Progress 10/01/13 to 09/30/17
Outputs
Target Audience:Results from this research were reported at the Food Research Institute Annual Meeting, University of Wisconsin - Madison, April 18-19 to an audience of researchers and food industry stakeholders. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Barak have directly mentored the graduate student, Grace Kwan, who has carried out all the metabolic experiments to date, along with the undergraduate researcher, Louise Pritcher. How have the results been disseminated to communities of interest?We have reported our results at public seminars at the University of Wisconsin - Madison, Department of Plant Pathology attended by researchers interested in plant - microbe interactions, to the international scientific community at the 5th American Society for Microbiology Salmonella Conference, the American Phytopathological Socitey, and the Food Research Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This research has produced a change in knowledge as to the genes and the specific conditions which influence which govern biofilm formation and growth of Salmonella enterica in the rhizosphere and differences that may affect Salmonella persistence in the phyllosphere. Salmonella enterica successfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the important S. enterica plant colonization factors are those involved in biofilm formation. S. enterica biofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. For objective 1, we examined the mechanisms used by S. enterica to colonize plants, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority of in vitro cellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated that adrA and colanic acid biosynthesis is associated with S. enterica desiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial for S. enterica survival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli. For objective 2, we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in the Salmonella-containing vacuole, we found that the presence of individual nutrients and the utilization of particular pathways differed between the two host niches but similar metabolic networks were required for S. enterica colonization of both hosts. Networks necessary for both included biosynthesis of amino acids, purines, and vitamins and catabolism of glycerol and glucose were necessary. Additionally, while both fatty acid biosynthesis and degradation contributed to S. enterica animal colonization, only fatty acid biosynthesis was required during plant colonization. We also found that a manA mutant is severely compromised in its ability to colonize plants, due to attachment and growth defects. Reduced attachment was linked to the inability to produce O-antigen whereas impaired growth was caused by a failure to produce a novel metabolic regulator, mannose-6-phosphate. Utilization of similar metabolic networks for different functions is a strategy that facilitates S. enterica exploitation of cross-kingdom hosts. For objective 3, we found that S. enterica may use a copper tolerance/resistance operon to persist on tomato. E. coli has two copper tolerance operons while S. enterica has three. This is an interesting finding as crops which have been linked to salmonellosis outbreaks, specifically tomato, are commonly sprayed with copper to reduce plant disease. The plant disease treatment is broad spectrum and likely alters the microbiome of the crop which could lead to the persistence or proliferation of Salmonella where E. coli would be removed.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2017
Citation:
Kwan, G., Cowles, K.N, Plagenz, B., Pisithkul, T., Amador-Noguez, D., and Barak, J.D. Comparison between Salmonella enterica plant and animal colonization factors reveals metabolic network division between lifestyles. Front. Microbiol.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Fresh produce a common vector of Salmonella, Food Research Institute Annual Meeting, University of Wisconsin Madison, April 18-19.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Results from this research were reported to an audience of researchers at the 5th American Society of Microbiology Salmonella Conference in Potsdam, Germany. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Barak have directly mentored the graduate student, Grace Kwan, who has carried out all the metabolic experiments to date, along with the undergraduate research, Louise Pritcher. How have the results been disseminated to communities of interest?We have reported our results at public seminars at the University of Wisconsin - Madison, Department of Plant Pathology, which was attended by researchers interested in plant - microbe interactions, to the international scientific community at the 5th American Society for Microbiology Salmonella Conference, and the Food Research Annual Meeting. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue our investigation of plant colonization by enteric human pathogens, metabolic differences between closely related enteric genera, and comparison between S. enterica strategies of animal and plant colonization.
Impacts
What was accomplished under these goals?
