A primary goal in our lab is to analyze the regulatory pathways that coordinate iron availability—a critical environmental factor—and virulence. Iron acquisition is a highly competitive contest between pathogen and host. Because iron is at very low concentration under most conditions, all organisms have evolved mechanisms to obtain this scarce nutrient. The question facing us is how Pseudomonas syringae responds to iron in the environment, and how this information is integrated with the regulation of genes involved in pathogenesis.
This question is deliberately posed in a broad way because we do not know many details yet concerning iron-related gene regulation. Our strategy is based on two perspectives. First, we design experiments that are as unbiased as possible—we often describe the approach as “letting the organism speak for itself”. Second, rather than concentrate efforts on single genes, our attention is drawn first to higher-level factors that influence the expression of many genes simultaneously and thus provide insights into global patterns and mechanisms underlying the cell’s regulatory “programs”.
Our three complementary research themes are summarized below.
Identification of ECF sigma factor regulons (Bryan Swingle)
The selection of promoters for transcription is arguably one of the most critical control points in gene regulation and is one of the most important mechanisms involved in responding to environmental signals, including iron availability. A regulon is a set of genes whose expression is coordinated by the cell through the mechanism of promoter selection. Promoter identification by DNA-directed RNA polymerase is accomplished by sigma factor subunits, for which there are only a small number (< 20) of genes in DC3000. We are in the process of characterizing the sigma factors in DC3000 and are matching them with their promoters through a combination of molecular experiments and bioinformatics.
Global transcription analysis and small RNAs (Melanie Filiatrault)
A closely related project involves the direct examination of the transcriptome—a term that encompasses all of the RNA molecules that are produced by RNA polymerase under specific conditions such as growth in the presence of limited iron. High-throughput sequencing methods can be used to characterize nearly all of the transcripts in the cell and map them directly onto the genomic sequence. This data permits us to infer transcription start sites (which are typically very close to the promoter) and also makes it possible to identify sets of genes (operons) that are transcribed from some promoters as a single mRNA molecule. By associating sigma factors with their cognate promoters, promoters with downstream transcriptional units, and transcriptional units with genes, we hope to infer many of the important regulatory events connecting environmental signals and virulence.
Iron-related gene expression (Phil Bronstein)
A third project directly addresses the identification of genes that are expressed in response to changes in iron availability. This project primarily involves microarray experiments to examine iron-related gene expression. In addition, we use transposon-based transcriptional reporter fusions that permit gene activity to be inferred by monitoring the production of the reporter (two examples of commonly-used reporters are lux and GFP). Expression analysis leads to the discovery of novel regulatory genes that are involved in the cell’s iron economy and integrates naturally with data obtained from the other two projects to produce a more complete picture of the role of iron in the biology of Pseudomonas syringae.
Our interdisciplinary approach has led us to collaborate with a number of groups on campus. These research relationships are vital to our work and are explained here.