Source: USDA-ARS, HORTICULTURAL CROPS RESEARCH LAB submitted to
ANTIBIOTICS AND THEIR BIOSYNTHETIC INTERMEDIATES: INTER-CELLULAR CHEMICAL MESSENGERS MEDIATING GENOME-WIDE TRANSCRIPTIONAL EFFECTS IN SOIL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0224769
Grant No.
2011-67019-30192
Project No.
OREW-2010-04973
Proposal No.
2010-04973
Multistate No.
(N/A)
Program Code
A1401
Project Start Date
Mar 15, 2011
Project End Date
Mar 14, 2016
Grant Year
2011
Project Director
Loper, J. E.
Recipient Organization
USDA-ARS, HORTICULTURAL CROPS RESEARCH LAB
3420 NW ORCHARD AVENUE
CORVALLIS,OR 97330
Performing Department
(N/A)
Non Technical Summary
Plant pathogens pose costly constraints to the yield of food, fiber and ornamental crops and damage or kill plants growing in nature and managed landscapes. Certain naturally-occurring bacteria serve as biological control agents, living on plant surfaces and protecting them from infection by plant pathogens. Biological control offers an attractive complement to existing practices for management of plant diseases, but its widespread use in agriculture is impeded by unexplained variations in efficacy. One source of variation is in the inconsistent expression of biocontrol traits by antagonists inhabiting soil or plant surfaces where they suppress target plant pathogens. This project focuses on the biological control agent Pseudomonas fluorescens Pf-5, which produces many secondary metabolites including antibiotics that are toxic to plant pathogenic fungi and Oomycetes. Our hypothesis states that secondary metabolites produced by Pf-5 can function as chemical messengers influencing the expression of many genes by the bacterium, including those required for biological control. We will test this hypothesis by evaluating the effect of specific antibiotics on the expression of all genes in the Pf-5 genome. Experiments will assess Pf-5 growing in laboratory cultures and on surfaces of seed, where the bacterium functions as a biological control agent. The project will explore the concept that the production of multiple secondary metabolites is coordinately regulated in P. fluorescens, and that the metabolites themselves function in this coordination. We anticipate that the results will highlight new factors controlling the production of antibiotics that are critical to the biological control of plant disease.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21540101160100%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1160 - Pathology;
Goals / Objectives
This project will test the hypothesis that secondary metabolites can function as chemical messengers in inter-cellular communication among bacteria, having broad effects on gene expression. The project focuses on Pseudomonas fluorescens Pf-5, a soil bacterium that produces many secondary metabolites and functions in the biological control of soilborne plant diseases. There are three major objectives of the project. 1) Characterize the transcriptome of P. fluorescens Pf-5 by strand-specific RNA sequencing to identify the genome-wide distribution of transcripts and small non-coding RNAs. 2) Determine the influence of specific secondary metabolites produced by Pf-5 on the transcriptome and metabolic profiles of the bacterium. 3) Evaluate the role of specific secondary metabolites in cell-to-cell communication between strains of P. fluorescens.
Project Methods
Strand-specific direct RNA sequencing (RNA-Seq) will be done to map all genes and non-coding RNAs in the genome of P. fluorescens Pf-5. We will then use that information to evaluate the influence of specific secondary metabolites on the transcriptome of Pf-5, assessed using quantitative RNA-Seq, and the metabolic profile of Pf-5, assessed by HPLC analysis. Using these approaches, we expect to gain a detailed view of the effects of certain secondary metabolites on genome-wide patterns of gene expression and metabolism of Pf-5. We will also explore mechanisms mediating the co-regulation of two or more secondary metabolite biosynthetic pathways in Pf-5. Site-specific mutations in selected biosynthetic and regulatory genes for secondary metabolite production will be generated and evaluated in spiking experiments, in which culture media are amended with known concentrations of a specific metabolite. These experiments will enable us to identify specific regulatory elements coordinating the production of secondary metabolites by Pf-5. Finally, the role of a specific secondary metabolite in inter-cellular communication between two co-inoculated strains of P. fluorescens will be explored in culture and on seed surfaces. Taken together, the proposed experiments provide a multipronged approach to test the concept that antibiotics function in chemical communication between bacterial cells, in addition to their accepted roles in direct toxicity or competition in natural habitats.

