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).
3. Progress Report:
The overarching objective of the project involving the grant was to use proteomic approaches to investigate mechanisms by which plant-beneficial bacteria, such as Pseudomonas fluorescens Pf-5, 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. Strain Pf-5 and a derivative strain, containing a mutation in the vfr gene, were grown on synthetic cucumber root exudate medium, and the resulting cells sent to Applied Biomics, Inc. for proteomic analysis. Scientists at Applied Biomics, Inc., isolated and separated proteins from both strains and subsequently identified proteins that were produced at different levels by the two strains. A number of proteins were differentially expressed in the two strains including transport proteins functioning in polyamine uptake and transport proteins functioning in uptake of compounds related to stress tolerance in bacteria. Polyamine uptake and stress tolerance have previously been identified as traits important to colonization of plant roots. This partially explains why the derivative strain, containing the mutation in vfr, is greatly impacted in root colonization.