Source: UNIVERSITY OF DELAWARE submitted to
PLANT RESPONSES TO FOOD-BORNE BACTERIA AND VIRUSES AND MECHANISMS USED BY PATHOGENS TO SURVIVE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0224932
Grant No.
2011-67018-30217
Project No.
DEL00698
Proposal No.
2010-03525
Multistate No.
(N/A)
Program Code
A1301
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2015
Grant Year
2011
Project Director
Kniel-Tolbert, K. E.
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
Animal And Food Sciences
Non Technical Summary
By studying concurrently three important pathogens that are also the main causes of foodborne illness associated with fresh produce we will gain a better understanding of how the plant and the microbe interact during the processes of association, attachment, internalization and survival. This is the main objective of this program. This unique research team is composed of a virologist and a bacteriologist who are experts in produce-specific food safety and a plant pathologist has significant experience in working with both phyto- and enteric pathogens. These three individuals can accomplish the objectives of the proposed research and as the main goal of the Food-borne Pathogens-Plant Interactions Foundational Program this research will generate fundamental information into the physical and molecular mechanisms that enable human pathogens to attach, internalize, grow and survive in and on fresh produce. Additionally this work will generate information on how plants interact with food-borne pathogens and if these associations affect the attachment and fate of human pathogens on fresh produce. With regards to potential long range improvement of U.S. agriculture and food systems the goals of this project will clearly be used to better enhance the safety of produce and reduce transmission of these pathogens in the field. Acquired data will be translated into applied farming practices and integrated into Good Agricultural Practices where applicable. In particular information concerning persistence and survival will impact growing and irrigation practices. Information gained by studying the plant defense response in relation to colonization by viruses and bacteria will also impact pre-harvest growing practices.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121430110014%
7121430110112%
7121460110014%
7121460110112%
7124010110012%
7124010110112%
7124030110012%
7124030110112%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1430 - Greens and leafy vegetables; 4010 - Bacteria; 4030 - Viruses; 1460 - Tomato;

Field Of Science
1101 - Virology; 1100 - Bacteriology;
Goals / Objectives
The ultimate long term goal of this project is to understand and characterize the mechanisms that allow human foodborne pathogens, specifically norovirus, pathogenic E. coli and Salmonella, to attach to, persist on/in, and colonize plants. By elucidating the mechanisms at the plant-pathogen interface using genetic and proteomic approaches, we will generate information that will be useful in inhibiting the attachment and persistence of enteric pathogens on leafy greens and other produce commodities. These results will lead to the more effective use and application of antimicrobials and good agricultural practices, ultimately reducing the number of illnesses and detrimental impact on public health. Three specific objectives are listed below. 1. Concurrently evaluate the persistence of three foodborne pathogens (E. coli O157:H7, Salmonella, and Norovirus) on foliar surfaces of plants (lettuce and spinach) under the same conditions to facilitate a direct comparison of the survival characteristics of these organisms in the phyllosphere. 2. Investigate plant defense pathways during attachment and internalization of E. coli O157:H7, Salmonella and noroviruses. Determine if plants respond to these human pathogens using one or more plant-specific defense strategies. 3. Evaluate the metabolic pathways used by pathogenic bacteria to attach to plants, specifically using a novel approach to identify the major proteins of pathogenic bacteria using proteomics.
Project Methods
Baby spinach and Romaine lettuce plants will be grown in BSL-2 growth chambers using previously utilized methods under dynamic temperature and light conditions. Plants at 14- or 21-day stages of growth will be inoculated with the respective pathogens grown in bovine fecal suspensions. Shoot tissue will be inoculated with respective pathogenic suspensions using multiple spot inoculations (10 &#956;l spots) on foliar surfaces so that a total of 100 spots (1 ml) will be applied to each plant on multiple leaves. Post-homogenization samples will be analyzed by plating or a modified Most Probably Number technique or RT-PCR. Plants at the second true leaf stage will be dip-inoculated with either an eight strain cocktail of Salmonella enterica or four strain cocktail of EHEC. Plants will be kept in a controlled-environment under a day and night cycle of 12 h with a temperature of 26C and 18C, respectively. Humidity will be constant at 75%. Four leaflets will be removed from each plant seven and 14 days post-inoculation and placed into individual tubes of 500 &#956;l water. The leaflet will be weighed, homogenized, aliquots serially diluted, and plated on selective media for population enumeration. Weights will be used as a normalization factor to determine the cfu/g of tissue. Samples will be taken 1, 7, 14, and 30 dpi. Populations will be log transformed and means will be tested for significance using Duncan's multiple range test by SAS. Internalized virus will be quantified using plaque assay in RAW cells to determine the amount of infectious virus and by qRT-PCR to determine the amount of genomic copies of nucleic acid present. Lettuce seeds will be grown for 20 days prior to being subjected to contaminated water containing 5x10e8 RT-qPCRU/ml MNV for 24 hr, followed by removal of virus solution and replaced with virus free nutrient solution every day for up to 5 days. Total RNA will be eluted with 60 &#956;l RNase-free water and a two step RT-qPCR will be used to quantify the virus concentration. Humidity is a major factor controlling plant transpiration and guttation. Plants will be grown under two conditions, one with high humidity and one with lower (<70%). Romaine lettuce at the rosette stage, ~14 days old, will be dip-inoculated. Leaves will be sampled 72 h post-inoculation. Proteins will be extracted with the All prep DNA/RNA/Protein kit, precipitated, and then dissolved in a kit-supplied sample buffer. Proteins will be separated by 1-dimensional SDS/PAGE and analyzed after tryptic digestion by reversed-phase high-performance liquid-chromatography coupled to electrospray-ionization tandem mass-spectrometry. MS data will be converted to peak lists and analyzed with 2 search algorithms and validated with Scaffold. MS/MS spectra will be searched against a database consisting of protein sequences obtained from RefSeq and a second database consisting of all protein sequences from the complete genomes of Salmonella enterica and EHEC. For protein identification, at least 2 peptide matches will be required. The minimum peptide identification probability will be 95% and protein identification probability will be 99%.

