Progress 10/01/05 to 09/30/09
Outputs
OUTPUTS: The outputs completed during this reporting period include conducting experiments to identify new secondary metabolites produced by Pseudomonas syringae pathovars and analyzing the resulting data. The information gained from these studies was presented at meetings and through publication in scientific journals. This provided fundamentally new information concerning secondary metabolite production by P. syringae pathovars. PARTICIPANTS: Andrew Berti, research assistant (graduate student) Yolande Chan, research assistant (graduate student) Donald Drott, research assistant (graduate stuent) TARGET AUDIENCES: The target audience is microbial physiologists, plant biologists, and natural product chemists. PROJECT MODIFICATIONS: None.
Impacts
The findings from these studies provided a proof-of-principle that genome "mining" could be used to identify metabolites produced by Pseudomonas syringae pathovars that were not known.
Publications
- Berti, A. D. and Thomas, M. G. Analysis of acrhomobactin biosynthesis by Pseudomonas syringae pv. syringae B728a. 2009. J. Bact. 191:4594-4604.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The outputs completed during this reporting period include conducting experiments to identify the genes that code for enzymes involved in the production of secondary metabolites in Pseudomonas syringae strains. The information obtained from these studies was disseminated to communities of interest through seminars presented at various academic institutions and at the 108th General Meeting for the American Society for Microbiology. This information provided fundamentally new information concerning secondary metabolite production by P. syringae strains. PARTICIPANTS: Andrew D. Berti, graduate student in the Microbiology Doctoral Training Program. Yolande A. Chan, graduate student in the Microbiology Doctoral Training Program. TARGET AUDIENCES: The target audience for these efforts are microbial physiologists, plant biologists, and natural product chemists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results from these studies provided additional proof that bioinformatic analysis of P. syringae genomes will lead to the identification of new secondary metabolites and new types of enzymology involved in the production of these metabolites. Furthermore, these data have provided important insights into the flexibility of the enzymology involved in the production of secondary metabolites and how unnatural derivatives of these metabolites can remain biologically active.
Publications
- No publications reported this period
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The outputs completed during this reporting period include conducting experiments to identify new secondary metabolites produced by Pseudomonas syringae pv. tomato DC3000 and analyzing the data from these studies. The information obtained from these studies was disseminated by a poster presentation at the 107th General Meeting of the American Society for Microbiology and a publication in the Journal of Bacteriology. This information provided fundamentally new information concerning secondary metabolite production by P. syringae pv. tomato DC3000.
PARTICIPANTS: Andrew D. Berti. Graduate student in Microbiology Doctoral Training Program. Nathan J. Greve. Undergraduate researcher in Biochemistry ('07). Quin H. Christensen. Undergraduate researcher in Bacteriology ('05).
TARGET AUDIENCES: The target audience for these efforts are microbial physiologists, plant biologists, and natural product chemists.
PROJECT MODIFICATIONS: There are no project modifications.
Impacts
The results from these studies provided a proof-of-principle that bioinformatics analysis of P. syringae genomes provides a means for identifying previously unknown metabolic potential. Furthermore, these data provided the first evidence that linear lipopeptides can be produced by P. syringae pathovars and these linear molecules of biologically active. Prior to this work, it was believed that the biologically active molecules were cyclic structures, not linear.
Publications
- Berti, A. D., Greve, N. J., Christensen, Q. H., and Thomas, M. G. (2007) Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility in Pseudomonas syringae pv. tomato DC3000. J. Bacteriology 189:6312-6323.
|
Progress 01/01/06 to 12/31/06
Outputs
Using bioinformatics approaches, we previously identified a set of biosynthetic gene clusters in Ps. syringae DC3000 that we predicted would produce previously uncharacterized secondary metabolites. Molecular genetic techniques were used to insert transcriptional reporter fusions into one gene in each gene cluster. Screening growth conditions found that expression of one of the transcriptional fusions was induced when Ps. syringae DC3000 reached entered stationary phase of growth. Growth conditions for the expression of the other clusters have yet to be determined. The one gene cluster that did express under our specific growth conditions was characterized further. Using molecular genetic techniques, a deletion of the first gene of the targeted gene cluster was accomplished. Characterization of this mutant strain found that the deletion disrupted the ability of DC3000 to have swarming motility. This suggests the biosynthetic gene cluster codes for the enzymes involved
in the production of a biosurfactant. Metabolite profiles of wild-type Ps. syringae DC3000 and the deletion strain identified a set of four metabolites that were present in the wild-type strain but absent in the mutant strain. A purification scheme was developed to obtain the four metabolites that were found in two separate fractions. Initial structural analysis of the four metabolites suggests the metabolites are structural analogs and they are all lipopeptides. This is consistent with the metabolites being biosurfactants. Further analysis of these metabolites is ongoing to complete the structural analysis. These will be new Ps. syringae biosurfactants that may play a role in virulence or can be exploited for applied purposes.
Impacts
The identification of new biosurfactants produced by Ps. syringae DC3000 gives significant insight into the physiology of the bacterium. This also suggests the inhibition of the production of these metabolites may be a mechanism to block the pathogenicity of this bacterium. Furthermore, biosurfactants can be exploited for more applied purposes such as antifungal agens, solvents, and detergents.
Publications
- No publications reported this period
|
Progress 01/01/05 to 12/31/05
Outputs
Our initial work targeted three different potential secondary metabolite biosynthetic gene clusters in Pseudomonas syringae pv. tomato DC3000. The introduction of reporter gene fusions into a gene from each cluster allowed us to screen for whether we could find growth conditions where the gene fusions are expressed. One of the reporter fusions was induced when Ps. syringae DC3000 entered the stationary phase of growth and we are targeting this gene cluster for further analysis. We are currently comparing metabolite profiles of culture supernatants of the wild-type strain versus a strain carrying a mutation in the targeted gene cluster in an attempt to identify the associated metabolite. We are also screening for whether the mutant strain has a phenotype in plant virulence assays, biofilm formation, swarming motility, and antifungal/antibacterial activity. We are also performing mutagenesis studies to identify transcriptional regulators of the targeted gene fusion and
have identified one gene coding a LuxR-like transcriptional regulator that when inactivated abolishes expression of the reporter gene.
Impacts
The ability of Pseudomonas syringae pathovars to cause disease in plants is, at times, dependent on their ability to produce a particular secondary metabolite. We are currently using a genomics approach to identify secondary metabolites that may be involved in the ability of Pseudomonas syrinae pathovars to cause disease in plants. Disruption of the production of such a metabolite may lessen the disease caused by a targeted pathovar. These metabolites may also show biological activities that may be exploited for other purposes (e.g. antifungal activity).
Publications
- No publications reported this period
|
|