THE ERWINIA CHRYSANTHEMI TYPE III SECRETION SYSTEM IS REQUIRED FOR MULTICELLULAR BEHAVIOR

Authors: YAP, MEE-NGAN - UNIV. OF WISC, MADISON; YANG, CHING-HONG - UNIV. OF WISC, MADISON; Barak Cunningham, Jeri; JAHN, COURTNEY - UNIV. OF WISC, MADISON; CHARKOWSKI, AMY - UNIV. OF WISC, MADISON

Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2004
Publication Date: 1/20/2005
Citation: Yap, M., Yang, C., Barak, J.D., Jahn, C., Charkowski, A.O. 2005. The erwinia chrysanthemi type III secretion system is required for multicellular behavior. Journal of Bacteriology. 187(2):639-648.

Interpretive Summary: Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of an extracellular polysaccharide, cellulose, and protein filaments, known as curli. Curli interact intimately with host cells, genetically linking aggregative multicellular behavior to bacterial virulence. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here we demonstrate that Erwinia chrysanthemi, which does not encode curli genes, requires the TTSS for pellicle formation in culture. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for aggregative multicellular behavior. We found that cellulose is a component of the E. chrysanthemi pellicle, but that pellicle formation still occurs in a strain with an insertion in a cellulase synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.

Technical Abstract: Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of an extracellular polysaccharide, cellulose, and protein filaments, known as curli. Curli interact intimately with host cells, genetically linking aggregative multicellular behavior to bacterial virulence. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here we demonstrate that Erwinia chrysanthemi, which does not encode curli genes, requires the TTSS for pellicle formation in culture. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a '54 dependent enhancer-binding protein (EPB) homolog. In turn, a functional hrpS gene is required for activity of the sigma factor homolog hrpL, which activates genes encoding the TTSS structural and secreted proteins. Mutations in hrpX and hrpY, which encode cognate sensor kinase and response regulator homologs, respectively, did not have identical phenotypes. The hrpY mutant did not form pellicles, while the hrpX mutant did, suggesting that the response regulator HrpY is phosphorylated by sensors in addition to HrpX. Pellicle formation was temperature dependent and pellicles did not form at 36ºC, although TTSS genes were expressed. We found that cellulose is a component of the E. chrysanthemi pellicle, but that pellicle formation still occurs in a strain with an insertion in a cellulase synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS