Publications by authors named "Chunxu Song"

25 Publications

  • Page 1 of 1

Designing a home for beneficial plant microbiomes.

Curr Opin Plant Biol 2021 Mar 5;62:102025. Epub 2021 Mar 5.

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, Netherlands; Institute of Biology, Leiden University, Leiden, Netherlands.

The plant microbiome comprises a highly diverse community of saprotrophic, mutualistic, and pathogenic microbes that can affect plant growth and plant health. There is substantial interest to exploit beneficial members of plant microbiomes for new sustainable management strategies in crop production. However, poor survival and colonization of plant tissues by introduced microbial isolates as well as lack of expression of the plant growth-promoting or disease-suppressive traits at the right time and place are still major limitations for successful implementation of microbiomes in future agricultural practices and plant breeding programs. Similar to building a home for humans, we discuss different strategies of building a home for beneficial plant microbiomes, here referred to as the 'MicrobiHome'.
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http://dx.doi.org/10.1016/j.pbi.2021.102025DOI Listing
March 2021

Antimicrobial activity screening of rhizosphere soil bacteria from tomato and genome-based analysis of their antimicrobial biosynthetic potential.

BMC Genomics 2021 Jan 7;22(1):29. Epub 2021 Jan 7.

Department of Molecular Genetics, University of Groningen, Groningen, The Netherlands.

Background: Tomato plant growth is frequently hampered by a high susceptibility to pests and diseases. Traditional chemical control causes a serious impact on both the environment and human health. Therefore, seeking environment-friendly and cost-effective green methods in agricultural production becomes crucial nowadays. Plant Growth Promoting Rhizobacteria (PGPR) can promote plant growth through biological activity. Their use is considered to be a promising sustainable approach for crop growth. Moreover, a vast number of biosynthetic gene clusters (BGCs) for secondary metabolite production are being revealed in PGPR, which helps to find potential anti-microbial activities for tomato disease control.

Results: We isolated 181 Bacillus-like strains from healthy tomato, rhizosphere soil, and tomato tissues. In vitro antagonistic assays revealed that 34 Bacillus strains have antimicrobial activity against Erwinia carotovora, Pseudomonas syringae; Rhizoctonia solani; Botrytis cinerea; Verticillium dahliae and Phytophthora infestans. The genomes of 10 Bacillus and Paenibacillus strains with good antagonistic activity were sequenced. Via genome mining approaches, we identified 120 BGCs encoding NRPs, PKs-NRPs, PKs, terpenes and bacteriocins, including known compounds such as fengycin, surfactin, bacillibactin, subtilin, etc. In addition, several novel BGCs were identified. We discovered that the NRPs and PKs-NRPs BGCs in Bacillus species are encoding highly conserved known compounds as well as various novel variants.

Conclusions: This study highlights the great number of varieties of BGCs in Bacillus strains. These findings pave the road for future usage of Bacillus strains as biocontrol agents for tomato disease control and are a resource arsenal for novel antimicrobial discovery.
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http://dx.doi.org/10.1186/s12864-020-07346-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789753PMC
January 2021

Novel Modifications of Nonribosomal Peptides from Brevibacillus laterosporus MG64 and Investigation of Their Mode of Action.

Appl Environ Microbiol 2020 11 24;86(24). Epub 2020 Nov 24.

Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands

Nonribosomal peptides (NRPs) are a class of secondary metabolites usually produced by microorganisms. They are of paramount importance in different applications, including biocontrol and pharmacy. spp. are a rich source of NRPs yet have received little attention. In this study, we characterize four novel bogorol variants (bogorols I to L, cationic linear lipopeptides) and four succilins (succilins I to L, containing a succinyl group that is attached to the Orn/Lys in bogorols I to L) from the biocontrol strain MG64. Further investigation revealed that the bogorol family of peptides employs an adenylation pathway for lipoinitiation, different from the usual pattern, which is based on an external ligase and coenzyme A. Moreover, the formation of valinol was proven to be mediated by a terminal reductase domain and a reductase encoded by the gene. Furthermore, succinylation, which is a novel type of modification in the family of bogorols, was discovered. Its occurrence requires a high concentration of the substrate (bogorols), but its responsible enzyme remains unknown. Bogorols display potent activity against both Gram-positive and Gram-negative bacteria. Investigation of their mode of action reveals that bogorols form pores in the cell membrane of both Gram-positive and Gram-negative bacteria. The combination of bogorols and relacidines, another class of NRPs produced by MG64, displays a synergistic effect on different pathogens, suggesting the great potential of both peptides as well as their producer MG64 for broad applications. Our study provides a further understanding of the bogorol family of peptides as well as their applications. NRPs form a class of secondary metabolites with biocontrol and pharmaceutical potential. This work describes the identification of novel bogorol variants and succinylated bogorols (namely, succilins) and further investigates their biosynthetic pathway and mode of action. Adenylation domain-mediated lipoinitiation of bogorols represents a novel pathway by which NRPs incorporate fatty acid tails. This pathway provides the possibility to engineer the lipid tail of NRPs without identifying a fatty acid coenzyme ligase, which is usually not present in the biosynthetic gene cluster. The terminal reductase domain (TD) and BogI-mediated valinol formation and their effect on the biological activity of bogorols are revealed. Succinylation, which is rarely reported in NRPs, was discovered in the bogorol family of peptides. We demonstrate that bogorols combat bacterial pathogens by forming pores in the cell membrane. We also report the synergistic effect of two natural products (relacidine B and bogorol K) produced by the same strain, which is relevant for competition for a niche.
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http://dx.doi.org/10.1128/AEM.01981-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688214PMC
November 2020

Beyond Plant Microbiome Composition: Exploiting Microbial Functions and Plant Traits via Integrated Approaches.

Front Bioeng Biotechnol 2020 7;8:896. Epub 2020 Aug 7.

Institute of Biology, Leiden University, Leiden, Netherlands.

Plants recruit specific microorganisms to live inside and outside their roots that provide essential functions for plant growth and health. The study of the microbial communities living in close association with plants helps in understanding the mechanisms involved in these beneficial interactions. Currently, most of the research in this field has been focusing on the description of the taxonomic composition of the microbiome. Therefore, a focus on the plant-associated microbiome functions is pivotal for the development of novel agricultural practices which, in turn, will increase plant fitness. Recent advances in microbiome research using model plant species started to shed light on the functions of specific microorganisms and the underlying mechanisms of plant-microbial interaction. Here, we review (1) microbiome-mediated functions associated with plant growth and protection, (2) insights from native and agricultural habitats that can be used to improve soil health and crop productivity, (3) current -omics and new approaches for studying the plant microbiome, and (4) challenges and future perspectives for exploiting the plant microbiome for beneficial outcomes. We posit that integrated approaches will help in translating fundamental knowledge into agricultural practices.
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http://dx.doi.org/10.3389/fbioe.2020.00896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426627PMC
August 2020

Characterization of two relacidines belonging to a novel class of circular lipopeptides that act against Gram-negative bacterial pathogens.

Environ Microbiol 2020 12 20;22(12):5125-5136. Epub 2020 Jul 20.

Department of Molecular Genetics, University of Groningen, Groningen, the Netherlands.

