Research
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.
Collaborations
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.
Cluster analysis of iron-responsive
genes in DC3000 using data from a microarray (P. Bronstein and C. Myers).
Light-emitting DC3000 colonies containing random luxCDABE transcriptional fusions (A. Chambers and P.
Bronstein).