2011 Annual Report
1a.Objectives (from AD-416)
Identify promoters and ECF sigma factors that control expression of known and suspected virulence factors.
Characterize the subset of the transcriptome related to growth in defined medium, the induction of virulence factors, and response to iron bioavailability.
Elucidate mechanisms leading to iron-dependent expression of operons encoding virulence factors and regulators.
1b.Approach (from AD-416)
Research will employ an interdisciplinary approach involving computational biology and laboratory methods for high-throughput functional genomics and genetics. Expression studies including the use of microarrays and high-throughput reporter screens will be used to characterize the components and behavior of virulence-related pathways, especially those related to iron homeostasis. Mutants in key regulatory proteins and gene reporter systems will be used to elucidate regulatory interactions. Computational methods will be used to identify regulatory motifs, detect statistically significant correlations in gene expression, and model selected pathways. We aggressively integrate laboratory and computational approaches to genome-scale problems in order to design and implement the most effective experiments and analytical methods.
During this reporting period, we have continued to exploit the development and deployment of high-throughput sequencing technology that we completed during previous reporting periods.
Pursuant to Objective 2A and improving upon the high-throughput sequencing techniques developed last year, we have identified, validated, and characterized the start of thousands of transcripts from DC3000. These results were used to refine the DC3000 genome annotation and for computational analysis to identify the regulatory sequence upstream of many genes within DC3000. A paper describing this work has been submitted for publication.
Pursuant to Objective 3A and 3B, we continued our effort to characterize the Fur regulon of DC3000 and to refine our model of Fur binding sites. This year we combined our high-throughput data sets generated in previous reporting periods with new laboratory data in order to confirm and characterize both known and novel Fur binding sites. A paper describing this work has been accepted for publication.
Pursuant to Objective 1, we studied PSPTO_1203, one of five IS ECF sigma factors. During the reporting period, we have performed a number of experiments to determine the 1203 regulon and the environment signals that trigger this system. Our work shows that the pathway involved in triggers of the 1203 system involves the triggering of other ECF sigma factors. A paper describing this work has been accepted for publication.
Wei, Y., Flores-Mireles, A.L., Costa, E.D., Ryan, G.T., Schneider, D.J., Winans, S.C. 2010. Saturation mutagenesis of a CepR binding site as a means to identify new quorum-regulated promoters in Burkholderia cenocepacia. Molecular Microbiology. 79(3):616-632.
Park, D., Mirabella, R., Bronstein, P., Preston, G.M., Lim, C., Collmer, A., Schuurink, R.C. 2010. Mutations in gamma-aminobutyric acid (GABA) transaminase genes in plants or Pseudomonas syringae reduce bacterial virulence. Plant Journal. 64(2):318-330.
Butcher, B.G., Bronstein, P., Myers, C., Stodghill, P., Bolton, J.J., Markel, E.J., Filiatrault, M.J., Swingle, B.M., Gaballa, A., Helmann, J.D., Schneider, D.J., Cartinhour, S.W. 2011. Characterization of the Fur regulon in Pseudomonas syringae pv. tomato DC3000. Journal of Bacteriology. 193(18):4598-4611. Available: http://www.ncbi.nlm.nih.gov/pubmed/21784947.
Wu, S., Lu, D., Kabbage, M., Wei, H., Swingle, B.M., Dickman, M., He, P., Shan, L. 2011. Bacterial effector HopF2 interacts with AvrPto and suppresses Arabidopsis innate immunity at the plasma membrane. Molecular Plant-Microbe Interactions. 24(5):585-593.
Moll, S., Schneider, D.J., Stodghill, P., Myers, C.R., Cartinhour, S.W., Filiatrault, M.J. 2010. Construction of an rsmX co-variance model and identification of five rsmX-like ncRNAs in Pseudomonas syringae pv. tomato DC3000. RNA Biology. 7(5):1-10.
Sebaihia, M., Bocsanczy, A.M., Biehl, B.S., Quail, M.A., Perna, N.T., Glasner, J.D., Declerck, G.A., Cartinhour, S.W., Schneider, D.J., Bentley, S.D., Parkhill, J., Beer, S.V. 2010. Complete genome sequence of the plant pathogen Erwinia amylovora strain ATCC 49946. Journal of Bacteriology. 192(7):2020-2021.
Swingle, B.M., Bao, Z., Markel, E.J., Chambers, A., Cartinhour, S.W. 2010. Recombineering using RecTE from Pseudomonas syringae. Applied and Environmental Microbiology. 76(15):4960-4968.
Schneider, D.J., Collmer, A. 2010. Studying plant-pathogen interactions in the genomics era: beyond molecular Koch’s postulates to systems biology. Annual Review of Phytopathology. 48:457-479.