The development of sustainable agriculture and the increasing antibiotic resistance of human pathogens call for novel antimicrobial compounds. Here, we describe the extraction and characterization of a class of cationic circular lipopeptides, for which we propose the name relacidines, from the soil bacterium Brevibacillus laterosporus MG64. Relacidines are composed of a fatty acid side chain (4-methylhexanoic acid) and 13 amino acid residues. A lactone ring is formed by the last five amino acid residues and three positively charged ornithines are located in the linear fragment. Relacidines selectively combat Gram-negative pathogens, including phytopathogens and human pathogens. Further investigation of the mode of action revealed that relacidine B binds to the lipopolysaccharides but does not form pores in the cell membrane. We also provide proof to show that relacidine B does not affect the biosynthesis of the cell wall and RNA. Instead, it affects the oxidative phosphorylation process of cells and diminishes the biosynthesis of ATP. Transcription of relacidines is induced by plant pathogens, which strengthens the potential of B. laterosporus MG64 to be used as a biocontrol agent. Thus, we identified a new group of potent antibiotic compounds for combating Gram-negative pathogens of plants or animals.
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http://dx.doi.org/10.1111/1462-2920.15145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818431PMC
December 2020

Characterization of plant growth-promoting rhizobacteria from perennial ryegrass and genome mining of novel antimicrobial gene clusters.

BMC Genomics 2020 Feb 12;21(1):157. Epub 2020 Feb 12.

Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.

Background: Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol.

Results: We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins.

Conclusion: Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens.
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http://dx.doi.org/10.1186/s12864-020-6563-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017464PMC
February 2020

Draft Genome Sequences of 10 Paenibacillus and Bacillus sp. Strains Isolated from Healthy Tomato Plants and Rhizosphere Soil.

Microbiol Resour Announc 2019 Mar 21;8(12). Epub 2019 Mar 21.

Department of Molecular Genetics, University of Groningen, Groningen, the Netherlands

In order to investigate the underlying interaction mechanisms between plants and Gram-positive bacteria, 10 and strains were isolated from healthy tomato rhizosphere and plant tissues.
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http://dx.doi.org/10.1128/MRA.00055-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430313PMC
March 2019

Draft Genome Sequences of Six Strains and One Strain Isolated from the Rhizosphere of Perennial Ryegrass (Lolium perenne).

Microbiol Resour Announc 2019 Jan 24;8(4). Epub 2019 Jan 24.

Department of Molecular Genetics, University of Groningen, Groningen, the Netherlands.

Seven strains of endospore-forming bacteria with antagonistic activity against a series of plant pathogens were sequenced in order to investigate their antimicrobial gene clusters and antimicrobial modes of action. The selected strains include six strains and one strain.
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http://dx.doi.org/10.1128/MRA.01586-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346203PMC
January 2019

The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi.

Microb Biotechnol 2019 05 7;12(3):459-471. Epub 2019 Jan 7.

Laboratory of Animal Ecology and Entomology, Institute of Zoology, University of Neuchâtel, CP 2, CH-2007, Neuchâtel, Switzerland.

Soil-dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN-bacteria-arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non-volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non-volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A. faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens.
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http://dx.doi.org/10.1111/1751-7915.13365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465237PMC
May 2019

Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system.

Environ Microbiol 2018 12 26;20(12):4245-4260. Epub 2018 Aug 26.

Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.

Bacillus subtilis HS3 and Bacillus mycoides EC18 are two rhizosphere-associated bacteria with plant growth-promoting activity. The CRISPR-Cas9 system was implemented to study various aspects of plant-microbe interaction mechanisms of these two environmental isolates. The results show that fengycin and surfactin are involved in the antifungal activity of B. subtilis HS3. Moreover, this strain emits several other volatile organic compounds than 2,3-butanediol, contributing to plant growth promotion. Confocal laser scanning microscopy observations of the GFP-labelled strain showed that HS3 selectively colonizes root hairs of grass (Lolium perenne) in a hydroponic system. For B. mycoides EC18, we found that the wild-type EC18 strain and a ΔasbA (petropectin-deficient) mutant, but not the ΔdhbB (bacillibactin-deficient) and ADKO (asbA and dhbB double knockout) mutants, can increase the plant biomass and total chlorophyll. All the mutant strains have a reduced colonization capability on Chinese cabbage (Brassica rapa) roots, at the root tip and root hair region compared with the wild-type strain. These results indicate that the siderophore, bacillibactin, is involved in the plant growth promoting activity and could affect the root colonization of B. mycoides. Collectively, the CRISPR-Cas9 system we developed for environmental isolates is broadly applicable and will facilitate deciphering the mechanisms of Bacillus-plant interactions. © 2018 The Authors.
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http://dx.doi.org/10.1111/1462-2920.14305DOI Listing
December 2018

BAGEL4: a user-friendly web server to thoroughly mine RiPPs and bacteriocins.

