Progress 05/01/21 to 04/30/22
Outputs
Target Audience:The target audience reached in this reporting period included plant pathologists and microbiologists of multiple ranks (graduate students, postdocs, professors, USDA ARS researchers) through a presentation at Plant Health 2021. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunities to two postdoctoral scholars, including allowing one postdoc to present a poster at Plant Health 2021 and give a departmental seminar. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through a peer-reviewed publication, a poster presentation at Plant Health 2021, and a departmental seminar. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess whether reduced toxin-antitoxin binding is the source of the higher persistence in the streptomycin high persisters. Objective 2: Objective complete. Objective 3: Assess whether the high persister mutants survive phyllosphere-associated stressors better, worse, or the same as the wildtype strain.
Impacts
What was accomplished under these goals?
This work has provided deeper insight into the prevelance and function of stress tolerance systems in plant-associated microbes. These research findings impact our knowledge of how to take advantage of these systems in two ways. First, it will help guide future efforts to block or circumvent the ability of plant pathogens to survive lethal stresses, which will aid in our efforts to reduce crop loss to plant disease. Second, it will aid us in developing strategies for promoting resiliance of agriculturally beneficial microbes under similar stresses. Objective 1: This objective is to understand the genetics that underlies streptomycin and tailocin persistence. Following up on the observation that a highly conserved TA system is the site of streptomycin high-persister mutations, we examined the total genomic TA content across a diverse collection of Pseudomonas syringae genomes. These results demonstrated that are 6 TA systems that are widely prevelant (found in >80% of examined genomes), 8 TA systems that are common (found in 20% - 80% of examined genomes), and 12 TA systems that are rare (found in <20% of examined genomes). In examining patterns of TA systems across the Pseudomonas syringae species complex, we found an average of ~15 TA systems per genome. Strains belonging to phylogroups 1, 3, 5, and 13 on average encode significantly more than the average, whereas phylogroups 2, 4, 9, and 10 encode significantly less than this average. The number of TA systems correlates with both the size of the genome and number of plasmids harbored by each organism. Taken together these results indicate that TA systems and potentially TA system-mediated stress tolerance is likely ecologically important in dynamic ways across the species. Objective 2: This objective is to understand teh physiological state of persister cells. As our previous results indicated that streptomycin persisters survive antibiotic exposure in a state of reduced metabolism, we tested whether such a reduced metabolic state would result in increased survival to other stressors. To test this, we exposed the streptomycin high persisters to extended starvation conditions. Unexpectedly, the streptomycin high persisters suffered greater mortality under starvation conditions. These results potentially indicate that plant-associated microbes have specific genetic and physiological mechanisms that cope with distinct stressors. Objective 3: Work has continued to be delayed on this objective as the postdoc working on this grant accepted a new job in industry. A new postdoc has recently been hired and will focus their efforts on addressing this objective.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Kandel PP, Naumova M, Fautt C, Patel RR, Triplett LR, and Hockett KL. Genome Mining Shows Ubiquiitous Presence and Extensive Diversity of Toxin-Antitoxin Systems in Pseudomonas syringae. Frontiers in Microbiology. doi:10.3389/fmicb.2021.815911
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:The target audience reached in this reporting period included plant pathologists and microbiologists of multiple ranks (graduate students, postdocs, professors, USDA ARS researchers) through peer-reviewed publication and presentation at Plant Health 2020. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunities for two postdoctoral scholars and one high school volunteer. Additionally, this project has allowed one postdoc to present a poster at Plant Health 2021. How have the results been disseminated to communities of interest?Results have been dissimenated to communities of interest through a peer-reviewed publication and a poster presentation at Plant Health 2021. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess the biochemical function of the identified TA system by assaying purified proteins and mutant strains for their ability to degrade or suppress NADP levels. Objective 2: The physiological effects of the TA system mutations is being assessed. Objective 3: Test the effect of the high persister mutations on phyllosphere-associated stressors.
Impacts
What was accomplished under these goals?
