Recipient Organization
University of Utah
201 S President Circle RM 408
Salt Lake City,UT 84112-9023
Performing Department
(N/A)
Non Technical Summary
The many microbial species that colonize plants are in competition with one another on the plant. To win this battle, pathogenic plant microbes employ diverse weapons capable of killing neighboring microbes that belong to other strains without harming microbes of the same strain. The goal of this project is to determine how a potent and common microbe-killing weapon is targeted to some bacteria and not others. If the mechanism of targeting is understood at a basic molecular level, it may become possible to engineer novel antibiotics that kill only certain pathogens without harming "good" bacteria. Broader Impacts: The project has significant potential to benefit society. Foremost is the security of food to feed people. The mechanisms discovered in this research will apply to food crops and the pathogens that destroy those crops. Discoveries about the evolution of pathogens and how they can be coerced into fighting each other will help agricultural scientists protect food crops of the future. Additionally, the molecules central to this plant-bacteria system are relevant to animals and humans. Understanding how plant pathogens identify and kill each other has the potential for precise control of human pathogens, even those that are rapidly developing resistance to traditional antibiotics. The proposed work will enroll trainees with the NSF-funded STEM Ambassador Program. Each trainee will identify scientifically underserved community groups, design and execute outreach activities, and measure the effectiveness to increase understanding and appreciation of science among nonscientists.In this project a team of scientists at two sites, in the US and the UK, will work together to unravel mechanisms that Pseudomonas pathogens of plants use to target and kill one another. Bacteria that invade and cause disease in plants make use of molecular killing machines that were derived originally from the tail apparatus of bacteriophages - the viruses of bacteria - and are therefore called tailocins. Tailocins exhibit high specificity in their killing - the tailocins made by a plant pathogen do not attack plant cells or structures, instead kill a subset of other competing bacteria, and somehow avoid killing bacteria of the same strain. Intellectual Merit: The hypothesis to be tested by this project is that killing specificity depends on a particular molecular receptor built into the lipopolysaccharide (LPS) component of the outer membrane. Differences in the carbohydrate composition of the LPS between bacterial strains and species render some strains susceptible to specific tailocins while others are resistant. In this project the LPS differences between bacterial strains that confer resistance and susceptibility to tailocins will be elucidated through a combination of synergistic approaches including plant infection studies, biochemistry, and bioinformatic analysis. In total, the project will discover mechanisms - evolutionary and structural - that suppress a common pathogen in plants and in so doing will address fundamental questions of how to prevent the spread of single strains of bacteria, and how to maintain microbial diversity.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Goals / Objectives
The many microbial species that colonize plants are in competition with one another on the plant. To win this battle, pathogenic plant microbes employ diverse weapons capable of killing neighboring microbes that belong to other strains without harming microbes of the same strain. The goal of this project is to determine how a potent and common microbe-killing weapon is targeted to some bacteria and not others. If the mechanism of targeting is understood at a basic molecular level, it may become possible to engineer novel antibiotics that kill only certain pathogens without harming "good" bacteria. Broader Impacts: The project has significant potential to benefit society. Foremost is the security of food to feed people. The mechanisms discovered in this research will apply to food crops and the pathogens that destroy those crops. Discoveries about the evolution of pathogens and how they can be coerced into fighting each other will help agricultural scientists protect food crops of the future. Additionally, the molecules central to this plant-bacteria system are relevant to animals and humans. Understanding how plant pathogens identify and kill each other has the potential for precise control of human pathogens, even those that are rapidly developing resistance to traditional antibiotics. The proposed work will enroll trainees with the NSF-funded STEM Ambassador Program. Each trainee will identify scientifically underserved community groups, design and execute outreach activities, and measure the effectiveness to increase understanding and appreciation of science among nonscientists.In this project a team of scientists at two sites, in the US and the UK, will work together to unravel mechanisms that Pseudomonas pathogens of plants use to target and kill one another. Bacteria that invade and cause disease in plants make use of molecular killing machines that were derived originally from the tail apparatus of bacteriophages - the viruses of bacteria - and are therefore called tailocins. Tailocins exhibit high specificity in their killing - the tailocins made by a plant pathogen do not attack plant cells or structures, instead kill a subset of other competing bacteria, and somehow avoid killing bacteria of the same strain. Intellectual Merit: The hypothesis to be tested by this project is that killing specificity depends on a particular molecular receptor built into the lipopolysaccharide (LPS) component of the outer membrane. Differences in the carbohydrate composition of the LPS between bacterial strains and species render some strains susceptible to specific tailocins while others are resistant. In this project the LPS differences between bacterial strains that confer resistance and susceptibility to tailocins will be elucidated through a combination of synergistic approaches including plant infection studies, biochemistry, and bioinformatic analysis. In total, the project will discover mechanisms - evolutionary and structural - that suppress a common pathogen in plants and in so doing will address fundamental questions of how to prevent the spread of single strains of bacteria, and how to maintain microbial diversity.