Progress 03/15/11 to 03/14/16

Outputs
Target Audience:The research from this project was presented to audiences at the following conferences or seminars: Rhizosphere 4, Maastricht, the Netherlands, (2015);American Society for Microbiology conference on Pseudomonas held in Washington D.C. (2015);10th International Plant Growth Promoting Rhizobacteria Workshop, Belgium, (2015);National meeting of the American Phytopathological Society, Pasadena, California, USA, (2015);Seminar in the Department of Botany and Plant Pathology, Oregon State University, Oregon (2016); theSpring Conference, Center for Genome Research and Biocomputing, Oregon State University, Oregon (2016); and the Symposium on Microbial and Plant Systems Modulated by Secondary Metabolites, The Joint Genome Institute of the US Department of Energy, Walnut Creek, California, USA, (2016). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Overall, this project provided opportunities for training and professional development of two postdoctoral fellows, two graduate students and three undergraduate students. The first postdoctoral fellow on this project attended an Illumina-sponsored working group for users of high-throughput sequencing technology. This workshop provided information as to the steps required for utilizing Illumina technology in DNA and RNA sequencing, from sample collection to data analysis. It provided insight into the various technologies available, including introduction of a cloud-based server on which to store and analyze sequencing data. She also attended a seminar at Oregon State University for an introduction to CLC Genomics Workbench, a software package that includes tools for analysis of next generation sequencing data. Thirdly, she participated in a post-doctoral seminar on teaching preparation at Oregon State University, a 5-meeting series that explored the basis for college-level teaching, such as discussing learning techniques, practice in course development, and providing options for various assessment strategies. In addition, the postdoctoral fellow on this project presented a poster summarizing our results at the annual meeting of the American Phytopathological Society in Austin, Texas in August of 2013, where she had the opportunity to interact with an international group of scientists working in the field of plant-microbe interactions. She also regularly attended seminars and workshops in the Department of Botany and Plant Pathology and the Center for Genome Research and Biocomputing at Oregon State University. She gained experience in working in a multidisciplinary collaborative environment. The second postdoctoral fellow on this project attended the conference "Phytobiomes 2015: Designing a New Paradigm for Crop Improvement" held in Washington D.C. in June of 2015. He also attended the American Society for Microbiology conference on Pseudomonas held in Washington D.C. in September of 2015 and the Symposium on Microbial and Plant Systems Modulated by Secondary Metabolites held in Joint Genome Institute of the US Department of Energy at Walnut Creek, California in May of 2016. In these conferences, he presented the results of this project and had the opportunity to interact with an international group of scientists working in the field of plant-microbe interactions. He also attended the Integrated Microbial Resource workshop at the Department of Energy's Joint Genome Institute in March of 2015 where he had an opportunity to learn methods for analysis of microbial genomes and metagenomes. He regularly attended seminars and workshops in the Department of Botany and Plant Pathology and the Center for Genome Research and Biocomputing at Oregon State University. He gave a seminar in the Department of Botany and Plant Pathology and a talk at the spring conference held by the Center for Genome Research and Biocomputing, at Oregon State University. This project provided one undergraduate student majoring in computer science and another undergraduate student majoring in biochemistry an experiential learning experience in biological research including cloning techniques. The grant also partially supported a graduate student in Molecular and Cellular Biology who obtained his PhD in 2013 and is now a postdoctoral researcher working in network biology, applying his skills in computational biology and knowledge in gene expression to model expression networks of plants. That student presented his work on Gene Counter, the RNAseq analysis method used in this project, at a conference at Cold Spring Harbor in 2012. This project also provided partial support and a collaborative experience for a graduate student in Pharmaceutical Sciences who quantified secondary metabolites by HPLC for this project. How have the results been disseminated to communities of interest?This research has been disseminated to the scientific community through presentations at the following national and international conferences and seminars: American Phytopathological Society Annual Conference, Honolulu, Hawaii, (2011). 13th International Meeting on Pseudomonas, Sydney, Australia, (2011). British Society of Plant Pathology, Cambridge, UK, (2011). Microbial Genome Sequencing and Microbial Observatories Programs' Awardee Workshop, International Plant and Animal Genome Conference, San Diego, California, (2012). 50th Anniversary Meeting of the Korean Society for Plant Pathology, Seoul, South Korea, (2012) 9th International Plant Growth Promoting Rhizobacteria Workshop, Medellín, Colombia,(2012). Joint meeting of the American Phytopathological Society and the Mycological Society, Austin, Texas, USA (2013). Society for General Microbiology, Manchester, England, (2013) Seminar in the Department of Plant Pathology, University of Minnesota, USA (2013) Seminar in the Department of Plant Pathology, University of California, Davis, USA (2013) The Jacques Monod Conference on Bacterial Fungal Interactions in Roscoff France, (2013) The joint meeting of the American Phytopathological Society and the Mycological Society, Austin, Texas, (August 2013). The Department of Plant Pathology at the University of Minnesota (2013), The Jacques Monod Conference on Bacterial Fungal Interactions in Roscoff France (2013), The National Institute for Agricultural Research (INRA) in Rennes, France (2013). International Congress on Molecular Plant-Microbe Interactions in Rhodes, Greece, (2014). Rhizosphere 4, Maastricht, the Netherlands, (2015). The conference "Phytobiomes 2015: Designing a New Paradigm for Crop Improvement" held in Washington D.C. (2015) American Society for Microbiology conference on Pseudomonas held in Washington D.C. (2015) 10th International Plant Growth Promoting Rhizobacteria Workshop, Belgium, (2015) National meeting of the American Phytopathological Society, Pasadena, California, USA, (2015) Seminar in the Department of Botany and Plant Pathology, Oregon State University, Oregon (2016) Spring Conference, Center for Genome Research and Biocomputing, Oregon State University, Oregon (2016) Symposium on Microbial and Plant Systems Modulated by Secondary Metabolites, The Joint Genome Institute of the US Department of Energy, Walnut Creek, California, USA, (2016) What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The most important impacts of this project were changes in knowledge of the roles and regulation of secondary metabolites produced by a plant-associated bacterium. Our primary accomplishments were discoveries of new roles for secondary metabolites produced by the bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) and new mechanisms for regulation of secondary metabolism. We discovered that nanomolar or micromolar concentrations of phloroglucinol, a biosynthetic intermediate of the antibiotic 2,4-diacetylphloroglucinol, influenced the expression of hundreds of genes in the producing bacterium, Pf-5. Above all others, genes for the biosynthesis of a second antibiotic, pyoluteorin, were highly regulated by phloroglucinol. Our results showed that phloroglucinol is converted, through the product of a gene in the pyoluteorin gene cluster, into two