Nucleic Acids Res 2018 07;46(W1):W278-W281

Molecular Genetics, GBB, University of Groningen, Groningen, 9747AG, the Netherlands.

Interest in secondary metabolites such as RiPPs (ribosomally synthesized and posttranslationally modified peptides) is increasing worldwide. To facilitate the research in this field we have updated our mining web server. BAGEL4 is faster than its predecessor and is now fully independent from ORF-calling. Gene clusters of interest are discovered using the core-peptide database and/or through HMM motifs that are present in associated context genes. The databases used for mining have been updated and extended with literature references and links to UniProt and NCBI. Additionally, we have included automated promoter and terminator prediction and the option to upload RNA expression data, which can be displayed along with the identified clusters. Further improvements include the annotation of the context genes, which is now based on a fast blast against the prokaryote part of the UniRef90 database, and the improved web-BLAST feature that dynamically loads structural data such as internal cross-linking from UniProt. Overall BAGEL4 provides the user with more information through a user-friendly web-interface which simplifies data evaluation. BAGEL4 is freely accessible at http://bagel4.molgenrug.nl.
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http://dx.doi.org/10.1093/nar/gky383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030817PMC
July 2018

Secondary Metabolism and Interspecific Competition Affect Accumulation of Spontaneous Mutants in the GacS-GacA Regulatory System in .

mBio 2018 01 16;9(1). Epub 2018 Jan 16.

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, USA

Secondary metabolites are synthesized by many microorganisms and provide a fitness benefit in the presence of competitors and predators. Secondary metabolism also can be costly, as it shunts energy and intermediates from primary metabolism. In spp., secondary metabolism is controlled by the GacS-GacA global regulatory system. Intriguingly, spontaneous mutations in or (Gac mutants) are commonly observed in laboratory cultures. Here we investigated the role of secondary metabolism in the accumulation of Gac mutants in strain Pf-5. Our results showed that secondary metabolism, specifically biosynthesis of the antimicrobial compound pyoluteorin, contributes significantly to the accumulation of Gac mutants. Pyoluteorin biosynthesis, which poses a metabolic burden on the producer cells, but not pyoluteorin itself, leads to the accumulation of the spontaneous mutants. Interspecific competition also influenced the accumulation of the Gac mutants: a reduced proportion of Gac mutants accumulated when Pf-5 was cocultured with than in pure cultures of strain Pf-5. Overall, our study associated a fitness trade-off with secondary metabolism, with metabolic costs versus competitive benefits of production influencing the evolution of , assessed by the accumulation of Gac mutants. Many microorganisms produce antibiotics, which contribute to ecologic fitness in natural environments where microbes constantly compete for resources with other organisms. However, biosynthesis of antibiotics is costly due to the metabolic burdens of the antibiotic-producing microorganism. Our results provide an example of the fitness trade-off associated with antibiotic production. Under noncompetitive conditions, antibiotic biosynthesis led to accumulation of spontaneous mutants lacking a master regulator of antibiotic production. However, relatively few of these spontaneous mutants accumulated when a competitor was present. Results from this work provide information on the evolution of antibiotic biosynthesis and provide a framework for their discovery and regulation.
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http://dx.doi.org/10.1128/mBio.01845-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770548PMC
January 2018

The Ecological Role of Volatile and Soluble Secondary Metabolites Produced by Soil Bacteria.

Trends Microbiol 2017 04 27;25(4):280-292. Epub 2016 Dec 27.