Project Methods
Methods/Research PlanAim 1 Tradeoffs - Do mutations that confer resistance to tailocins affect the ability of Pseudomonas to colonize the plant?Rationale: In our previous research we determined that co-occurring Pseudomonas strains differ in their susceptibility to co-occurring tailocins and that several strains were broadly resistant to all tailocins tested. Despite this resistance, fully resistant strains are found at low frequencies in the Pseudomonas pathogen population (11%). Why have the more resistant genetic backgrounds not risen to prominence (or fixed) in the Pseudomonas population? One hypothesis is that there are tradeoffs to evolving broad-scale resistance -- the LPS variants and other resistance loci that are best able to resist tailocins are less fit in other capacities. Indeed, previous research on the evolution of phage resistance in Pseudomonas found phage-resistant strains to have significant general growth defects when grown in the absence of phage35. In an insect colonization model, Heiman et al.24 found the tailocin-resistance mutations in a Pseudomonas strain had a negative fitness effect upon colonization -- the insect immune system recognized the tailocin-resistant strain (with a mutated LPS) better than it did the susceptible strain.We have identified a large suite (70 genes) of tailocin-resistance mutations1. In this Aim, we will identify which of these resistance mutations show the strongest resistance phenotypes, and which (if any) of these mutations compromise the growth of the P. viridiflava strain. First, we will generate an ordered single mutant library of the P. viridiflava genome. We will then test each of the different resistance variants for their growth capabilities in the plant and plant-related environments. Lastly, we will test for plant immune detection of the different LPS variants.Aim 2: Biochemistry - How does variation in the tail fiber affect killing specificity?Rationale: Molecular interactions important for targeting tailocins to kill "other" bacteria and thereby avoid "self" bacteria will be revealed through structure determination and structure-guided biochemical analysis. We will find the host-specific glycan receptors, visualize the relevant molecular interactions, and test the importance of specificity determinants through mutational analysis. Discoveries from these activities will reveal the code for specificity and thereby biochemical constraints for coevolution.Aim 3. Evolution - How are variants of the tailocin and LPS evolving across populations and over time?Rationale: In our previous analysis of both historical and contemporary P. viridiflava genomes, we uncovered the genetic continuity of the same bacterial pathogenic metapopulation across two centuries in Southern Germany. Furthermore, we observed the maintenance of a finite and defined set of TFP variants, which co-evolved with their receptors in the LPS during the same timeframe1. What remains unknown is the evolutionary durability of various TFP variants. How frequently do these variants (re)-emerge in the metapopulation?, through which mechanisms? And at what rate can resistance to TFP variants arise in the LPS carbohydrate profiles? To address these questions, we have sequenced 338 herbarium-derived A. thaliana metagenomes spanning about two centuries (Fig 6). The broad geographical distribution of this expanded dataset will enable distinguishing between population continuity and replacement at different locations. Moreover, the wider sampling period (heterochronous nature) of this new dataset, in contrast to our previous analysis, will generate populations that exhibit detectable amounts of de novo evolutionary change across genetic sequences sampled at different time points, termed "measurably evolving populations" (MEP) 84,85. The Pseudomonas MEP will facilitate the calculation of the rate at which substitutions accrue over time (substitution rate) in wild populations of P. viridiflava. This information, in turn, will enable the estimation of divergence times of different sublineages and to study the interplay between susceptibility and resistance across the timed P. viridiflava phylogeny.