Location:2010 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, we have continued to exploit rapid advances in sequencing technology. Improving upon the high-throughput sequencing techniques developed last year, we have identified, validated, and characterized the expression of dozens of small RNAs. The high-throughput methods that were used to study small RNAs are being used to produce refined transcript and operon maps of DC3000. In order to improve our model for Fur binding sites, we developed improved laboratory and computational methods that allowed us to discover additional Fur binding sites that were not evident from previous results, further expanding the putative Fur regulon. Finally, we have developed a model for the promoter recognized by PSPTO_1203, one of five IS ECF sigma factors. We have also identified many of its regulon members. The tools and techniques that we have developed are being used by other research scientists for studying gene regulation in other bacteria.
1. A new strategy providing 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, ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY 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. A new DNA sequencing strategy facilitates the detection of gene regulation. Gene regulation is often mediated by the binding of proteins to specific locations on the bacterial chromosome. To identify these regions, ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY 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.
3. Recombineering phytopathogenic microbes. Making specific changes to the genomes of certain bacteria has been extremely difficult. ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY discovered that some bacteria already possess mechanisms that can allow foreign DNA to be incorporated into their genome. We found that by providing the bacteria with specific DNA fragments, we can make desired changes to bacterial genomes much more efficiently. Many previously intractable experiments on a wide range of bacteria are now possible.
4. High-throughput mapping of transcript ends in microbes. Developing a transcript map requires knowing the beginning and end of each mRNA molecule. ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY developed new strategies for capturing the ends of millions of mRNAs. We have precisely mapped hundreds of ends and have greatly refined the DC3000 genome annotation. The refined transcript map and annotations provide a better understanding of the transcriptional organization of this important plant pathogen.
5. Discovery and mapping of key regulatory small RNA's in a phytopathogenic microbe. Small RNAs are key components in the regulation of genes involved with virulence and other important bacterial functions, however very few have been described in plant-pathogenic bacteria. ARS scientists at the Robert W. Holley Center for Agriculture & Health in Ithaca, NY have developed a pipeline that combines laboratory and computational methods in order to identify, classify, and characterize novel small RNAs. Using this pipeline, we have identified over 75 putative small RNAs in DC3000, including a number that are conserved in closely related species. Many of these may have important roles not only in plant-pathogenesis but also bio-control, bio-remediation, and human-pathogenesis.
Swingle, B.M., Markel, E.J., Costantino, N., Bubunenko, M., Cartinhour, S.W., Court, D. 2010. Oligonucleotide recombination in gram negative bacteria. Molecular Microbiology. 75(1):138-148.