TheErwinia chrysanthemiType III Secretion System Is Required for Multicellular Behavior

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 extracellular polysaccharide, cellulose, and protein filaments, known as curli. 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 carry curli genes, requires the TTSS for pellicle formation. 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 homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36°C, even though TTSS genes were expressed at this temperature. 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 cellulose 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.Erwinia chrysanthemi is an economically important enterobacterial plant pathogen that causes soft rot and wilt diseases on numerous species of plants. Aggregative multicellular behavior, which results in the formation of large cell aggregates on the culture surface known as pellicles, was demonstrated in E. chrysanthemi over 40 years ago (32). In related species, pellicle formation requires cellulose and protein filaments, known as aggregative fimbriae or curli (37,45,49,52). The recently sequenced E. chrysanthemi 3937 (Ech 3937) genome revealed homologs of genes required for cellulose production in related enterobacteria, but no homologs of genes required for curli synthesis (N. Perna, personal communication). Ech 3937 also does not encode csgD, a regulatory protein that controls curli and cellulose production in related species (18). Thus, the regulatory proteins controlling pellicle formation and whether or not there are protein filaments that play an analogous role to curli in formation of E. chrysanthemi pellicles were unknown.In plant-pathogenic bacteria, the type III secretion system (TTSS) is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hypersensitive response conserved) genes. The TTSS functions as a molecular syringe, injecting virulence proteins into host cells; some of these proteins may interfere with host defense machinery (9,17,23). The hrc genes, many of which are hom...

Harpin Mediates Cell Aggregation in Erwinia chrysanthemi 3937

“…Pellicle formation in 3937 requires a functional type III secretion system (T3SS) (23). A similar phenomenon has been observed in Escherichia coli, where a degenerate T3SS is required for both virulence and bacterial aggregation (10).…”

“…The EC16 HrpN produced in King's B medium was sensitive to proteinase K, further evidence that the EC16 protein is localized to the outside of the bacterial cell and that localization of HrpN to the outside of the bacterial cell is not sufficient for aggregation (data not shown). Similarly, we have found that strain 3937 produces HrpN at 36°C even though no pellicle forms at this temperature (23).…”

“…Cellulose, which is a major constituent of both the 3937 pellicle and plant cell walls, is not a likely candidate since a strain 3937 cellulose synthase mutant is still able to form bacterial aggregates (23). Therefore, the basis of the interaction between HrpN 3937 and other surface molecules on bacterial cells and host cells remains to be discovered.…”

“…This was inferred from the observation that the addition of proteinase K into the pellicle-inducing medium, SOBG medium (23), prevents pellicle formation but not bacterial growth (Fig. 1A), suggesting that the aggregative factor is extracellular protein.…”

“…Pellicles are a type of biofilm that forms at the air-liquid interface. In 3937, pellicles are genetically distinct from the biofilm that forms at the surface-liquid-air interface (23). (A) Exogenous addition of proteinase K (Invitrogen, Carlsbad, CA) inhibited pellicle formation in 3937.…”