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Proteomic analysis of wild type and soil-colonization deficient strains of Pseudomonas fluorescens

EMSL Project ID


Proteomic analysis of wild type and soil-colonization deficient strains of Pseudomonas fluorescens A significant step towards developing effective genetically modified-microbes for bioremediation purposes is understanding the ecology of release, growth and establishment of populations in soil. In Pseudomonas fluorescens, a microbe widely used for enhancing soil characteristics, our laboratory identified a locus (adnA) affecting several cell processes important for its behavior in soil. Mutants deficient in adnA are deficient in biofilm formation, conjugative ability, motility, and attachment to sand and seeds. Of more importance, mutants affected in adnA did not colonize and persist in soil as well as the wild type (Marshall, 2001). AdnA shows homology to FleQ, from Pseudomonas aeruginosa, a transcription factor of the NtrC/NifA family that is involved in flagella synthesis (Casaz, 2001). This type of regulators is Sigma 54-dependent, known to activate transcription upon changing environmental conditions. Therefore, by identifying traits affected by AdnA we will gain insights on how to improve soil colonization, persistence and what environmental conditions favor these processes. We propose to identify peptides and their genetic determinants affecting soil colonization by comparing proteomes from the wild type and an adnA-deficient mutant.Objectives:1) To identify peptide signatures affected by the AdnA transcription factor.In order to identify gene products affected by AdnA, we will compare protein profiles from Pseudomonas fluorescens strains that differ in their ability to colonize and persist in soil. We will use Pf0-1, our wild type strain first isolated by Compeau et al. (1988), and Pf2x, a strain with an Omega cassette in the middle of adnA. Pf2x strain was constructed by marker exchange using a non-replicative vector containing sacB as a counterselective marker. Insertion of the omega cassette abolishes AdnA synthesis and renders cells non-motile. Thus, protein extracts from Pf2x are likely to contain no peptides activated and/or peptides that are repressed by the AdnA transcription factor. Briefly, protein extraction, digestion, separation and sequencing will be carried out at the William R. Wiley Environmental Molecular Sciences Laboratory at the Pacific Northwest Laboratory. Protein extracts will be digested and processed by capillary liquid chromatography to obtain accurate mass tags as described by Smith et al. (2001).2) To determine cellular location of peptides affected by AdnA.In order to determine where AdnA-affected peptides are localized, we will fractionate cells into membranes and cytosols and compare peptide profiles in both cell fractions from Pf0-1 and Pf2x. As its homology to FleQ suggests, it is likely that we will identify peptides that are structurally- or regulatory-involved in flagella synthesis, biofilm formation, and soil colonization. 3) To identify genetic determinants of peptides affected by AdnA and determine their effects in soil colonization.To confirm the role of these peptides in soil behavior and fulfill Koch's postulates, the specific sequence of the peptides identified as Adna-dependent will be reversed translated and used to search the Pf0-1 genome sequence for putative open reading frames. Open reading frames will be submitted for blasts searches and used to design primers to amplify the coding regions for these peptides. The role of these proteins in soil colonization will be confirmed by insertional disruption or creation of in frame-deletion mutants. These mutants will be assayed for their behavior in soil and compared to the wild type. This objective will be carried out at the Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology and Microbiology at the School of Medicine, Tufts University. Microbiol. 2001.147: 355-361AEM. 2001. 67: 852-857AEM. 1988. 54: 2432-2438NABIR-PI workshop abstracts p 45. 2001. March 12-14, Warrenton, Va.

Project Details

Project type
Exploratory Research
Start Date
End Date


Principal Investigator

Stuart Levy
Tufts University School of Medicine

Team Members

Mary Lipton
Environmental Molecular Sciences Laboratory

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