Progress 08/15/12 to 08/14/14
Outputs
Target Audience: Outreach efforts to reach a broad target of high school and undergraduate students include: 1. An invited seminar in the Microbiology Department at the University of Georgia (July 2013), hosted by the Summer Research Experience for Undergraduates (REU) program. Here, I presented data from the NIFA-funded project to undergraduate and graduate students, as well as met with undergraduate REU students to discuss graduate school and careers in science. 2. An invited seminar in the Biology Department at the University of North Carolina Asheville (April 2014) , a solely undergraduate liberal arts university with a strong focus on undergraduate research. Here, I presented data supported by the NIFA-funded project as well as taught a lecture on microbial pathogenesis. 3. Served as a volunteer and judge for the Massachusetts State Science and Engineering Fair, held at MIT (May 2014). Changes/Problems: Changes: During the award term, publications highlighting a new mass spectrometry method were published that allow monitoring of bacterial metabolite production in real-time using live bacterial colonies. This approach was perfectly-suited to assess antifungal compound production (and thus biocontrol potential) from mixedPseudomonas andPedobactercolonies; thus, the technique was implemented to complete the aims of the proposal, although it was not outlined in the original proposal. In the original proposal, travel money was requested to attend annual NIFA Fellowship Project Directors meetings in Washington DC. These meetings were not held by NIFA, and thus that travel money was used to attend and present data from the NIFA-funded project at a national meeting of the American Society for Microbiology (May 2014). What opportunities for training and professional development has the project provided? Funding from the NIFA fellowship allowed the PI to present data related to this project at regional and national meetings, including poster presentations at the Boston Bacterial Meeting (June 2013, June 2014), the American Society for Microbiology General Meeting (May 2013), and the American Society for Microbiology meeting on Bacterial Cell-Cell Communication (abstract accepted for presentation, October 2014). Funding also allowed presentation of the NIFA-funded project as an invited Young Investigators Oral Presentation at the American Society for Microbiology General Meeting (June 2014). Funding from the NIFA fellowship allowed the PI to join Pieter Dorrestein's laboratory at the University of California San Diego as a visiting scientist, and receive training on cutting mass spectrometry methods applied to bacterial metabolite production. Addtional in-house training on RNA-seq method development and Illumina RNA-Seq data management and analysis were provided in 3 day workshop hosted by the Computational Biology Initiative at Tufts University Medical School. How have the results been disseminated to communities of interest? Oral Presentations: "Mixing it up: Co-culturing ofPseudomasandPedobacterelicits novel phenotypes" (July 2013, Department of Microbiology, University of Georgia): Invited seminar presented to a primarily undergraduate audience as part of the NSF Research Experience for Undergraduates program. "Co-culturing ofPseudomonasandPedobacterdrives a novel social motility" (April 2014, Department of Biology, University of North Carolina Asheville): Invited seminar presented to science and non-science majors at an undergraduate liberal art college. "Reciprocal genetic screens identify factors involved inPseudomonas-Pedobacter social motility." (May 2014, American Society for Microbiology, General Meeting. Young Investigators Oral Presentation. Poster Presentations: McCully, LM,Seaton, SC,Bitzer, AS, SB Levy and MW Silby. (Poster) 20th Annual Boston Bacterial Meeting. June 2014, Boston, Massachusetts. Seaton, SC, AS Bitzer, MW Silby and SB Levy. (Poster) 19th Annual Boston Bacterial Meeting (BBM2013), June 2013, Boston, Massachusetts Bitzer, AS, SC Seaton, SB Levy and MW Silby. (Poster) 113th American Society of Microbiology General Meeting, May 2013, Denver, Colorado. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Continued studies of thePseudomonas-Pedobactermodel interaction focused on an exciting interaction-dependent motility phenotype that arises following physical contact between these two phylogenetically-unrelated soil bacteria. The social, or cooperative, motility occurs rapidly across solid surfaces, under conditions in which neither organism is motile alone. Using a comprehensive and reciprocal genetic screening approach, multiple genetic factors, from both the psuedomonad andPedobacterpartners, were linked to this social motility trait. The screen revealed that known motility structures, including flagella and pili, are not required for the observed social motility, and instead the social motility phenotype is enhanced in the absence of these surface structures. Mutations inPseudomonasType VI secretion inhibit social motilty, and thus indicate a potential role for this system in mediating thePseudomonas-Pedobacterinteraction. A screen ofPedobactermutants reveal a cluster of genes encoding extracellular polysaccharides that alter social motility, indicating a role for secreted polysaccharides/surfactants in mediating the interaction-induced social motility. Pedobacter genes predicted to encode a polyketide antibiotic are absolutely required for the observed social motility; thus, we are currently exploring the role of these genes and the antibiotic on chemical communication and signaling between these two organisms. To assess the link between the observed social motility and plant colonization/biocontrol, a series of experiments were completed to assess the dual species consortia under natural conditions, including in soil and during plant root colonization. Soil colonization studies reveal that initial colonization and long-term persistence of both organisms in natural soil is robust. WhilePseudomonasshowed high dissemination is soil (likely due to flagellar motility),Pedobacterwas virtually non-motile in soil dissemination assays both in pure culture and during interaction withPseudomonas. The co-culture did not have enhanced plant colonization potential or enhanced plant protection during fungal challenge, thus the potential of the mixed culture for biocontrol is unclear. In a series of experiments not originally outlined in the proposal, direct chemical communication betweenPseudomonas andPedobacterand antibiotic production by the consortia, was assessed using a recently-developed nano-DESI mass spectrometry method. This technique allows direct monitoring of metabolite production by living bacterial colonies. Funding from the NIFA fellowship allowed the PI the opportunity to complete the mass spectrometry work (during a 4-week visit) at the University of California San Diego, under the direction or Pieter Dorrestein. These data and the associated training experience added greatly to the fellowship outcomes.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Seaton, S.C. and M. W. Silby (2014) Genetics and Genomics of the Pseudomonas fluorescens Group. In Genomics of Plant-Associated Bacteria. Eds. Gross, Lichens-Park and Kole. Springer.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Seaton, S.C., M.W. Silby and S.B. Levy (2013) Pleiotropic effects of GacA on Pseudomonas fluorescens Pf0-1 in vitro and in soil. Applied and Environmental Microbiology. 79: 5405-5410.
