PROTEOMICS ANALYSIS OF PLANT-BENEFICIAL BACTERIA
Sustainable Agricultural Systems Laboratory
Project Number: 1245-21220-179-01
General Cooperative Agreement
Start Date: Sep 28, 2010
End Date: Jul 14, 2013
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.
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).