signaling molecules that control the expression of pyoluteorin biosynthesis genes. To our knowledge, this co-regulation of two antibiotic biosynthesis pathways, mediated by an intermediate in one pathway that is converted into signaling molecules by an enzyme encoded in the second pathway, has no precedent in the scientific literature. To enhance knowledge of factors influencing antibiotic production in Pseudomonas spp., we analyzed the genomic sequence of Pf-5 for codon usage patterns and observed that the six rarest codons in the genome are present in all seven known antibiotic biosynthesis gene clusters. By altering the codon usage pattern of a key regulator for one antibiotic, pyoluteorin, we demonstrated that a bias in codon usage regulates antibiotic production in P. protegens Pf-5. A bias in codon usage has been linked to the regulation of many phenotypes in eukaryotes and prokaryotes but, to our knowledge, this is the first example of the role of a rare codon in the regulation of antibiotic production by a Gram-negative bacterium. These results highlight new factors controlling the production of antibiotics that are critical to the biological control of plant disease. We also discovered that strains in two defined lineages of Pseudomonas spp. are toxic to insects representing two diverse orders, Lepidoptera and Diptera. A previously-described insect toxin called FitD is produced by some of the Pseudomonas strains we tested, and was responsible for some but not all of the insect toxicity of these bacteria. A secondary metabolite called rhizoxin, which is known to be toxic to fungi and to cause root deformity of plants, is produced by one of the Pseudomonas strains we tested. Rhizoxin production was responsible for the toxicity of that strain to the dipteran insect Drosophila melanogaster. This work identified a new factor, rhizoxin, involved in insect toxicity by bacteria and shows that a single bacterium, P. protegens Pf-5, has two distinct ways of killing insects in different orders. Pseudomonas spp. were known previously to protect plants from certain plant diseases and this work shows that these bacteria also have potential to protect plants from insect pests. This study also resulted in new knowledge about bacteria that cause brown blotch disease of mushrooms, which is an important problem in commercial mushroom production. We discovered that P. protegens Pf-5 causes brown blotch symptoms on mushrooms due to its production of the anti-fungal compounds, pyoluteorin and 2,4-diacetylphloroglucinol. Previously, the toxin tolaasin was the primary factor associated with brown blotch of mushrooms. The discovery that bacteria that produce pyoluteorin and 2,4-diacetylphloroglucinol can cause this disease is significant and can be used to enhance the diagnosis and management of this disease in commercial mushroom production systems. Finally, we discovered that P. protegens Pf-5 can produce trace levels of toxoflavin, a compound toxic to many bacteria and fungi as well as plants. We used a combination of molecular and analytical chemistry approaches to evaluate toxoflavin biosynthesis by Pf-5, which led us to propose a new biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also discovered a new gene for toxoflavin degradation in Pf-5 and showed that toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria. The accomplishments of this project are summarized by three objectives as follows: 1) Characterize the transcriptome of P. fluorescens Pf-5 by strand-specific RNA sequencing to identify the genome-wide distribution of transcripts and small non-coding RNAs. We completed, analyzed, and published the results from RNAseq experiments that characterized the transcriptome of P. protegens Pf-5. 2) Determine the influence of specific secondary metabolites produced by Pf-5 on the transcriptome and metabolic profiles of the bacterium. We completed experiments designed to evaluate the influence of phloroglucinol, an intermediate in 2,4-diacetylphloroglucinol biosynthesis, on the transcriptome of P. protegens Pf-5. Phloroglucinol had a concentration-dependent influence on the expression of hundreds of genes with diverse roles in bacterial physiology. These results supported our hypothesis that phloroglucinol serves as a chemical messenger having broad effects on the Pf-5 transcriptome. A manuscript describing RNAseq experiments showing the influence of phloroglucinol on the Pf-5 transcriptome was published. Of the hundreds of genes influenced by phloroglucinol, pyoluteorin biosynthesis genes were influenced the most. To determine the mechanism by which phloroglucinol influences the transcription of pyoluteorin biosynthesis genes, we constructed a transcriptional fusion of pltL, the first gene in the pyoluteorin biosynthesis gene operon, to a green fluorescent protein (GFP) reporter gene (pltL::gfp), and monitored GFP fluorescence in cultures of Pf-5 mutants grown with or without phloroglucinol. We discovered that phloroglucinol is required for activation of pltL::gfp by PltR, a positive transcriptional regulator of pyoluteorin biosynthesis genes. We also discovered that pltM, which encodes a putative halogenase, is required for the phloroglucinol-mediated activation of pyoluteorin biosynthetic genes. To understand the role of pltM in the pyoluteorin production, we purified the PltM protein and found that purified PltM processes phloroglucinol into two novel compounds that interact with PltR to activate the expression of pltL::gfp. To identify the novel compounds, we extracted the crude signal(s) from the cultures of a Pf-5 and determined their structures by liquid chromatography and mass spectroscopy (LC-MS) to be mono- and di-chlorinated phloroglucinols. Next, we showed that purified mono- and di-chlorinated phloroglucinols induced the expression of pltL::gfp in the presence of PltR. Our results also demonstrated that these two chlorinated phloroglucinols can be released by cells and sensed by neighboring cells to induce pyoluteorin production and antibiosis against fungal pathogen. 3) Evaluate the role of specific secondary metabolites in cell-to-cell communication between strains of P. fluorescens. We demonstrated that phloroglucinol as well as chlorinated phloroglucinols produced by Pf-5 could induce pyoluteorin production by a co-inoculated strain in culture, thereby causing that strain to suppress a target plant pathogen. These results indicate that both phloroglucinol and chlorinated phloroglucinols can function as intercellular signals inducing the expression of pyoluteorin genes in neighboring bacterial cells. Overall, our research established a new mechanism of crosstalk between separate antibiotic clusters that functions in both intracellular and extracellular bacterial communication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ara�jo, W.L., Creason, A., L., Emy T. Mano, E.T., Neves, A.A.C., Minami, S.N., Chang, J.H., and Loper, J.E. 2016. Genome sequencing and transposon mutagenesis of Burkholderia seminalis strain TC3.4.2R3 identify genes contributing to suppression of orchid necrosis caused by Burkholderia gladioli. Molecular Plant Microbe Interactions 29:435-446.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Yan, Q., Philmus, B., Hesse, C., Kohen, M., Chang, J.H., and Loper, J.E. 2016. The rare codon AGA is involved in regulation of pyoluteorin biosynthesis in Pseudomonas protegens Pf-5. Frontiers in Microbiology 7:497.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Loper, J.E., Henkels, M.D., Rangel, L.I., Olcott, M.H., Rosen, K.L., Walker, F.L., Kidarsa, T.A., Sneh, B., Taylor, B.J. (2016) Rhizoxin analogs, orfamide A, and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster. Environ Microbiol doi: 10.1111/1462-2920.13369.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Rangel, L.I, Henkels, M.D., Shaffer, B.T., Walker, F. L., Davis, E.W. II, Stockwell, V.O., Bruck, D., Taylor, B.J., Loper, J.E. 2016. Characterization of toxin complex gene clusters and insect toxicity of bacteria representing four subgroups of Pseudomonas fluorescens. PLoS One (in press).


