Assembly and adhesive properties of curli : a stationary phase-specific surface organelle in Gram-negative enteric bacteria

The natural environments of bacteria are most often characterized by suboptimal conditions for growth, e. g., limited supply of nutrients. Consequently, periods of dormancy or negligible growth are the rule rather than the exception. Starvation leads to profound developmental changes. The most dramatic response to starvation is the generation of dormant spores by many species of gram-positive bacteria and the generation of multicellular fruiting bodies by myxobacteria. In contrast, many gram negative bacteria, e. g.,enteric bacteria like Escherichia coli and Salmonella enterica, do not enter a dormant state but maintain a low level of metabolism combined with an increased resistance to environmental stresses. Although not considered a typical starvation-induced response, bacteria express a variety of surface structures in response to diverse environmental conditions. Often, these structures are proteinaceous filaments extending out from the bacterial cell surface. These structures serve the purpose of mediating contact between the bacterium and a eukaryotic cell surface, a tissue matrix or serum protein, or to other bacteria, conspecific or of other species. These interactions are often the comitted steps leading to subsequent colonization of an epithelial surface, entry into a host cell, exchange of DNA between bacteria, or development of a bacterial community organized as biofilms, colonies or multicellular fruiting bodies. In Escherichia coli, a novel type of fimbriae-like structure denoted curli was recently discovered. This surface organelle is characterized by a distinct morphology, a high binding affinity for many eukaryotic proteins, and a unique pattern of stationary phase-dependent expression. To further understand the mechanisms of curli biogenesis and the regulation of curli expression, a genetic analysis was performed to identify the genetic determinants involved in these processes.

This thesis presents the identification of six genes, csgDEFG and csgBA, the products of which are specifically involved in curli production. CsgA is the major subunit protein of the curli filament. CsgB is a surface-exposed protein required for polymerization of CsgA subunits into curli filaments and for anchoring of these to the cell surface. CsgG is an outer membrane-anchored periplasmic lipoprotein which stabilizes the CsgB and CsgA proteins. CsgF, together with CsgB, participates in nucleation of CsgA subunits. A cell deficient in nucleation function secretes soluble CsgA monomers or oligomers to the growth medium. These CsgA subunits are polymerization-competent, i. e., they spontaneously polymerize to curli fibers on the surface of any cell presenting a functional nucleator (CsgB). This process of extracellular formation of curli indicates a novel pathway of fimbriae biogenesis, the extracellular nucleation-precipitation pathway. The addition of subunits to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted subunits and the growing curli tip. The adhesive properties of curli are dependent on CsgA subunits polymerized in the presence of CsgE. Thus, inactivation of the csgEgene results in curli deficient in binding. A similar type of adhesion-deficient curli is produced by cells devoid of Nacetylglucosamine-6-phosphate deacetylase. Several regulatory proteins controlling the expression of curli were identified. CsgD is required for transcription of the csgBA operon. OmpR, a transcriptional activator responding to osmolarity, and d, a stationary phase-specific sigma factor, activates transcription of the csgDEFG operon. Mutations in several other loci affect transcription of the csg genes, indicating a complex regulation of curli expression where different signals are integrated.