Objective 1: This objective is to understand the genetics that underlies streptomycin and tailocin persistence. The alleles associated with streptomycin high persistence have been confirmed through molecular genetics to be causative for the high-persistence phenotype. We demonstrated that this toxin and the mutant variants significantly decrease growth of E. coli when heterologously expressed. Our data indicates that the mutations result from increased recovery from dormancy rather than higher frequency of entering into a dormant state. These results have the potential to contribute to a change in knowledge of how the frequency of persister cells is regulated. Objective 2: This objective is to understand the physiological state of tailocin survivors and how it compares to that of antibiotics. By performing culturing and cell-counting with combinations of four fluorescent physiological stains (Redox Sensor Green, propidium iodide,DRAQ7, and Hoescht to assess redox activity, compromised membranes, cell death, and nucleic acid content, respectively) we were able to quantify the populations occupying five distinct physiological states after inhibitory streptomycin and tailocin exposure. We observed that the antibiotic-treated populations undergo a diversification of pathogen physiological states. This includes an increased variability in redox activity among living cells, and the emergence of significant populations of living membrane compromised cells and redox-inactive membrane intact cells. Cell counts indicate that onlya tiny proportion of living cells are culturable.Conversely, most cells die rapidly following tailocin exposure, with the living population physiologically homogenous and highly culturable. Using fluorescence-activated cell sorting (FACS), we have observed that the culturable population between the two treatments is distinct: the majority of culturable cells remaining after antibiotic exposure come from the cell fraction exhibiting a low level of redox activity as determined by green intensity, while the culturable cells surviving tailocin had a moderate level of redox activity. Only the fraction containing culturable cells were infectious on the plant, while unculturable cells in an elevated redox state were noninfectious. These findings provide the first indication that distinct subpopulations confer survival to different types of antimicrobial treatments, which will aid us in determining how best to combat stress-tolerant cells in agricultural settings. This was further supported by our observation that a combination or sequential treatment of tailocin and streptomycin resulted in complete sterilization of Pph, whereas either treatment alone resulted in culturable and infectious population. Other standard antibiotics were not as effective as tailocin in killing streptomycin persisters, and streptomycin performed better than other antibiotics in killing tailocin persisters, suggesting synergism between aminoglycosides and membrane disruptors. The finding that a simultaneous streptomycin and tailocin treatment effectively sterilizes cultures is promising for understanding how to develop combination treatments for bacterial control. The results have been reported in mBio. Objective 3: Work delayed because of covid-19 related restrictions on access to laboratory and research facilities.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Patel RR, Kandel PP, Traverso E, Hockett KL, and Triplett LR. Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence to Survive Bacteriocin and Streptomycin Treatments. mBio. 12(2) e00161-21. doi:10.1128/mBio.00161-21
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Progress 05/01/19 to 04/30/20
Outputs
Target Audience:The target audience reached in this reporting period included plant pathologists and microbiologists of multiple ranks (graduate students, postdocs, professors, USDA ARS researchers) through peer-reviewed publication. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training opportunities for two postdoctoral scholars, and one high school volunteer. Additionally, this project has allowed one postdoc to present a talk at Plant Health 2020. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through a peer-reviewed publication and an oral presentation at Plant Healthy 2020. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The identified TA system will be cloned for molecular genetic and in vitro characterization. Objective 2: A manuscript describing the results reported above is currently in preparation. Objective 3: Now that we know which populations are culturable (from objective 2), we will now initiate selecting persister sub-populations through FACS or antimicrobial treatment to inoculated onto plants.
Impacts
What was accomplished under these goals?
Crop pests, including microbial pathogens, are a major impediment to efficient crop production in the US and worldwide. To control these pests, both chemicals and biologicals are routinely sprayed. These control methods often breakdown for multiple reasons, one of which being that the crop pathogens are able to temporarily tolerate either chemical or biological treatment. This problem of temporary tolerance may likely create additional problems for crop production in that it may allow the pathogen to 'overwinter', where it persists in a field from one season to the next, thereby inciting additional disease in the subsequent growing season. To begin to address this problem, we have begun to investigate the mechanisms that allows pests to achieve temporary tolerance using multiple approaches. To date, we have identified several mechanisms that promote tolerance. Significantly, we have found that certain combinations of treatments can completely eliminate temporary tolerance within a laboratory setting. If these treatments translate into the agricultural field setting, they have the potentiall to significantly enhance the efficiency of controlling crop diseases, thereby reducing the cost of production and amount of chemical applications needed. This would translate into better economic outcomes for both producers and consumers. Objective 1: Collections of both P. syringae pv. phaseolicola (Pph) tailocin and streptomycin high-persisters were recovered and whole-genome sequenced. The underlying mutations were identified. The tailocin high-persister mutations were confirmed to be causative for the phenotype using molecular genetic methods. In total, X tailocin and Y streptomycin persisters have been recovered to date. Regarding the tailocin high persister mutations, one hypothetical gene has been identified as influencing this phenotype. This gene is found in other Pseudomonas pathogens, but is not widely distributed in other plant pathogenic bacteria. Regarding the streptomycin high persister mutations, a toxin-antitoxin (TA) system has been identified which is widely distributed among P. syringae phytopathogens. In total, these results have led to a change in knowledge surrounding the genes that contribute to antimicrobial tolerance in an imporant bacterial phytopathogen. Objective 3: Nothing to report for this goal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Kandel PP, Baltrus DA, Hockett KL. Pseudomonas Can Survive Tailocin Killing via Persistence-like and Heterogenous Resistance Mechanisms. Journal of Bacteriology. 202:e00142-20. doi:10.1128/JB.00142-20
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