Progress 03/15/14 to 03/14/15

Outputs
Target Audience:The results of this project were presented at the International Congress on Molecular Plant-Microbe Interactions in Rhodes, Greece in July of 2014. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoctoral fellow on this project attended the conference "Phytobiomes 2015: Designing a New Paradigm for Crop Improvement" to be held in Washington D.C. in June of 2015 and presented a poster on this work. There, he had the opportunity to interact with an international group of scientists working in the field of plant-microbe interactions. He also attendedthe Integrated Microbial Resource workshop at the Department of Energy's Joint Genome Institute in March of 2015 where he had an opportunity to learn methods for analysis of microbial genomes and metagenomes. He also regularly attended seminars and workshops in the Department of Botany and Plant Pathology and the Center for Genome Research and Biocomputing at Oregon State University. How have the results been disseminated to communities of interest?The postdoctoral scientist on this projectpresented a poster entitled "Identification of a signal that mediates the crosstalk between biosynthetic gene clusters for the antibiotics 2,4-diacetylphloroglucinol and pyoluteorin in Pseudomonas protegens Pf-5" at the conference "Phytobiomes 2015: Designing a New Paradigm for Crop Improvement"held in Washington D.C. in late June. This work was also presented by the Project Director at the International Congress onMolecular Plant-Microbe Interactions, Rhodes, Greece, July 2014. What do you plan to do during the next reporting period to accomplish the goals?We intend to complete experiments investigating the mechanisms mediating cross-talk between phloroglucinol and pyoluteorin biosynthesis gene clusters of Pf-5.