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Progress 08/15/12 to 08/14/13
Outputs
Target Audience: Project results were disseminated to the scientific community (both in academic, industry and government research labs) through a peer-reviewed publication, multiple scientific posters presented at local and national meetings, and an invited seminar. The invited seminar was given to, among others, a group of undergraduate researchers participating in a summer Research Experience for Undergraduates program at the University of Georgia. Changes/Problems: While previously published studies report that Pseudomonas-Pedobacter mixed cultures show enhanced antifungal biocontrol activity, this phenotype is not consistently seen in our hands. Thus, it is unclear if the in planta studies described in Aim 3 of this proposal will be informative. However during our initial studies of the Pseudomonas-Pedobacter interaction, a novel motility phenotype was exhibited by the mixed culture that is not apparent in pure cultures of either organism. This phenotype is of extreme interest to the overall goals of the proposal, due to the important role that motilty plays both in survival and dessimination of biocontrol bacteria in the field and in plant root colonization. Thus, we seek to understand the nature of this motility phenotype including its mechanism, the Pseudomonas and Pedobacter genes required, and the effect, if any, the motility may have in soil and on plant roots. This provides an additional resource for analyzing the Pseudomonas-Pedobacter interaction. What opportunities for training and professional development has the project provided? Spent 5 weeks as a Visiting Scientist at University of California at San Diego (laboratory of Pieter Dorrestrein) to learn mass spectrometry techniques, and to collect data. Took a one week course to learn the basics of analysis of RNA-Seq data. The training course was offered through Tufts Computational Biology Initiative. Attend weekly research seminars in the Tufts University Department of Molecular Microbiology. How have the results been disseminated to communities of interest? Peer-reviewed research publication Poster presentation at the American Society of Microbiology Annual Meeting Poster presentation at the Boston Bacterial Meeting Invited seminar presentation for the University of Georgia, Department of Microbiology What do you plan to do during the next reporting period to accomplish the goals? Complete RNA-Seq data analysis, and identify major genes involved in Pseudomonas-Pedobacter interaction. Continue analysis of Pseudomonas-Pedobacter interaction motility. This unexpected phenotype affords many opportunities for analysis of genes (both Pseudomonas and Pedobacter) that mediate the novel motility. Begin an analysis of the ability of Pseudomonas-Pedobacter mixed cultures to colonize plant roots and ultimately enable biocontrol of fungal plant pathogens.
Impacts
What was accomplished under these goals?
First, the biocontrol potential of our wild-type Pseudomonas fluorescens strain and the same strain with the addition of a functional gacA allele was assessed. It was determined that the Gac+ strain exhibits enhanced antifungal biocontrol activity, enhanced swarming motility, and enhanced biofilm formation confirming that our wild-type Pseudomonas strain harbors a defective GacS/GacA regulatory cascade. The role of GacA on bacteria-bacteria interactions in the rhizosphere, and specifically with the Pedobacter strain of interest, is currently being explored. Second, to analyze global changes in gene expression during Pseudomonas-Pedobacter interaction, an analysis of the growth and survival of dual cultures was assessed in natural soil. At Day 15 following inoculation in soil, sufficient cell numbers of both organisms were present to allow RNA extraction from the soil for transcriptomic analysis. Following RNA extraction, transcriptome libraries were generated for pure cultures of Pseudomonas, pure cultures of Pedobacter, and the Pseudomonas-Pedobacter mixed culture. Transcriptome libraries are currently being sequenced. Third, during studies of the Pseudomonas-Pedobacter interaction of solid medium, a novel type of interaction-dependent motility was observed. Because of the ciritical role of motility in rhizosphere adaptation and biocontrol efficacy, study of the motility phenotype is being pursued. Important Pseudomonas genes involved in the motility have been identified. Fourth, a newly developed mass spectrometry technology was used in an attempt to identify small molecule metabolites that may mediate the ability of Pseudomonas and Pedobacter to sense and respond to one another. Mass spectrometry data collected at the University of California at San Diego is currently being analyzed.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Seaton, S. C., M. W. Silby, and S. B. Levy. 2013. Pleiotropic effects of GacA on Pseudomonas fluorescens Pf0-1 in vitro and in soil. Appl and Environ. Microbiol. 79: 5405-5410.
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