This research has produced a change in knowledge as to the genes and the specific conditions which influence the govern biofilm formation and growth of Salmonella enterica in the rhizosphere. Salmonella enterica successfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the important S. enterica plant colonization factors are those involved in biofilm formation. S. enterica biofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. For objective 1, we recently examined the mechanisms used by S. enterica to colonize plants, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority of in vitro cellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated that adrA and colanic acid biosynthesis is associated with S. enterica desiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial for S. enterica survival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli. For objective 2:To determine the S. enterica metabolic requirements for plant colonization, we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in the Salmonella-containing vacuole, we found that the presence of individual nutrients and the utilization of particular pathways differed between the two host niches but similar metabolic networks were required for S. enterica colonization of both hosts. Networks necessary for both included biosynthesis of amino acids, purines, and vitamins and catabolism of glycerol and glucose were necessary. Additionally, while both fatty acid biosynthesis and degradation contributed to S. enterica animal colonization, only fatty acid biosynthesis was required during plant colonization. We also found that a manA mutant is severely compromised in its ability to colonize plants, due to attachment and growth defects. Reduced attachment was linked to the inability to produce O-antigen whereas impaired growth was caused by a failure to produce a novel metabolic regulator, mannose-6-phosphate. Utilization of similar metabolic networks for different functions is a strategy that facilitates S. enterica exploitation of cross-kingdom hosts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Cowles KN, Willis DK, Engel TN, Jones JB, and Barak J.D. 2016. Diguanylate cyclases, AdrA and STM1987, regulate Salmonella enterica exopolysaccharide production during plant colonization in an environment-dependent manner. Appl Environ Microbiol. 82(4): 1237-1248
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Produce not where and what you expected, Food Research Institute Annual Meeting, University of Wisconsin Madison, April 18-19
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Results from this research were reported to an audience of microbiologist and plant science specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Dr. Barak has directly mentored the graduate student, Grace Kwan, who has carried out all the metabolic experiments to date. How have the results been disseminated to communities of interest?We have reported our results at public seminars at the University of Wisconsin - Madison, which is Department of Plant Pathology attended by researchers interested in plant - microbe interactions, and to the international scientific community at Rhizosphere 4 held in the Netherlands, June 2015. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue our investigation of plant colonization by enteric human pathogens, metabolic differences between closely related enteric genera, and comparison between S. enterica strategies of animal and plant colonization.
Impacts
What was accomplished under these goals?
This research has produced a change in knowledge as to the genes and the specific conditions which influence which govern biofilm formation and growth of Salmonella enterica in the rhizosphere. Salmonella enterica successfully persists on plants, utilizing fresh produce as a vector to animal hosts. Among the important S. enterica plant colonization factors are those involved in biofilm formation. S. enterica biofilm formation is controlled by the signaling molecule cyclic di-GMP and represents a sessile lifestyle on surfaces that protects the bacterium from environmental factors. Thus, the transition from a motile, planktonic lifestyle to a sessile lifestyle may represent a vital step in bacterial success. For objective 1, we recently examined the mechanisms used by S. enterica to colonize plants, including the role of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), the enzymes involved in cyclic di-GMP metabolism. We found that two biofilm components, cellulose and curli, are differentially required at distinct stages in root colonization and that the DGC STM1987 regulates cellulose production in this environment independent of AdrA, the DGC that controls the majority of in vitro cellulose production. In addition, we identified a new function for AdrA in the transcriptional regulation of colanic acid and demonstrated that adrA and colanic acid biosynthesis is associated with S. enterica desiccation tolerance on the leaf surface. Finally, two PDEs with known roles in motility, STM1344 and STM1697, had competitive defects in the phyllosphere, suggesting that regulation of motility is crucial for S. enterica survival in this niche. Our results indicate that specific conditions influence the contribution of individual DGCs and PDEs to bacterial success, perhaps reflective of differential responses to environmental stimuli. Growth is essential for successful colonization of a niche. S. enterica has an infectious dose to humans of approximately 1,000 cells. Since environmental S. enterica populations are small, the bacterium must grow on plants to reach an infectious dose. Understanding plant-specific S. enterica metabolism may suggest ways to control pathogen growth on produce. For objective 2, we found that over one-quarter of the known metabolic reactions for S. enterica are active during growth in root exudates. Specific amino acids are quantitatively limiting for optimal S. enterica growth, indicating that amino acid biosynthesis is required for optimal growth in association with roots. The relative importance of amino acid biosynthesis or transport is dependent on the amino acid and/or growth stage of the bacterium. We conclude that an emerging paradigm of bacterial rhizosphere competence is metabolic robustness. To achieve dramatic reductions in bacterial growth in this environment, central metabolic networks, rather than dedicated pathways, may need to be targeted. The metabolic robustness of S. enterica that makes metabolic manipulation difficult is the same trait that contributes to its success as a seed and root colonist.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Cowles, K. and Barak, J.D. 2015. Environmentally-dependent regulation of root colonization factors in Salmonella enterica. Rhizosphere 4. Maastricht, The Netherlands.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Dr. Barak has mentored the graduate student, Grace Kwan, who has carried out all the experiments to date. How have the results been disseminated to communities of interest? We have reported our results at public seminars at the University of Wisconsin - Madison. Researchers from the department of Plant Pathology attended and were interested in plant - microbe interactions. What do you plan to do during the next reporting period to accomplish the goals? We plan to continue our investigation of plant colonization by enteric human pathogens and metabolic differences between closely related enteric genera.