Impacts
What was accomplished under these goals? Objective 1. Characterize the transcriptome of P. protegens Pf-5 (previously called P. fluorescens Pf-5) to identify the genome-wide distribution of transcripts and small non-coding RNAs This objective was completed previously. Objective 2. Determine the influence of phloroglucinol and other metabolites of Pf-5 on the transcriptome and antibiotic-production profiles of the bacterium Objective 2a. Evaluate the effect of phloroglucinol on the Pf-5 transcriptome. A manuscript describing RNAseq experiments showing the influence of phloroglucinol on the Pf-5 transcriptome has been accepted for publication. Objective 2b: Identify the mechanism by which phloroglucinol influences pyoluteorin production by Pf-5. To determine the mechanism by which phloroglucinol influences the production of pyoluteorin, we constructed a transcriptional fusion of the pyoluteorin biosynthesis genes pltL and pltA to a green fluorescent protein (GFP) reporter gene (pltLA::gfp), and monitored GFP fluorescence in cultures of Pf-5 mutants grown with or without phloroglucinol. We confirmed that phloroglucinol influences pyoluteorin production at the transcriptional level, and the LysR family regulator PltR is a positive transcriptional regulator required for the expression of pltLA::gfp. We also discovered that phloroglucinol is required for activation of pltLA::gfp by PltR, suggesting that phloroglucinol regulates pyoluteorin production, at least in part, through the action of PltR. In an effort to investigate if phloroglucinol functions as a signal directly, we tested the expression of pltLA::gfp transcriptional fusion in a strain of P. fluorescens that does not have gene clusters for either phloroglucinol or pyoluteorin. We found that pltM, which encodes a putative halogenase, is required for the phloroglucinol-mediated activation of pyoluteorin biosynthetic genes. Furthermore, we made a pltM mutant and found that the pltM mutant does not produce pyoluteorin, indicating that pltM plays an important role in pyoluteorin production. The role of pltM in the pyoluteorin biosynthesis is intriguing because there are two halogenases in the pyoluteorin gene cluster, pltM and pltA, but PltA is known to add both chlorines to pyoluteorin. To understand the role of pltM in the pyoluteorin production, we purified the PltM protein and found that the purified PltM processes phloroglucinol into novel compounds that activate the expression of pltLA::gfp in the presence of pltR. These data suggested that PltM transforms phloroglucinol into a signal that interacts with the PltR regulator and then activates the expression of pyoluteorin biosynthetic genes. To identify the novel signal(s), we extracted the crude signal(s) from the cultures of a Pf-5 gacA mutant containing a plasmid-borne pltM gene expressed from a constitutive promoter. LC-MS analysis elucidated the chemical structure of two signals as mono-chlorinated phloroglucinol and dichlorinated phloroglucinol, respectively. Our data indicate that phloroglucinol, an intermediate of 2,4-diacetylphloroglucinol, is processed by PltM into novel compounds that function as signals activating the expression of pyoluteorin biosynthetic genes. To our knowledge, this is the first example of co-regulation of two antibiotics mediated by transforming an intermediate of one pathway into signals that activate the second pathway. Objective 2c. Determine the effects of a spectrum of secondary metabolites produced by Pf-5 on its secondary metabolome. One of the challenges of genomics-guided natural product discovery is the difficulty of isolating unknown secondary metabolites synthesized from orphan gene clusters that are expressed at low levels. We discovered that a seven-fold mutant of strain Pf-5, which has knockouts in each of the seven known secondary metabolite gene clusters in the genome, overproduced a product of an orphan gene cluster. Consequently, we were able to establish that the product of that gene cluster is toxoflavin because the seven-fold mutant produced toxoflavin in high enough levels for chemical analysis. In contrast, wildtype Pf-5 did not produce adequate levels of the compound for structure elucidation. These results suggest that cross-talk among the many secondary metabolite gene clusters of Pf-5 influence the expression of biosynthetic genes in the toxoflavin gene cluster, in addition to genes for pyoluteorin biosynthesis described above. Due to the remarkable metabolic diversity of Pf-5, the seven-fold mutant also could serve as a vehicle for the expression of orphan gene clusters introduced from other organisms. Objective 3. Evaluate the role of phloroglucinol in cell-to-cell communication between strains of P.protegens. We demonstrated that phloroglucinol produced by cells of Pf-5 could induce pyoluteorin production by a co-inoculated strain in culture, thereby causing that strain to suppress a target plant pathogen. These results demonstrate that phloroglucinol can function as an intercellular chemical messenger influencing both secondary metabolism and antibiosis of Pseudomonas. The role of phloroglucinol in the intercellular crosstalk was also confirmed by using a pltLA::gfp transcriptional fusion. Co-culture of a phlD mutant with a pltM mutant containing a pltLA::gfp transcriptional fusion assay showed that phloroglucinol produced by pltM mutant can be processed into a chlorinated phloroglucinol derivative in the phlD mutant. Our data suggest that the chlorinated phloroglucinol is secreted by the phlD mutant and transported back to the pltM mutant to activate transcription of the pltLA::gfp fusion. To our knowledge, this is the first example showing a function of intermediates in antibiotic biosynthesis in bacterial cell to cell communication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Philmus BJ, Shaffer BT, Kidarsa TA, Yan Q, Raaijmakers JM, et al. (2015) Investigations into the biosynthesis, regulation and self-resistance of toxoflavin in Pseudomonas protegens Pf-5. ChemBioChem 16: 1782-1790.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Quecine MC, Kidarsa TA, Goebel NC, Shaffer BT, Henkels MD, Zabriskie TM, and Loper JE (2016) An Interspecies signaling system mediated by fusaric acid has parallel effects on antifungal metabolite production by Pseudomonas protegens Pf-5 and antibiosis of Fusarium spp. Appl Environ Microbiol 82: 1372-1382.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Clifford JC, Buchanan A, Vining O, Kidarsa TA, Chang JH, McPhail KL, and Loper JE (2016) Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens. Environ Microbiol.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Yan Q, Philmus B, Hesse C, Kohen M, Chang JH, and Loper JE. 2016. The rare codon AGA is involved in regulation of pyoluteorin biosynthesis in Pseudomonas protegens Pf-5. Frontiers in Microbiology