Progress 04/01/11 to 03/31/15

Outputs
Target Audience:The target audience served by this project includes academics and industry professionals who will be interested in the scientific outcomes of the project. This is a cross-disciplinary audience including members of the food science and plant science communities. Growers of fruits and vegetables and extension specialists working with these communities will also be interested in the scientific outcomes that add to the knowledge of survival and interaction of human pathogens on plants. Science-based educational programs including work from this program have been shared through formal classroom instruction at the University of Delaware and the University of Wisconsin-Madison. Scientific information was also shared through extension and outreach less formal educational situations at the University of Delaware and at the USDA-ARS in Beltsville, MD. Changes/Problems:There were no major changes in the original scientific plan of the project. There were some delays in the research due to mechanical issues with growth chambers and due to the government shutdown. We are continuing to conduct some research that is related to this project to complete an interesting aspect of the study, which is assessment of salicylic acid and jasmonic acid on the persistence of E.coli , Salmonella, and norovirus, being conducted at the University of Delaware. What opportunities for training and professional development has the project provided?Two graduate students and 7 undergraduate students benefited from training and professional development through the funding made available through this project. These students conducted research and participated in dissemination of the research findings at the University of Delaware, University of Wisconsin-Madison, and at the USDA-ARS in Beltsville, MD. These students represented diverse backgrounds and cultures. At least two of the undergraduate students have gone on to continue studying food science and plant science in graduate school and one other has retained a position at the USDA-ARS and another has gone into research with a food company. The other undergraduate students are still in school for their undergraduate degrees. The two graduate students at the Universities have graduated and have successfully received positions of emplyment. How have the results been disseminated to communities of interest?Results have been disseminated through both poster and oral presentations at annual scientific meetings, including those of the International Association for Food Protection and the American Phytopathological Society. Results and information garnered from this project have been disseminated to the gerenal community and to growers at outreach meetings of the Mid-Atlantic Growers, USDA-ARS Poster Day, and in formal classroom teaching at the University of Delaware and the University of Wisconsin-Madison. 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 project is unique in that it addresses three important foodborne pathogens and their interactions with fresh produce, including leafy greens and alfalfa. The results from this project have caused a change in knowledge of what plant exudates are required for Salmonella growth. Reducing these exudates could reduce Salmonella growth and restrict the human pathogen below infectious doses. Additional results caused a change in knowledge that Salmonella can mask plant disease, rotting tissue, in bagged salads. Interactions between soft rot plant pathogens and human pathogens can lead to higher populations in human pathogens in what appears to be healthy looking leafy greens. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h post-inoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over one-quarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth. Regarding the interactions of E.coli on the spinach leaf, as a model, it appears that the rpoS gene system may not be as important as others in the persistence in produce. This research represents the firs study to concurrently and comparatively examine the persistence of E. coli O157:H7, S. Typhimurium, and the murine norovirus (MNV), a norovirus surrogate, on the spinach foliar surfaces. Populations of wild-type (wt) E. coli O157:H7 declined significantly ( P &lt; 0.05) more slowly (2.9 log MPN/plant) than those of rpoS-deficient E. coli O157:H7 (3.7 log MPN/plant) on foliar surfaces of 5-week old spinach plants over 8 days, indicating that the presence of the rpoS gene is important to the ability of E. coli O157:H7 to survive on foliar surfaces. There were no significant differences in the population declines between wild-type (wt) S. Typhimurium and rpoS-deficient S. Typhimurium on 5-week old spinach plants over 8 days, indicating that the rpoS gene may not be as important to the persistence of S. Typhimurium on foliar surfaces as to the persistence E. coli O157:H7. The MNV in this same situation declined only slightly by 0.6 log genome copies. Plant over the 8 day study, with no survival between days 4 and 8. Norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus in order to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid and jasmonic acid plant defense pathways were measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants were inoculated with either MNV, TV, human norovirus GII.4 or HBSS (control). Genes involving both the salicylic acid and jasmonic acid pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, studies including gene expression of SA- and JA-deficient A. thaliana mutant lines suggest that the jasmonic acid pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the pre-harvest environment.

