Progress 01/01/12 to 09/30/15
Outputs
Target Audience:As a direct result of this project the PI was contacted by Professor Zhongli Cui, PhD, Department of Microbiology, Nanjing Agricultural University, China, to visit his institution. He accepted this invitation and conducted a one week trip to Nanjing, China, in October 2015. There he presented two seminars and held smaller focused discussions on the use of myxobacteria as biocontrol agents to protect crops against fungal pathogens. In total about 30 graduate students and microbiologists attended all of the meetings/seminars. Due to mutual scientific interests and findings the PI and Dr. Cui initiated plans to consider a future collaboration. Arup Dey, graduate student, presented his work on mechanisms that myxobacteria use to kill other microbes. His work was orally presented as at the 42nd International Conference of the Biology of Myxobacteria held at Estes Park, CO, July 12-15, 2015. The PI organized this meeting and around 55 microbiologist attended. Vera Troselj, graduate student, presented a poster at the Rocky Mountain Branch ASM meeting held at Colorado Christian University, Denver, CO, October 17, 2015. She presented her work on how myxobacteria used type VI secretion systems to kill bacteria. Vera received a 1st place award for her poster presentation. Around 50 students/microbiologists attended the meeting. Due to a one year no cost extension, target audiences that werereached in 2014 are alsolisted below. Arup Dey, graduate student, presented his work on mechanisms that myxobacteria use to kill other microbes. His work was presented (poster) at the ASM conference on Cell-Cell Communication in Bacteria in San Antonio, TX, October 18-22, 2014. Austin Conklin, undergraduate, was a co-author on the poster. The PI gave an oral presentation that included this work at the same meeting. Around 200 microbiologists attended this international meeting. These presentations were highlighted in a meeting review publication in the Journal of Bacteriology (A Social Medium: ASM's 5th Cell-Cell Communication in Bacteria Meeting in Review, Gibbs & Federle, July 2015, 197:2084). Arup Dey gave an oral presentation at the Rocky Mountain Branch ASM meeting at Colorado College, Colorado Springs, CO, on October 25, 2014. Austin Conklin, undergraduate, presenteda poster at this same meeting. The PI served as the Rocky Mountain Branch ASM president and organized this meeting were about 60 students and microbiologist attended. The PI presented work on myxobacterial predation at the 41st International Conference of the Biology of Myxobacteria held at Frejus, France May 25-28, 2014. Around 40 microbiologists attended. Changes/Problems:The direction of this project changed to a more focused examination of mechanisms used by myxobacteria to kill other microbes. This effort is encompassed within Object 3 of the proposal and directly connects to biocontrol since biocontrol involves antagonistic to killing functions of the agent against crop pathogens. The mechanism of killing studied depended on cell-cell contact for the delivery on toxin to target cells. This effort results in a publication (conditionally accepted) in the Journal of Bacteriology. Part of the reason for a shift in project emphasis was the untimely death of the co-PI, whose expertise was in crop pathogens. What opportunities for training and professional development has the project provided?This project has involved training of graduate and undergraduate students. During this reporting cycle graduate students Arup Dey and Vera Troselj were trained by the PI and other senior researchers (e.g. Dr. Xueming Wei, postdoc) in the lab. In addition, Arup provided daily supervision of an undergraduate, Austin Conklin, who worked full time over the summer and part-time during the semesters. Arup Dey successfully defended his PhD dissertation in 2015. All team members met with the PI regularly (daily/weekly) and participated in weekly lab meetings where individuals presented their findings. As outlined under Target Audiences graduate students and an undergraduate presented their findings at two regional (ASM branch) and two international microbiology meetings. How have the results been disseminated to communities of interest?The results have been disseminated through scientific channels as outlined under Target Audience. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
There are over 2 million farms in the U.S. that generate around $400 billion in agricultural sales per year. The U.S. agricultural industry thus provides essential food for itself and the world and this sector is a major economic driver to the U.S. economy. One ongoing threat to the agricultural industry are pathogens that destroy crops. One category of pathogens are microbes, which cause tens of billions of dollars in crop damage annually. A number of methods are used to control crop disease and chemicals such as fungicides are a major strategy used by farmers. Although generally effective, the use of chemicals is problematic. For instance, they are expensive, harmful to the environment and humans and pathogens can develop resistance, thus rendering them ineffective. An alternative approach is biocontrol, where naturally occurring microbes are used to control crop diseases. Biocontrol stems from the long known observation that some soils are naturally suppressive