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), PO Box 50, 6700 AB, Wageningen, The Netherlands. Electronic address:

The rich diversity of secondary metabolites produced by soil bacteria has been appreciated for over a century, and advances in chemical analysis and genome sequencing continue to greatly advance our understanding of this biochemical complexity. However, we are just at the beginning of understanding the physicochemical properties of bacterial metabolites, the factors that govern their production and ecological roles. Interspecific interactions and competitor sensing are among the main biotic factors affecting the production of bacterial secondary metabolites. Many soil bacteria produce both volatile and soluble compounds. In contrast to soluble compounds, volatile organic compounds can diffuse easily through air- and gas-filled pores in the soil and likely play an important role in long-distance microbial interactions. In this review we provide an overview of the most important soluble and volatile classes of secondary metabolites produced by soil bacteria, their ecological roles, and their possible synergistic effects.
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http://dx.doi.org/10.1016/j.tim.2016.12.002DOI Listing
April 2017

The prey's scent - Volatile organic compound mediated interactions between soil bacteria and their protist predators.

ISME J 2017 03 2;11(3):817-820. Epub 2016 Dec 2.

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, Netherlands.

Protists are major predators of bacteria in soils. However, it remains unknown how protists sense their prey in this highly complex environment. Here, we investigated whether volatile organic compounds (VOCs) of six phylogenetic distinct soil bacteria affect the performance of three different soil protists and how that relates to direct feeding interactions. We observed that most bacteria affected protist activity by VOCs. However, the response of protists to the VOCs was strongly dependent on both the bacterial and protist interacting partner. Stimulation of protist activity by volatiles and in direct trophic interaction assays often coincided, suggesting that VOCs serve as signals for protists to sense suitable prey. Furthermore, bacterial terpene synthase mutants lost the ability to affect protists, indicating that terpenes represent key components of VOC-mediated communication. Overall, we demonstrate that volatiles are directly involved in protist-bacterial predator-prey interactions.
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http://dx.doi.org/10.1038/ismej.2016.144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322296PMC
March 2017

Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility.

Environ Microbiol 2016 10 28;18(10):3453-3465. Epub 2016 Apr 28.

Department of Microbial Ecology, Netherlands Institute of Ecology, Droevendaalsesteeg 10, 6708, PB, Wageningen, The Netherlands.

Swarming motility is a flagella-driven multicellular behaviour that allows bacteria to colonize new niches and escape competition. Here, we investigated the evolution of specific mutations in the GacS/GacA two-component regulatory system in swarming colonies of Pseudomonas protegens Pf-5. Experimental evolution assays showed that repeated rounds of swarming by wildtype Pf-5 drives the accumulation of gacS/gacA spontaneous mutants on the swarming edge. These mutants cannot swarm on their own because they lack production of the biosurfactant orfamide A, but they do co-swarm with orfamide-producing wildtype Pf-5. These co-swarming assays further demonstrated that ΔgacA mutant cells indeed predominate on the edge and that initial ΔgacA:wildtype Pf-5 ratios of at least 2:1 lead to a collapse of the swarming colony. Subsequent whole-genome transcriptome analyses revealed that genes associated with motility, resource acquisition, chemotaxis and efflux were significantly upregulated in ΔgacA mutant on swarming medium. Moreover, transmission electron microscopy showed that ΔgacA mutant cells were longer and more flagellated than wildtype cells, which may explain their predominance on the swarming edge. We postulate that adaptive evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these mutations during dispersal of bacteria into new territories are frequency-dependent.
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http://dx.doi.org/10.1111/1462-2920.13288DOI Listing
October 2016

Exploring the genomic traits of fungus-feeding bacterial genus Collimonas.

BMC Genomics 2015 Dec 24;16:1103. Epub 2015 Dec 24.

Netherlands Institute of Ecology, Department of Microbial Ecology, Droevendaalsesteeg 10, Wageningen, 6708 PB, The Netherlands.