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: Markland, S.M. and K.E. Kniel. 2015. Human Pathogen-Plant Interactions: Concerns for Food Safety. In H. Bais and J. Sherrier (Ed.) Advances in Botanical Research: Plant Microbe Interactions. Elsevier. In Press.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: M. Sharma, Invited Presentation "E.coli in Wonderland (Through the looking glass&)". IAFP 2014 Annual Meeting, Indianapolis, IN.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S.M. Markland, H. Bais, K. E. Kniel. Effects of Plant Growth Promoting Rhizobacteria on the Immune Response of Romaine Lettuce and Spinach toward Human Bacterial Pathogens, IAFP Annual Meeting, Indianapolis, IN, August 2014, P1-85
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S. M. Markland, H. P. Bais, and K. E. Kniel. Investigation of biocontrol and plant response to reduce interactions of enteric pathogens on leafy greens, American Phytopathological Society, Austin, TX, August 2013, 718-P.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: E. Handy, R. Reynnells, M.T. Callahan, C. Roberts, S. Markland, K. Kniel, and M. Sharma. Survival of Escherichia coli O157:H7, Salmonella Typhimurium, Murine Norovirus, and Tulane Virus on Foliar Surfaces of Spinach Plants. July 2015. IAFP Annual Meeting, Portland, OR. P1-185.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: S. Markland, S. Craighead, K. Kniel. Plant-microbe Interactions Associated with Noroviruses and Leafy Greens. July 2015. IAFP Annual Meeting, Portland, OR. P1-186.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kwan, G., Pisithkul, T., Amador-Noguez, D., and Barak, J.D. 2015. De novo amino acid biosynthesis contributes to Salmonella enterica growth in alfalfa seedling exudates. Appl. Environ. Microbiol. 81(3): 861-873
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kwan, G., Charkowski, A.O., and Barak, J.D. 2013. Salmonella enterica moderates Pectobacterium carotovorum populations and virulence on lettuce. mBio. 4: e00557-12.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Kwan, G. and Barak, J.D. 2012. Proteomic analysis of Salmonella enterica plant colonization: insights into the metabolic requirements of human pathogens on plants. American Phytopathological Society Annual Meeting, Providence, RI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Kwan, G. and Barak, J.D. 2014. Investigating the metabolic requirements of Salmonella enterica during plant-associated growth: the role of amino acids. Food Research Institute Annual Meeting, Madison, WI.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Marklamd, S.M., Ferrelli, A.M., Craighead, S.A., Bais, H., and Kniel, K.E. Application of Bacillus subtilis to the roots of leafy greens, in the presence of Listeria innocua and Salmonella Newport, induces closure of stomata. Foodborne Pathogens and Disease. 2015. In press.
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Manuscript in preparation - S. Markland, M. Sharma, K. Kniel. HUMAN NOROVIRUS AND ITS SURROGATES INDUCE PLANT IMMUNE RESPONSE IN ARABIDOPSIS THALIANA AND LACTUCA SATIVA


