Location:2009 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.
3. Progress Report
During this reporting period, rapid advances in sequencing technology, in conjunction with bioinformatics, have made it possible to examine gene regulation in Pseudomonas syringae in new and cost effective ways. High-throughput sequencing was exploited to discover and catalog small RNAs expressed under specific conditions. Other experiments relying on this technology were conducted to detect binding sites for Fur, a major regulator for iron metabolism, across the genome, and also identify putative promoters recognized by four iron-starvation type sigma factors, obviating the need for laborious promoter-trap screens. The advantage of these methods is that they make it possible to move quickly from global discoveries to hypotheses about specific genes and regulatory mechanisms. For example, sigma factor binding sites identified by sequencing-based methods are being tested for activity under different conditions. Significant progress has also been made in improving methods for mutagenesis by adapting a phage recombination system from Pseudomonas syringae B728a to enhance recombination rates. This will make it much easier to generate mutants to test the role of specific genes and regulatory sites.
1. High-throughput RNA sequencing strategies provide direct characterization of gene expression. A detailed analysis of gene expression requires the ability to associate every RNA in the cell with its corresponding gene. To address this need, we developed a new RNA sequencing protocol in collaboration with Illumina, Inc. The sequence data were analyzed using novel statistical procedures and custom software and was carefully validated by other experiments. The final result is a “map” that shows the RNA levels corresponding to every position in the genome and provides a global view of the biochemical activities in the cell. The new method is applicable to any other bacterial species that can be grown in the laboratory, including many pathogens of plants and animals, and will help identify genes that are important in the disease process.
2. High-throughput DNA sequencing strategies facilitate the detection of promoters and protein-DNA binding sites. Gene regulation is often mediated by the binding of proteins to specific locations on the bacterial chromosome. To identify these regions, we exploited a technology that captures protein-DNA associations in the living cell and then uses DNA sequencing to inventory the captured DNAs. The analysis localizes proteins along the chromosome with high resolution and reveals promoters and other kinds of regulatory sites. These methods are easily adapted to target different proteins and can be used to probe the binding of proteins under different environmental conditions, including those related to the onset of pathogenesis.
Felise, H.B., Nguyen, H.V., Pfuetzner, R.A., Barry, K.C., Jackson, S.R., Blanc, M.P., Kline, T., Bronstein, P., Miller, S.I. 2008. An Inhibitor of Gram-Negative Bacterial Virulence Protein Secretion. Cell Host and Microbe. 4(4):325-336.