Background: Collimonas is a genus belonging to the class of Betaproteobacteria and consists mostly of soil bacteria with the ability to exploit living fungi as food source (mycophagy). Collimonas strains differ in a range of activities, including swimming motility, quorum sensing, extracellular protease activity, siderophore production, and antimicrobial activities.

Results: In order to reveal ecological traits possibly related to Collimonas lifestyle and secondary metabolites production, we performed a comparative genomics analysis based on whole-genome sequencing of six strains representing 3 recognized species. The analysis revealed that the core genome represents 43.1 to 52.7% of the genomes of the six individual strains. These include genes coding for extracellular enzymes (chitinase, peptidase, phospholipase), iron acquisition and type II secretion systems. In the variable genome, differences were found in genes coding for secondary metabolites (e.g. tripropeptin A and volatile terpenes), several unknown orphan polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), nonribosomal peptide synthetase (NRPS) gene clusters, a new lipopeptide and type III and type VI secretion systems. Potential roles of the latter genes in the interaction with other organisms were investigated. Mutation of a gene involved in tripropeptin A biosynthesis strongly reduced the antibacterial activity against Staphylococcus aureus, while disruption of a gene involved in the biosynthesis of the new lipopeptide had a large effect on the antifungal/oomycetal activities.

Conclusions: Overall our results indicated that Collimonas genomes harbour many genes encoding for novel enzymes and secondary metabolites (including terpenes) important for interactions with other organisms and revealed genomic plasticity, which reflect the behaviour, antimicrobial activity and lifestylesof Collimonas spp.
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http://dx.doi.org/10.1186/s12864-015-2289-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690342PMC
December 2015

Molecular and chemical dialogues in bacteria-protozoa interactions.

Sci Rep 2015 Aug 6;5:12837. Epub 2015 Aug 6.

1] Laboratory of Phytopathology, Wageningen University, 6708 PB Wageningen, the Netherlands [2] Microbial Ecology Department, Netherlands Institute of Ecology (NIOO-KNAW), 6708 PB Wageningen, the Netherlands.

Protozoan predation of bacteria can significantly affect soil microbial community composition and ecosystem functioning. Bacteria possess diverse defense strategies to resist or evade protozoan predation. For soil-dwelling Pseudomonas species, several secondary metabolites were proposed to provide protection against different protozoan genera. By combining whole-genome transcriptome analyses with (live) imaging mass spectrometry (IMS), we observed multiple changes in the molecular and chemical dialogues between Pseudomonas fluorescens and the protist Naegleria americana. Lipopeptide (LP) biosynthesis was induced in Pseudomonas upon protozoan grazing and LP accumulation transitioned from homogeneous distributions across bacterial colonies to site-specific accumulation at the bacteria-protist interface. Also putrescine biosynthesis was upregulated in P. fluorescens upon predation. We demonstrated that putrescine induces protozoan trophozoite encystment and adversely affects cyst viability. This multifaceted study provides new insights in common and strain-specific responses in bacteria-protozoa interactions, including responses that contribute to bacterial survival in highly competitive soil and rhizosphere environments.
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http://dx.doi.org/10.1038/srep12837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542665PMC
August 2015

Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP.

BMC Microbiol 2015 Feb 14;15:29. Epub 2015 Feb 14.

Laboratory of Phytopathology, Wageningen University, 6708 PB, Wageningen, the Netherlands.

Background: Lipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis.

Results: This study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.

Conclusions: Combining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.
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http://dx.doi.org/10.1186/s12866-015-0367-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332742PMC
February 2015

The Novel Lipopeptide Poaeamide of the Endophyte Pseudomonas poae RE*1-1-14 Is Involved in Pathogen Suppression and Root Colonization.

Mol Plant Microbe Interact 2015 Jul 9;28(7):800-10. Epub 2015 Jul 9.

