|Butcher, Bronwyn -|
|Bronstein, Philip -|
|Myers, Christopher -|
|Gaballa, Ahmed -|
|Helmann, John -|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 2011
Publication Date: July 22, 2011
Citation: 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. Interpretive Summary: Microbes must manage iron very carefully. On the one hand, iron is required for critical metabolic processes. On the other, unless properly sequestered, iron can damage, and even kill, the cell. In the model plant pathogen, Pseudomonas syringae pv. tomato DC3000 the protein, Fur, is a critical component in the iron regulation network. In high iron conditions, Fur binds to DNA and prevents the expression of critical iron regulating genes. In low iron conditions, Fur releases from the DNA and allows expression of these genes to occur. In order to better understand the iron regulation network, we conducted a study in which we discovered the positions within the DC3000 genome where Fur binds under high iron conditions. Our results confirmed that Fur binds to the genome in many places that had previously been predicted. In addition, we found that Fur binds in a number of surprising locations. These new results suggest that the established model of Fur binding in high iron and stopping gene expression is more nuanced, and there appear to be other mechanisms by which Fur controls iron regulating genes. Since iron regulation is an essential cellular function, our results are helpful in understanding this function in other bacterial species. Since iron acquisition is central to virulence, our results will be especially important to researchers studying plant and animal pathogens.
Technical Abstract: The plant pathogen Pseudomonas syringae pv. tomato DC3000 is found in a wide variety of environments and as a result must monitor and respond to various environmental signals. In previous studies, we investigated the transcriptional response of DC3000 to iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator) and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) 312 regions were highly enriched by immunoprecipitation with a C-terminally tagged Fur protein. Integration of this data with previous microarray and global transcriptome analysis allowed us to expand the known DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Intriguingly, 37% of the in vivo Fur binding sites identified by ChIP-seq are located within genes. Using non-radioactive DNaseI footprinting we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855) and a two-component system (TCS) (PSPTO_3382/PSPTO_3381/PSPTO_3380). Deletion of the TCS resulted in decreased virulence on Arabidopsis providing evidence that Fur may be involved in regulating virulence factors in DC3000.