Progress 04/01/12 to 03/31/13

Outputs
Target Audience: Target audiences during this time frame have included formal classroom instruction, laboratory instruction or undergraduate and graduate student researcchers, and members of the scientific community through outreach. Dr. Kniel teaches two undergraduate courses where this research is included in discussions on produce safety and interactions of pathogens with plants. Presentations to the scientific community included food scientists, microbiologists, and plant pathologists. Dr. Sharma provided invited presentations including results from his work associated with this project to various visiting groups to the USDA-ARS EMSL laboratory. Dr. Barak teaches an upper division undergraduate/graduate courses focused on plant-associated bacteria. The results from this project have been delivered to students at the University of Wisconsin-Madison via Dr. Barak&rsquo;s course. Dr. Barak was invited to speak at the Food Research Institute&rsquo;s annual meeting where she shared results from this project. Changes/Problems: A continuing challenge with this project is difficulty with primer choice for assessing the immune response of spinach or lettuce plants. As an alternative and to better evaluate the action of norovirus on a plant, we are first assessing the immune response of Arabidopsis thaliana to norovirus surrogate MNV-1. What opportunities for training and professional development has the project provided? Several graduate and undergraduate students have been trained at the three locations within this project. Students from the UW-Madison Undergraduate Research Scholars program have been trained during this project. Undergraduate summer scholars at the University of Delaware have worked on this project, including McNairs scholars. Undergraduate and graduate students in the Washington DC area have been trained at the USDA-ARS facility in Beltsville, MD. How have the results been disseminated to communities of interest? Results have been published as peer-reviewed abstracts in the annual meeting abstract booklets for professional societies of the International Association for Food Protection and the American Society of Phytopathologists. What do you plan to do during the next reporting period to accomplish the goals? Up through March 2013, the researchers remained focused on method development. In the next reporting period the focus will be on utilization of these methods to determine the differences of interaction of the three pathogens on plants as well as interaction of the three pathogens with the plant itself.

Impacts
What was accomplished under these goals? We conducted experiments to determine metabolic strategies used by Salmonella enterica to colonize plants. We took a metaproteomic approach to identify essential metabolic pathways used by the human pathogen S. enterica. In addition, we examined the growth of S. enterica in the presence of the plant pathogen, Pectobacterium carotovorum subsp. carotovorum. A comparison of the survival of E .coli O157:H7, Salmonella Typhimurium, and murine norovirus (MNV) under the same conditions on spinach plants was performed.Five week-old spinach (Menorca, semi-savoy) plants were co-inoculated with 6 logCFU/plant of each of the following strains: wild-type (wtO157) E. coli O157:H7, wt S. Typhimurium (wtST), and rpoS-deficient mutants of wt E. coli O157:H7 (rpoSO157) and S. Typhimurium (rpoSST). MNV was inoculated on the same spinach plants at 10^6 PFU/plants. Wt and rpoS-deficient strains were enumerated from plant homogenates on selective media either directly or by MPN. Five plants per day were analyzed on days 0&ndash;4. The presence of MNV was also determined on day 0 and day 3. Initial populations of all bacterial strains were 4.3 &ndash;4.5 logCFU/plant on day 0. Populations of wtST and wtO157 declined by 3.1 and 3.9 logCFU/plant, respectively; rpoSO157 and rpoSST declined by 3.9 and 4.7 logCFU/plant, respectively over 4 days. Populations of rpoSO157:H7 and rpoSST were 2 logCFU/plant lower than paired wtO157 and wtST on day 1. Populations of wtO157 strains were significantly (p&lt; 0.05) higher than those of both rpoS-deficient strains on day 2 and significantly higher than wtST, rpoSO157, and rpoSST on day 3. MNV were assayed and recovered on day 0 and day 3.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: K. Hirneisen, S. Markland, Q. Wang, K.E. Kniel. Enteric Virus Survival on Alfalfa Seeds and Sprouts. APS Human Pathogens on Plants Workshop, Hyattsville, MD, February 2012.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: K. Hirneisen and K.E. Kniel. Comparative Uptake of Enteric Viruses into Spinach and Green Onions. APS Human Pathogens on Plants Workshop, Hyattsville, MD, February 2012.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Kwan, G., Charkowski, A. O., and Barak, J. D., 2013. Salmonella enterica moderates Pectobacteirum carotovorum populations and virulence on lettuce. mBio. 4:e00557-12.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: J. Barak. Hanging on and hanging out: Salmonella entericas life on plants. 2013. University of Florida
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: J. Barak. Probing the life of Salmonella enterica on plants. 2013. Iowa State University
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: J. Barak. Dissecting Salmonellas life in the roots. 2012. Virginia Tech
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: J. Barak. "Plant Pathology: A Key Discipline for Food Safety of Fresh Produce. 2012 Korean Plant Pathology Society Annual Meeting. Seoul, Korea.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: J. Barak. Salmonellas life in the roots. 2012. 26th Annual Kenneth B. Raper Symposium on Microbiological Research, University of Wisconsin-Madison, Madison, WI


