1a.Objectives (from AD-416)
SASL is conducting research into the mechanisms by which plant-beneficial bacteria, such as Pseudomonas fluorescens PF5, colonize subterranean plant parts and suppress plant disease. Currently we are studying the role of the global regulatory molecule Vfr in these two processes. As this is a global regulatory molecule a system-wide or "omics" approach is required to determine all of the proteins in the Vfr regulatory cascade. One of the most cost-effective methods to do this is via proteomics, where the total protein profiles of the wild-type strain (PF5) and a strain with a mutation in vfr (and thus a non-producer of Vfr) are identified and compared under identical growth conditions. Proteins that are present in different concentrations in the two protein profiles are directly or indirectly regulated by Vfr. SASL does not have the infrastructure, equipment, or expertise to perform the protein separation and identification procedures on this scale. The cooperator has the technology, personnel, and infrastructure necessary to complete this analysis in a timely and cost-effective manner.
1b.Approach (from AD-416)
Grow the wildtype (PF5) and mutant, containing a mutation in the vfr gene, under identical conditions in a synthetic cucumber root exudate medium. Harvest cultures from both bacterial strains at three separate time points and freeze on dry ice. Total intracellular protein from bacterial biomass from these three time points is differentially stained by strain and then separated by two-dimensional gel electrophoresis. Individual proteins differentially expressed in the two strains are individually harvested and identified by mass spectroscopy (MALDI-TOF).
The overarching objective of the grant was to use proteomics approaches to investigate mechanisms by which plant-beneficial bacteria, such as Pseudomonas fluorescens PF5, colonize subterranean plant parts and suppress plant disease. The specific objective of the grant was to determine the role of the regulatory protein Vfr in these processes. Technical problems were encountered during attempts to scale up growth of strains used in this project. The end result was that materials (total cellular protein from these strains) needed by Applied Biomics, Inc. to perform the work on the grant was not supplied by the ADODR. The grant was extended one year so that technical problems can be resolved and materials made available to Applied Biomics, Inc. to perform the required work. All work on the grant to date has been conducted in the laboratory of the ADODR. Therefore, no formal controls regarding project progress were required.