3 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands;

Endophytic Pseudomonas poae strain RE*1-1-14 was originally isolated from internal root tissue of sugar beet plants and shown to suppress growth of the fungal pathogen Rhizoctonia solani both in vitro and in the field. To identify genes involved in its biocontrol activity, RE*1-1-14 random mutagenesis and sequencing led to the identification of a nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode a lipopeptide (LP) with a 10-amino-acid peptide moiety. The two unlinked gene clusters consisted of three NRPS genes, designated poaA (cluster 1) and poaB and poaC (cluster 2), spanning approximately 33.7 kb. In silico analysis followed by chemical analyses revealed that the encoded LP, designated poaeamide, is a structurally new member of the orfamide family. Poaeamide inhibited mycelial growth of R. solani and different oomycetes, including Phytophthora capsici, P. infestans, and Pythium ultimum. The novel LP was shown to be essential for swarming motility of strain RE*1-1-14 and had an impact on root colonization of sugar beet seedlings The poaeamide-deficient mutant colonized the rhizosphere and upper plant cortex at higher densities and with more scattered colonization patterns than the wild type. Collectively, these results indicate that Pseudomonas poae RE*1-1-14 produces a structurally new LP that is relevant for its antagonistic activity against soilborne plant pathogens and for colonization of sugar beet roots.
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http://dx.doi.org/10.1094/MPMI-12-14-0406-RDOI Listing
July 2015

The Rsm regulon of plant growth-promoting Pseudomonas fluorescens SS101: role of small RNAs in regulation of lipopeptide biosynthesis.

Microb Biotechnol 2015 Mar 9;8(2):296-310. Epub 2014 Dec 9.

Laboratory of Phytopathology, Wageningen University, 6708 PD, Wageningen, the Netherlands; Department of Microbial Ecology, Netherlands Institute of Ecology, 6708 PB, Wageningen, the Netherlands.

The rhizobacterium Pseudomonas fluorescens SS101 inhibits growth of oomycete and fungal pathogens, and induces resistance in plants against pathogens and insects. To unravel regulatory pathways of secondary metabolite production in SS101, we conducted a genome-wide search for sRNAs and performed transcriptomic analyses to identify genes associated with the Rsm (repressor of secondary metabolites) regulon. In silico analysis led to the identification of 16 putative sRNAs in the SS101 genome. In frame deletion of the sRNAs rsmY and rsmZ showed that the Rsm system regulates the biosynthesis of the lipopeptide massetolide A and involves the two repressor proteins RsmA and RsmE, with the LuxR-type transcriptional regulator MassAR as their most likely target. Transcriptome analyses of the rsmYZ mutant further revealed that genes associated with iron acquisition, motility and chemotaxis were significantly upregulated, whereas genes of the type VI secretion system were downregulated. Comparative transcriptomic analyses showed that most, but not all, of the genes controlled by RsmY/RsmZ are also controlled by the GacS/GacA two-component system. We conclude that the Rsm regulon of P. fluorescens SS101 plays a critical role in the regulation of lipopeptide biosynthesis and controls the expression of other genes involved in motility, competition and survival in the plant rhizosphere.
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http://dx.doi.org/10.1111/1751-7915.12190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353343PMC
March 2015

Discovery of new regulatory genes of lipopeptide biosynthesis in Pseudomonas fluorescens.