Progress 03/15/13 to 03/14/14

Outputs
Target Audience: The results of this project were presented at the joint meeting of the American Phytopathological Society and the Mycological Society, Austin, Texas (August 2013),in a seminar in the Department of Plant Pathology at the University of Minnesota (May 2013), the Jaques Monod Conference on Bacterial Fungal Interactions in Roscoff France (December, 2013), and the National Institute for Agricultural Research (INRA) in Rennes, France (December 2013). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The postdoctoral fellow on this project presented a poster summarizing our results at the annual meeting of the American Phytopathological Society in Austin, Texas in August of 2013, where she had the opportunity to interact with an international group of scientists working in the field of plant-microbe interactions.She also regularly attended seminars and workshops in the Department of Botany and Plant Pathology and the Center for Genome Research and Biocomputing at Oregon State University. She gained experience in working in a multidisciplinary collaborative environment. This project provided one undergraduate student majoring in computer science an experiential learning experience in biological research. The grant also partially supported a graduate student in Molecular and Cellular Biology who obtained his PhD in 2013 and is now a postdoctoral researcher working in network biology, applying his skills in computational biology and knowledge in gene expression to model expression networks of plants. That student presented his work on Gene Counter, the RNAseq analysis method used in this project, at a conference at Cold Spring Harbor in 2012. This project also provided partial support and a collaborative experience for a graduate student in Pharmaceutical Sciences who quantified secondary metabolites by HPLC for this project. How have the results been disseminated to communities of interest? The postdoctoral scientist on this project presented a poster entitled ‘Using next-generation sequencing to determine the influence of metabolic intermediates on the Pseudomonas protegens transcriptome’ at the joint meeting of the American Phytopathological Society and the Mycological Society, Austin, Texas, August 2013. This work was also presented in a seminar in the Department of Plant Pathology at the University of Minnesota (May 2013), the Jaques Monod Conference on Bacterial Fungal Interactions in Roscoff France (December, 2013), and the National Institute for Agricultural Research (INRA) in Rennes, France (December 2013). What do you plan to do during the next reporting period to accomplish the goals? We intend to complete experiments investigating the mechanisms mediating cross-talk between phloroglucinol and pyoluteorin biosynthesis gene clusters of P. protegens Pf-5.

Impacts
What was accomplished under these goals? Objective 2a. Evaluate the effect of phloroglucinol on the Pf-5 transcriptome. RNAseq experiments completed last year were analyzed, revealing that phloroglucinol, an intermediate in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol, influences the transcription of hundreds of genes in the biological control bacterium Pseudomonas protegens Pf-5. Experiments evaluated the influence of 100 nanomolar and 100 micromolar concentrations of phloroglucinol on the transcriptome of Pf-5 and derivative strains lacking phloroglucinol and/or 2,4-diacetylphloroglucinol production. Phloroglucinol influenced the transcription of 140 genes by at least four-fold. Of the genes influenced by phloroglucinol, transcripts of the pyoluteorin biosynthesis genes exhibited the greatest fold changes. These data suggest that specific mechanisms exist for the coregulation of 2,4-diacetylphloroglucinol and pyoluteorin, which were explored in Objective 2b. Other genes regulated by phloroglucinol fall in functional role categories for amino acid biosynthesis, energy metabolism, transport, regulatory, and unknown functions. These results support our hypothesis that phloroglucinol serves as a chemical messenger having broad effects on the Pf-5 transcriptome. A manuscript describing these results is in preparation and when completed will meet the aims of Objective 2a. One of the genes regulated by phloroglucinol is predicted to encode a TDT-family transporter, the only one of its type in the Pf-5 genome. We constructed a mutant of Pf-5 with a deletion in the TDT-transporter gene and compared the mutant to wildtype Pf-5 for a range of phenotypes. Experimental results suggest that this transporter does not play a role in the production or efflux of pyoluteorin or 2,4-diacetylphloroglucinol but is involved in sulfite tolerance of Pf-5. The mechanism of sulfite tolerance is still unknown, but could be related to sulfite ion efflux exhibited by certain TDT-family transporters in other microorganisms. Objective 2b: Identify the mechanism by which phloroglucinol influences pyoluteorin production by Pf-5. To determine the mechanism by which phloroglucinol influences the production of pyoluteorin, we constructed a transcriptional fusion of the pyoluteorin biosynthesis genes pltL and pltA to a green fluorescent protein (GFP) reporter gene, and monitored GFP fluorescence in cultures of Pf-5 mutants grown with or without phloroglucinol. We confirmed that phloroglucinol influences pyoluteorin production at the transcriptional level, and the regulatory gene PltR is a positive transcriptional regulator of pltL and pltA. We also discovered that phloroglucinol is required for induction of pltL and pltA by PltR, suggesting that phloroglucinol regulates pyoluteorin production, at least in part, through the action of PltR, although pltR is not differentially expressed at the transcriptional level in response to phloroglucinol treatment. Objective 2c. Determine the effects of a spectrum of secondary metabolites produced by Pf-5 on its secondary metabolome. We published a manuscript describing the derivation and characterization of a panel of Pf-5 mutants having deletions in the biosynthesis genes for one to seven known secondary metabolites. Culture extracts were analyzed by HPLC to determine the effects of each mutation on the secondary metabolome of Pf-5. Of the seven secondary metabolites produced by Pf-5, only 2,4-diacetylphloroglucinol and pyoluteorin appeared to be tightly co-regulated. Deletion of a gene for hydrogen cyanide biosynthesis (hcnB) enhanced the production of all other compounds, which is likely due to the enhanced growth of the hcnB mutant relative to wildtype Pf-5. The panel of mutants has been very useful in assessing the roles of specific metabolites in the lifestyle of Pf-5. For example, we used the mutant set to establish that 2,4-diacetylphloroglucinol and pyoluteorin production contribute to the brown blotch symptoms caused by Pf-5 on caps of the button mushroom (Agaricus bisporus). Purified 2,4-diacetylphloroglucinol and pyoluteorin mimicked the discoloration and pitting symptoms caused by Pf-5 on mushroom caps, and the compounds were isolated from mushroom caps inoculated with Pf-5, confirming their in situ production. These data extend knowledge of factors contributing to mushroom diseases caused by Pseudomonas spp. beyond tolaasin and related lipopeptides, which were the primary factors previously known to cause brown blotch of mushrooms. Objective 3. Evaluate the role of phloroglucinol in cell-to-cell communication between strains of P.protegens. We demonstrated that phloroglucinol produced by cells of Pf-5 could induce pyoluteorin production by a co-inoculated strain in culture, thereby causing that strain to suppress a target plant pathogen. These results demonstrate that phloroglucinol can function as an intercellular chemical messenger influencing antibiotic production byP. protegens as well as the bacterium's antagonism against the plant pathogenic oomycete Pythium ultimum. To our knowledge, this is the first example showing a function of intermediates in antibiotic biosynthesis in bacterial cell to cell communication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Henkels, M.D., Kidarsa, T.A., Shaffer, B.T., Goebel, N.C., Burlinson, P., Mavrodi, D.V., Bentley, M.A., Rangel, L.I., Davis II, E.W., Thomashow, L.S., Zabriskie, T.M., Preston, G.M., and Loper, J.E. 2014. Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites. Molecular Plant-Microbe Interactions 27:733-746
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Clifford, J., Kidarsa, T., Buchanan, A., Chang, J. H., and Loper, J. E. 2013. Using next-generation sequencing to determine the influence of metabolic intermediates on the Pseudomonas protegens transcriptome. Phytopathology 103:S2.28