Progress 04/01/11 to 03/31/12

Outputs
OUTPUTS: The three co-investigators for the project have established types of seeds that will be used across the studies. The co-investigators have met at two different meetings to discuss plans and objectives and continue to meet virtually when they cannot meet face-to-face. To address the goal of understanding the plant response to viruses; Arabidopsis is being used as a model plant for investigating norovirus surrogates (murine norovirus), along with pathogenic E. coli and Salmonella strains. In this work two plant immune system pathways are being analyzed: the Induced Systemic Resistance (ISR) response pathway and the Systemic Acquired Resistance (SAR) response pathway. The gene markers being studied in these two pathways include PR-1, Jin, Jar, PDF1 -2, and the housekeeping gene for ubiquitin-1. One group has obtained a parent, wild-type and rpoS-deficient mutant of the Salmonella Typhimurium from another co-investigator. To conduct our experiments, the wild-type strain was made resistant to nalidixic acid in order to differentiate from other E. coli and Salmonella strain from spinach foliar surfaces. Different recovery media have been tested for these strains to optimized recovery of wild-type E. coli O157:H7 and Salmonella and rpoS-deficient E. coli and Salmonella from leafy green surfaces. Salmonella enetrica proteome was assesed during plant colonization. The four hundred most abundant proteins were identified. The majority of proteins were involved in metabolism and transport functions. The proteome suggests active metabolic pathways versus inactive or those more energetically costly to the bacterium. Consistent and abundant presence of proteins of putative or function unknown suggests little prior knowledge exists about key functions for adaptation to plant environments. PARTICIPANTS: Post-bacculerate employees and graduate students were hired for the project and receiving mentoring in food and environmental microbiological techniques at all 3 institutions. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Initial experiments compared the survival of wild-type and rpoS-deficient E. coli on the leaves of growing spinach plants over three days. Results indicated that wild-type and rpoS-deficient cells survive at similar levels on day 0 and 1; however the populations begin to diverge on Day 2. Populations of the rpoS-deficient E. coli O157:H7 strain were 1.2 and 1.6 log CfU/plant lower than the wt E. coli O157:H7 on day 2 and 3 on growing spinach plants. These results indicate that the rpoS gene may play a yet uncharacterized role in the survival of E. coli O157:H7 to survive on foliar surfaces. Parts of the project have focused for the most part on method development due to the novelty of this project and the fact that this work has not yet been conducted with virus. As mentioned, the Arabadopsis plant is currently being used and methods ar being developed for how to best study the immune response of food crops. Assays are under development to study plant growth promoting rhizobacteria and their inhibition of foodborne pathogens and their function with the immune responses of the plant hosts. Mutational analysis confirms active metabolic networks. Based on the metabolic costs associated with gene expression and protein synthesis, we formed the assumption that if a protein is present, the corresponding metabolic reaction occurs. Organisms typically make enzymes when the corresponding reaction is needed. Our initial validation of essential metabolic networks whose products were among the abundant proteins confirmed the need for S. enterica to synthesize glycine, proline, isoleucine, and arginine. For example, IlvA and IlvD were in the proteomic data and S. enterica ilvA and ilvD mutants were significantly reduced in growth compared to wildtype. Additional growth studies confirmed an abundance of serine, methionine, and threonine in planta. The biosynthetic proteins for these amino acids were not among the most abundant proteins and deletion of the biosynthetic genes had no effect on replication compared to wildtype. In addition to amino acid auxotrophs, we are investigating the transporters identified in our proteomic data.

Publications

  • None at this time; however, abstracts will be submitted for presentation at the 2013 Annual Meetings of the International Association for Food Protection and the American Society of Phytopathologists.