower taxonomic diversity in the gut microbiota of obese individuals. By comparing the BGCs of known SM between obese and nonobese samples, we found that menaquinone produced by Enterobacter cloacae showed the highest correlation with BMI, in agreement with a recent study on human adipose tissue composition. Furthermore, an obesity-related nonribosomal peptide synthetase (NRPS) was negatively associated with Bacteroidetes, indicating that the SMs produced by intestinal microbes in obese individuals can change the microbiome structure. This is the first systemic study of the association between gut microbiome BGCs and obesity and provides new insights into the causes of obesity.

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Genomic Assemblies of Members of Burkholderia and Related Genera as a Resource for Natural Product Discovery

Microbiology Resource Announcements ◽

10.1128/mra.00485-20 ◽

2020 ◽

Vol 9 (42) ◽

Author(s):

Alex J. Mullins ◽

Cerith Jones ◽

Matthew J. Bull ◽

Gordon Webster ◽

Julian Parkhill ◽

...

Keyword(s):

Natural Product ◽

Genome Mining ◽

Genomic Analysis ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Natural Product Discovery

ABSTRACT The genomes of 450 members of Burkholderiaceae, isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

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The Significance of Bacillus spp. in Disease Suppression and Growth Promotion of Field and Vegetable Crops

Microorganisms ◽

10.3390/microorganisms8071037 ◽

2020 ◽

Vol 8 (7) ◽

pp. 1037 ◽

Cited By ~ 1

Author(s):

Dragana Miljaković ◽

Jelena Marinković ◽

Svetlana Balešević-Tubić

Keyword(s):

Plant Growth ◽

Plant Pathogens ◽

Growth Promotion ◽

Disease Suppression ◽

Biocontrol Agents ◽

Systemic Resistance ◽

Steady Increase ◽

Vegetable Crops ◽

Wide Range ◽

Bacillus Spp

Bacillus spp. produce a variety of compounds involved in the biocontrol of plant pathogens and promotion of plant growth, which makes them potential candidates for most agricultural and biotechnological applications. Bacilli exhibit antagonistic activity by excreting extracellular metabolites such as antibiotics, cell wall hydrolases, and siderophores. Additionally, Bacillus spp. improve plant response to pathogen attack by triggering induced systemic resistance (ISR). Besides being the most promising biocontrol agents, Bacillus spp. promote plant growth via nitrogen fixation, phosphate solubilization, and phytohormone production. Antagonistic and plant growth-promoting strains of Bacillus spp. might be useful in formulating new preparations. Numerous studies of a wide range of plant species revealed a steady increase in the number of Bacillus spp. identified as potential biocontrol agents and plant growth promoters. Among different mechanisms of action, it remains unclear which individual or combined traits could be used as predictors in the selection of the best strains for crop productivity improvement. Due to numerous factors that influence the successful application of Bacillus spp., it is necessary to understand how different strains function in biological control and plant growth promotion, and distinctly define the factors that contribute to their more efficient use in the field.

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Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903161116 ◽

2019 ◽

Vol 116 (40) ◽

pp. 19805-19814 ◽

Cited By ~ 8

Author(s):

Zachary L. Reitz ◽

Clifford D. Hardy ◽

Jaewon Suk ◽

Jean Bouvet ◽

Alison Butler

Keyword(s):

Predictive Power ◽

Genome Mining ◽

Genomic Analysis ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Peptide Synthetase ◽

Condensation Domain

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization—existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)—and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate–dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.

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