Progress 03/15/12 to 03/14/13

Outputs
Target Audience: TARGET AUDIENCES: Presentations at international conferences: Society for General Microbiology, Manchester, England, March 2013; 50th Anniversary Meeting of the Korean Society for Plant Pathology, Seoul, South Korea, October 2012; 9th International Plant Growth Promoting Rhizobacteria Workshop, Medellín, Colombia, May 2012. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The postdoctoral fellow on this project attended an Illumina-sponsored working group for users of high-throughput sequencing technology. This workshop provided information as to the steps required for utilizing Illumina technology in DNA and RNA sequencing, from sample collection to data analysis. It provided insight into the various technologies available, including introduction of a cloud-based server on which to store and analyze sequencing data. She also attended a seminar at Oregon State University for an introduction to CLC Genomics Workbench, a software package that includes tools for analysis of next-generation sequencing data. Thirdly, she participated in a post-doctoral seminar on teaching preparation at Oregon State University, a 5-meeting series that explored the basis for college-level teaching, such as discussing learning techniques, practice in course development, and providing options for various assessment strategies. This project also provided one undergraduate student majoring in computer science an experiential learning experience in biological research. The student has contributed to data processing, analysis, and presentation. One graduate student has served as his primary mentor, giving the former mentoring experience. How have the results been disseminated to communities of interest? We submitted an abstract entitled ‘Using next-generation sequencing to determine the influence of metabolic intermediates on the Pseudomonas protegens transcriptome’ for presentation at the national meeting of the American Phytopathological Society in August of 2013. What do you plan to do during the next reporting period to accomplish the goals? 1. Complete manuscript describing the influence of phloroglucinol on the Pf-5 transcriptome for submission to a peer-reviewed journal 2. Complete experiments evaluating the role of the aforementioned candidate gene in the cross-talk between phloroglucinol and pyoluteorin production by Pf-5. 3. Begin experiments aimed toward understanding the role of phloroglucinol as a signaling molecule in cell-cell communication between strains of P. protegens.

Impacts
What was accomplished under these goals? 1. Characterize the transcriptome of P. protegens Pf-5 (previously called P. fluorescens Pf-5) to identify the genome-wide distribution of transcripts and small non-coding RNAs We completed and analyzed two RNAseq experiments to characterize the transcriptome of P. protegens Pf-5. The program GeneCounter was used to identify transcripts that were differentially expressed by Pf-5 or specific mutants under varied cultural conditions. A selection of differentially-expressed genes was tested in a second experiment by RT-PCR thereby validating the RNAseq results. Using GENE-counter’s findTUs.pl program, empirical analysis of the RNA-Seq coverage was performed to predict small non-coding RNAs. This approach identified many putative non-coding RNAs that were differentially expressed within the treatments, almost all of which had not been previously detected in Pf-5. 2. Determine the influence of phloroglucinol and other metabolites of Pf-5 on the transcriptome and antibiotic-production profiles of the bacterium Objective 2a. Evaluate the effect of phloroglucinol on the Pf-5 transcriptome. We completed an RNA-Seq experiment designed to evaluate the influence of phloroglucinol on the transcriptomes of two Pf-5 derivative strains: Pf-5phlAG (a mutant lacking2,4-diacetylphloroglucinol (DAPG) production but retaining theability to synthesize the DAPG precursor phloroglucinol) and Pf-5phlADG (a mutant lacking both phloroglucinol and DAPG production). Data analysis generated a list of hundreds of genes differentially expressed among the treatments. As expected from previous results, expression of many genes in the pyoluteorin biosynthetic gene cluster is highly regulated by phloroglucinol. This study identified a large number of other genes in the Pf-5 genome that are also regulated by phloroglucinol, which supports our hypothesis that this compound serves as a signal having broad effects on the transcriptome of Pf-5. Objective 2b: Identify the mechanism by which phloroglucinol influences pyoluteorin production by Pf-5. To explore the possible role of pltZ, a negative regulator of pyoluteorin production, in the cross-talk between the phloroglucinol and pyoluteorin biosynthetic gene clusters, we constructed a deletion mutant in pltZ in wildtype and phloroglucinol-deficient backgrounds of Pf-5 and evaluated it for pyoluteorin production. These experiments confirmed the role of pltZ in pyoluteorin production but did not provide conclusive evidence for a role of pltZ in the cross-talk between the phloroglucinol and pyoluteorin biosynthetic gene clusters. We also constructed a deletion in phlH, a regulator of 2,4-diacetylphloroglucinol biosynthesis, and found that it plays a minor role in pyoluteorin production. Thirdly, we are examining a gene encoding a protein belonging to the TDT- family of transporters that was identified in our RNAseq experiments as regulated by phloroglucinol. We constructed a mutant having a deletion in this gene. Experiments aimed at determining the role of the TDT-family transporter in secondary metabolite production arein progress. Objective 2c. Determine the effects of a spectrum of secondary metabolites produced by Pf-5 on its secondary metabolome. We evaluated the influence of salicylic acid, an intermediate in the biosynthesis of the siderophore enantio-pyochelin, on secondary metabolite production by Pf-5 and a phcA mutant, which lacks salicylate and enantio-pyochelin production. We detected no significant influence of salicylic acid on production by Pf-5 of four antibiotics, but did detect a major influence on the production of a fifth antibiotic. We are following up on these results to determine the mechanism by which salicylic acid influences antibiotic production, which could include signaling or regulation by iron. 3. Evaluate the role of phloroglucinol in cell-to-cell communication between strains of P. protegens on seed surfaces. No progress.