to crop pathogens, while other soils are conductive to diseases. The explanation for these different outcomes is the makeup of the microbes that naturally reside in those soils. In other words, soil contain tens of thousands different microbial species and some of those species inhibit the growth or kill crop pathogens. Thus the complex microbial communities found in soil is analogous to the recent discoveries being made about the human microbiome. That is, scientists have found that the composition of the human microbiome has major impacts on human health. Some microbial species are harmful, while other species are helpful. These findings have sparked the use of probiotics, where helpful microbes are added to our intestinal tract to improve health. Similarly, the soil microbiome is complex and consists of both beneficial and harmful microbes toward crops. Farmers have been cognizant of this fact for some time and in this vain they use commercial biocontrol products to protect their crops. Although these biocontrol agents are useful, their overall effectiveness is stymied because little is known about these agents. For example, fundamental questions that remain unanswered include what microbial species/strains are most effective against particular diseases and by what mechanisms do biocontrol agents protect crops? This project was initiated under the hypothesis that myxobacteria can be used as effective biocontrol agents to protect crops from microbial pathogens. This idea was based on the fact that myxobacteria are natural soil predators that kill and consume other microbes while not harming plants. Thus the basic premise is that biocontrol works when the control agent outcompetes or kills the pathogen(s). In this regard it is noteworthy that of the known bacterial species myxobacteria can be reasonably argued as one of the most effective soil predators. They kill a broad range prey species, are motile in soil which allows their dispersion, and are thought to produce a wide range of weapons for predation. Thus myxobacteria biocontrol would occur when they are in close proximity to crops. In the soil they reside near or on plant roots, a zone that is called the rhizosphere, and possibly on plant stems and leaves. In these strategic locations myxobacteria can defend crops. In order to understand their potential utility in biocontrol we initiated three objectives in this project. The outcomes of the objectives varied; in some cases major discoveries were made while in other cases little advancement was achieved. For objective one we surveyed a panel of myxobacteria environmental isolates against a panel of known crop pathogens. From these studies we found that some myxobacterial isolates showed stronger antagonism against fungal and bacterial pathogens than others. In some cases the antagonistic interactions depended on diffusible compounds produced by the myxobacteria that blocked pathogen growth. In one case a possible secondary metabolite (natural product) produced by a myxobacterium was identified by a genetics approach as the causative agent of antagonism. In other cases antagonistic interactions depended on cell-cell contract. Here the molecular basis of antagonism may not depend on the secretion of a toxin into the environment, but instead may require cell-cell contact for inhibition to occur. For objective two there are no major findings to report (see project changes) and instead personnel resources were directed to objective three. For objective three we sought to identify genetic determinants and mechanisms by which myxobacteria may antagonize pathogens. This objective is important because if one seeks to use a microbe as a biocontrol agent it helps to understand how it works so it can be engineered and improved. In this regard we discovered a new mechanism for how myxobacteria kill. This mechanism involves cell-cell contact where a toxin is delivered to a target cell. Here, we identified and manipulated a myxobacterial targeting system that finds susceptible cells. Through genetic engineering we changed cell types that are targeted. We have also discovered three new toxins. The toxins are proteins that act as enzymatic poisons once they are delivered into the cytoplasm of the inflicted cell. The delivery system is based, in part, in a process we call outer membrane exchange. We hypothesize that for cargo delivery to reach the cytoplasm there is a second stage delivery mechanism that remains to be elucidated. This work has broadened our understanding into how myxobacteria kill and has added insight into how they might be manipulated to serve as improved biocontrol agents. This work was conditionally accepted for publication in the Journal of Bacteriology (ASM, American Society for Microbiology publication). In related on-going work we have characterized a different cell-cell contact dependent system used for killing. This is called the type VI secretion system and is found in many different species of gram-negative bacteria. In these studies we have discovered how the system is activated for killing. The actual toxin remains unknown, but currently we are testing one candidate toxin. In summary, we have advanced the field's mechanistic understanding for how myxobacteria kill competing microbes. Killing depends on both cell-cell contact and secreted diffusible factors. The ability to manipulate these systems may allow us to optimize myxobacteria as effective biocontrol agents. During the course of this project a patent was issued/granted: UW 08-090 CON (a continuation patent application related to Methods for Natural Product Optimization) by the United States Patent and Trademark Office on June 16, 2015 under patent number 9,057,089. The patent pertains to how natural product compounds, such as antibiotics and antifungals, can be improved or optimized from growth in a producer strain such as myxobacteria. In turn such optimized strains may be more effective biocontrol agents.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2016
Citation:
Sibling rivalry in Myxococcus xanthus is mediated by kin recognition and a polyploid prophage. Journal of Bacteriology (conditionally accepted)
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: As described under plans for next reporting period, our current efforts are focused at understanding the mechanisms of killing andtarget cell recognition. We have made significant progress in these topics as we have identified a novel cell recognition system and are currently working on several toxin and toxin delivery systems in M. xanthus. We hope to be able to understand these procesess and extend those findings to pathogen killing. What opportunities for training and professional development has the project provided? Personnel in the lab continued to be trained on other projects as this project was on hold. A PhD student, Yao Xiao, successfully completed her dissertation and defended in the summer of 2013. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Our lab is focused on how the social and soil-dwelling bacterium Myxococcus xanthus interacts with other cells. These cell interactions may be between kin cells or other soil species involving antagonistic or predatory interactions. We are currently seeking to understand how myxobacteria recognize neighboring cells, whether friend, foe or food. We have identified a cell surface receptor involved in direct cell-cell contact dependent recognition. We have also found that diffusible factors, such as antibiotics (e.g. TA),produced by myxobacteria can lead to discriminatory or predatory interactions with prey cells. We are also focused in understandingmechanistic details for how M. xanthus kills other cells and how it decides which cells to kill. Addressing these fundamental questions will then allow us to direct this knowledge toward predatory killing behaviors. That is, we seek to control how myxobacteria chooses prey cells and the type of cellular weapons it employs to kill. Understanding these processes and being able to engineer desired properties and outcomes holds great potential for using myxobacteria as a biocontrol agent to diminish and degrade plant pathogens that harm crops.
Impacts
What was accomplished under these goals?
The project was placed on hold this past reporting period and funds were deferred for a year. This was done because of personnel turnover in the lab and budget matters concering other myxobacterial projects.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yao Xiao, Daniel Wall. 2014. Genetic redundancy, proximity, and functionality of lspA, the target of antibiotic TA, in the Myxococcus xanthus producer strain. Journal of Bacteriology 196:1174-1183.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Yao Xiao, a graduate student on the project, presented her work at a University of Wyoming wide dissertation defense held within the Molecular Biology Department. Yao also presented her work at the 40th International Conference of the Biology of Myxobacteria held in Beijing China. Changes/Problems: As indicated above, due to personnel changes, i.e. students graduating, and also demands for another federally funded project in the lab, the project director has requested a one year no cost extension. In essence no funds are requested for the 2013-14 funding cycle as those funds will instead be deferred to the following year. Progress on the project will consequently be delayed by one year. What opportunities for training and professional development has the project provided? This project has involved the training of students working with the project director. Specifically, Cameron Finley, an undergraduate, was trained in microbiology and microbe-plant interactions in the laboratory. Cameron has now graduated from the University of Wyoming and is attending graduate school at Texas A&M University where he is studying plant-microbe interactions as it relates to crops. Chris Vassallo and Yao Xiao, graduate students, have also worked on this project and have received training in molecular biology, microbiology and plants. Dr. Yao Xiao graduated this past summer with a PhD degree and is now a postdoc at the University of Michigan studying the human microbiome. Chris Vassallo is continuing his studies in the lab. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? Due to personnel changes in the project the current plan is to delay activities for one year through a no cost extension.
Impacts
(N/A)
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Xiao, Y. and Wall, D. 2014. Genetic Redundancy, Proximity, and Functionality of lspA, the Target of Antibiotic TA, in the Myxococcus xanthus Producer Strain. J. Bacteriol. On-line version doi:10.1128/JB.01361-13
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