Impacts
What was accomplished under these goals?
This research has produced a change in knowledge as to the genes and the specific conditions which influence which govern biofilm formation in Salmonella enterica. For objective 1, we identified potential environment specificities amongst S. enterica GGDEF-containing proteins (Gcps), characterizing their role in colonization of and persistence in two host niches, roots and leaves. In competition with wildtype, an yhdA mutant was at a disadvantage in both the rhizosphere and phyllosphere, while two other gcp mutants adrA and yhjK were less competitive in the phyllosphere, but displayed an advantage over wildtype in the rhizosphere. A STM1987 mutant was defective for rhizosphere colonization in individual strain and competition assays while a yegE mutant had reduced rhizosphere colonization in individual inoculation, but competed equally well with wildtype. STM1987 and yegE mutant defects in rhizosphere colonization were alleviated by increased temperature. In addition to the plant phenotypes, an yhjK mutant also produced abnormal biofilms. Pellicles formed by an yhjK mutant appeared more fragile than wild-type pellicles and were cellulase-sensitive, indicating a potential defect in the production of proteinaceous curli. Additionally, colony morphology development was delayed in an yhjK mutant, suggesting temporal regulation of cellulose production by YhjK. Our results indicate that specific conditions, including plant niche, temperature, and time, alter the contribution of individual Gcps to bacterial success, and perhaps reflective of differential responses to environmental stimuli and not simply functional redundancy amongst Gcps. For objective 2, we conducted experiments to determine active metabolic pathways of Salmonella enterica. We found that over one-quarter of the known metabolic reactions for S. enterica are active during growth in sprouting seed exudates. Specific amino acids are quantitatively limiting for optimal S. enterica growth, indicating that amino acid biosynthesis is required for optimal growth in association with roots. The relative importance of amino acid biosynthesis or transport is amino acid and growth stage dependent. We conclude that an emerging paradigm of bacterial rhizosphere competence is metabolic robustness. To achieve dramatic reductions in bacterial growth in this environment, central metabolic networks, rather than dedicated pathways, may need to be targeted. The metabolic robustness of S. enterica that makes metabolic manipulation difficult is the same trait that contributes to its success as a seed and root colonist. For objective 3, we have initiated a project to identify genus-specific metabolic strategies of the closely related bacteria, S. enterica and Escherichia coli. This project builds off our original finding that these two enteric pathogens grow differently in lettuce root exudates. Our preliminary findings are that S. enterica has either a prolonged lag phase or fails to grow in the initially released root exudates. We have also found that, in competition, E. coli outcompetes S. enterica for the first 24 h following seed imbibing. However, following that time period, S. enterica populations begin to rise as E. coli populations proportionally decline and, thus, S. enterica must outcompete E. coli at 32-38 h. We continue to examine species-specific metabolic strategies between these enteric human pathogens with the hope of understanding differences in human outbreak data where E. coli causes more outbreaks from contaminated lettuce than S. enterica, even though both are common in the crop production environment. Studies under objective 3 are ongoing.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Kwan, G., Pisithkul, T., Amador-Noguez, D., and Barak, J.D. De novo amino acid biosynthesis contributes to Salmonella enterica growth in alfalfa seedling exudates. Applied Environmental Microbiology
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