FEMS Microbiol Lett 2014 Jul;356(2):166-75

Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands

Pseudomonas fluorescens SS101 produces the cyclic lipopeptide massetolide with diverse functions in antimicrobial activity, motility, and biofilm formation. To understand how massetolide biosynthesis is genetically regulated in SS101, c. 8000 random plasposon mutants were screened for reduced or loss of massetolide production. Of a total of 58 putative mutants, 45 had a mutation in one of the three massetolide biosynthesis genes massA, massB, or massC. For five mutants, the insertions were located in the known regulatory genes gacS, gacA, and clpP. For the remaining eight mutants, insertions were located in clpA, encoding the ClpP chaperone, in phgdh, encoding D-3-phosphoglycerate dehydrogenase, in the heat shock protein-encoding dnaK, or in the transmembrane regulatory gene prtR. Genetic, chemical, and phenotypic analyses showed that phgdh, dnaK, and prtR are indeed involved in the regulation of massetolide biosynthesis, most likely by transcriptional repression of the LuxR-type regulator genes massAR and massBCR. In addition to their role in massetolide biosynthesis, dnaK and prtR were found to affect siderophore and extracellular protease(s) production, respectively. The identification of new regulatory genes substantially extended insights into the signal transduction pathways of lipopeptide biosynthesis in P. fluorescens and into regulation of other traits that may contribute to its life-style in the rhizosphere.
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http://dx.doi.org/10.1111/1574-6968.12404DOI Listing
July 2014

Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions.

PLoS Genet 2012 Jul 5;8(7):e1002784. Epub 2012 Jul 5.

Agricultural Research Service, US Department of Agriculture, Corvallis, Oregon, United States of America.

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.
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http://dx.doi.org/10.1371/journal.pgen.1002784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390384PMC
July 2012

[Prediction, overexpression and activity confirmation of adenylation domain in Zwittermicin A biosynthesis gene cluster].

Wei Sheng Wu Xue Bao 2008 Sep;48(9):1260-5

State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.

Objective: The adenylation domain is required for the substrate activation of non-ribosomal peptide synthesis. The objective of this research was to prove that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.

Methods: We cloned the adenylation domain in the Zwittermicin A synthesis cluster of Bacillus thuringiensis strain YBT-1520 with PCR amplification. After a series of enzyme digestions and subclonings, new expression vectors pBMB1312 was obtained. In order to detect the proper condition for overexpression, we tried different Isopropyl beta-D-1-thiogalactopyranoside (IPTG) concentration and temperature during overepression.

Results: The overexpression protein of this domain could be purified under 20 degrees C, 0.1 mmol/L Isopropyl beta-D-1-thiogalactopyranoside (IPTG), BL21 codon plus RP (DE3) as the host strain. Then, PPi release assay indicated that 2, 3-diaminopropionate, the presume precursor of Zwittermicin A, could be adenylated by the adenylation domain.

Conclusion: This research confirmed that 2, 3-diaminopropionate is one of the presume precursors of Zwittermicin A biosynthesis.
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September 2008

L-2,3-diaminopropionate: one of the building blocks for the biosynthesis of Zwittermicin A in Bacillus thuringiensis subsp. kurstaki strain YBT-1520.

FEBS Lett 2008 Sep 8;582(20):3125-31. Epub 2008 Aug 8.

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.

Zwittermicin A (ZwA) is a hybrid polyketide-non-ribosomal peptide that is thought to be biosynthesized from five proposed building blocks, including the 2,3-diaminopropionate. Candidate genes for de novo biosynthesis of 2,3-diaminopropionate, zwa5A and zwa5B, have been identified in a previous study. In this research, zwa5A was interrupted and chemically synthesized 2,3-diaminopropionate was used to feed the zwa5A(-) mutant. Results showed that feeding with 2,3-diaminopropionate restored the ability of the zwa5A(-) mutant to produce ZwA. Another non-ribosomal peptide synthase gene, designated orf3, was identified. Amino acid dependent PPi release assay showed that the adenylation domain ZWAA2 of ORF3 acyl-adenylated l-2,3-diaminopropionate effectively. Taken together, it can be concluded that l-2,3-diaminopropionate is indeed one of the building blocks for the biosynthesis of Zwittermicin A.
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http://dx.doi.org/10.1016/j.febslet.2008.07.054DOI Listing
September 2008

Identification of three Zwittermicin A biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520.

Arch Microbiol 2007 Apr 16;187(4):313-9. Epub 2007 Jan 16.

College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.
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http://dx.doi.org/10.1007/s00203-006-0196-3DOI Listing
April 2007