Publications


    Progress 03/15/11 to 03/14/12

    Outputs
    OUTPUTS: The soil bacterium Pseudomonas fluorescens strain Pf-5 suppresses plant diseases caused by certain soilborne pathogens and produces a spectrum of secondary metabolites, including antibiotics toxic to plant pathogenic bacteria, oomycetes, and fungi. Two of the antibiotics that contribute to biological control are 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin. We previously demonstrated that phloroglucinol, an intermediate in DAPG biosynthesis, serves as a signaling molecule influencing the production of pyoluteorin by Pf-5 and other strains of Pseudomonas. We constructed deletion mutants in pltZ and pltR, genes in the pyoluteorin biosynthetic gene cluster that encode transcriptional regulators, and confirmed that PltZ inhibits pyoluteorin production and PltR activates pyoluteorin production in Pf-5. We found that PltZ is involved in the inhibition of pyoluteorin production by high concentrations of phloroglucinol. We also constructed a series of 12 point mutations in pltR, which will be tested for changes in the activation of pyoluteorin production by phloroglucinol and the autoinduction of pyoluteorin production by pyoluteorin. We completed one RNAseq experiment evaluating the influence of phloroglucinol on the transcriptomes of Pf-5 wild-type and a phlD mutant (deficient in phloroglucinol production). In a preliminary experiment evaluating the role of phloroglucinol on gene expression by Pf-5, we performed quantitative PCR on key genes for pyoluteorin biosynthesis and transport. As expected, we found that, in Pf-5, the addition of phloroglucinol decreased expression of pyoluteorin biosynthesis genes, represented by pltB and pltL, and had little effect on pltI, a gene involved in pyoluteorin transport. We also optimized protocols for RNA extraction from cultures of Pf-5 and rRNA depletion for enrichment of mRNA, thereby obtaining mRNA of adequate quality for direct sequencing and library construction for RNAseq. We are now analyzing the data from the first RNAseq experiment. PARTICIPANTS: Joyce E. Loper, Project Director, USDA-ARS Corvallis, Oregon; Jeff Chang, Principal Investigator, Oregon State University; Mark Zabriskie, Principal Investigator, Oregon State University. Training and professional development: Postdoctoral training, Jennifer Clifford and Teresa Kidarsa, Postdoctoral Research Affiliates, USDA-ARS, Corvallis, Oregon; Graduate student training, Jeff Kimbrel and Neal Goebel, Oregon State University. Undergraduate researchers: Ashley Bixenstein and Jennifer Lee, Oregon State University. TARGET AUDIENCES: This work was presented at the following national or international conferences: American Phytopathological Society, Honolulu, Hawaii, August 2011; 13th International Meeting on Pseudomonas, Sydney, Australia, September, 2011; British Society of Plant Pathology, Cambridge, UK, December 2011. It was also presented at the Microbial Genome Sequencing and Microbial Observatories Programs' Awardee Workshop, International Plant and Animal Genome Conference, San Diego, California, 2012. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    Biological control offers an attractive complement to existing practices for management of plant disease and therefore can contribute to the productivity and sustainability of U.S. agriculture in the future. Unfortunately, its widespread use in agriculture is impeded by unexplained variations in the efficacy of biocontrol organisms. One source of this variation is in the inconsistent expression of biocontrol traits by antagonists inhabiting soil or plant surfaces where they suppress target plant pathogens. The proposed research will explore factors controlling antibiotic production, a predominant trait contributing to biological control, in the soil bacterium Pseudomonas fluorescens Pf-5. The project builds upon a body of work establishing that the in situ expression of specific biocontrol traits by P. fluorescens is influenced by compounds produced by its co-inhabitants in the rhizosphere. The proposed research will extend this concept by evaluating the effect of antibiotics on the genome-wide expression profiles of Pf-5 in culture and on seed surfaces.

    Publications

    • No publications reported this period