Curli fiber assembly occurs by a process termed nucleation-precipitation, where soluble CsgA and CsgB interact at the cell surface to form insoluble amyloid fiber aggregates. A key event preceding nucleation-precipitation is the transportation of CsgA and CsgB to, and across, the outer membrane. CsgA and CsgB stability and secretion depend on the outer membrane-localized lipoprotein CsgG (21
). Here, we report that CsgG is spatially restricted on the cell surface and that other csg
-encoded proteins are required for organization of CsgG around the cell.
We found that CsgG formed SDS-resistant multimers and was clustered into spatially discrete foci that were exposed to the extracellular surface. Furthermore, we observed that curli fibers emanated from spatially discrete regions of WT cells (Fig. ). Immunogold labeling revealed that CsgG was most abundant at the point(s) of the cell where curli emanated from the surface (Fig. ). Additionally, we observed some binding of the gold-labeled anti-CsgG antibodies to the fibers themselves, suggesting that CsgG may become dislodged from the membrane during curli assembly. However, fiber-associated CsgG is likely only a minor fraction of the population, since CsgG-His is not recognized by an anti-His antibody added to the outside of cells (Fig. ). The spatial restriction of CsgG into foci around curli fibers suggests a model where CsgA and CsgB fiber assembly is coordinated with CsgG spatial organization.
What mechanisms may be responsible for the clustering of CsgG around curli fibers? One possibility is that CsgG is spatially restricted prior to CsgA or CsgB secretion. In this scenario, CsgA and CsgB are secreted to the same location of the cell surface, perhaps facilitating the high efficiency of curli assembly. Another possibility is that CsgG is not restricted into foci until after secretion of CsgA and CsgB begins. The postsecretion aggregation of CsgA and CsgB into fibers may itself result in the clustering of CsgG around the fibers. Our data favor the later model. First, we did not observe spatially clustered CsgG foci in CsgG-expressing strains deficient in curlin secretion or curli assembly (Fig. ). Further, CsgG foci were not evident when we transformed csgA
cells with a plasmid encoding CsgA-ΔR1, a CsgA mutant protein that is stable and secreted but is defective in fiber assembly (32
) (Fig. ).
We also found that the loss of CsgE, CsgF, CsgA, or CsgB resulted in loss of spatially clustered CsgG foci, with little change in CsgG surface exposure or targeting to the outer membrane (Fig. ). Stable CsgG oligomers likely form independently of other csg
-encoded proteins, since slow-migrating SDS-resistant CsgG species were detected in all strains expressing CsgG (Fig. ). However, the CsgG complexes formed in WT cells were slower migrating than the CsgG complexes observed in strains lacking CsgE, CsgF, CsgB, or CsgA (Fig. ). Overexpression of CsgG in the absence of all of the csg
proteins did not increase the relative amount of high-molecular-mass CsgG, as overexpressed CsgG migrated predominately as the intermediate-molecular-mass species (see Fig. S1 in the supplemental material). Therefore, some parameter besides CsgG concentration determines formation of the highest-molecular-mass species. Taken collectively, our data suggest two distinct phases of CsgG assembly and organization: (i) CsgG is targeted to the outer membrane and exposes the surface-accessible domain in the absence of any or all of the other csg-
encoded proteins, and (ii) spatial restriction of CsgG into microdomains and assembly of the highest-molecular-mass CsgG complexes requires curli fiber polymerization supported by the other csg-
encoded proteins. Since CsgG physically interacts with CsgE, CsgF, and CsgA (28
), any or all of these proteins may contribute to CsgG spatial restriction. However, genetic analyses to determine exactly which of these CsgG-interacting proteins is required for either the assembly of CsgG multimers or spatial restriction of CsgG were difficult, as deletion of any single csg-
encoded protein results in the loss of multiple other csg
-encoded proteins from the cell surface (8
Previous results indicated that the N-terminal cysteine of CsgG was lipidated and that lipidation was required for the transport of CsgG to the outer membrane (21
). We showed that CsgG contained a domain exposed to the cell surface (Fig. ), and a previous study indicated that CsgG had a periplasmic domain (21
). Surface-exposed lipoproteins have been identified in several bacterial species, including Escherichia
, and Neisseria
spp. Membrane-spanning lipoprotein translocons are not unprecedented. For example, the lipoprotein Wza is an E. coli
polysaccharide translocon that spans the outer membrane (11
). CsgG and Wza are topologically similar, as both have periplasmic and surface-exposed domains (Fig. ) (11
), and are also functionally similar, as both have been implicated as conduits for secretion across the outer membrane (10
). Importantly, CsgG lacks any apparent amino acid sequence similarity with either Wza or any other family of proteins (data not shown), and the architecture of the CsgG membrane-spanning domain(s) remains unclear. Future exploration of the domain architecture of CsgG and definition of the residues governing the contacts between CsgG and its many interacting proteins will help clarify the biology of this unique lipoprotein.
One outstanding question about the biosynthesis of functional amyloids is how cells control amyloid fiber aggregation without any apparent cellular toxicity. In the curli biogenesis system, the activities of nucleation and polymerization are separated into two different proteins, CsgB and CsgA, respectively. This suggests that the cell can avoid unregulated fiber polymerization by keeping CsgA and CsgB separated until they reach the cell surface. The fiber-dependent spatial clustering of CsgG suggests an elegant mechanism to regulate the segregation of CsgA and CsgB: only when the csg-encoded proteins interact with CsgG do spatial restriction and CsgA and CsgB interaction occur. These results support a model where CsgG is the center of a curli assembly platform, although very little is known about the molecular nature of the protein-protein interactions that facilitate CsgG ultrastructural changes. In particular, the mechanisms preventing CsgA and CsgB amyloid assembly on the periplasmic face of the spatially restricted assembly complex remain to be elucidated. Clarification of how the curli assembly platform forms will help further unravel the mechanism of coordinated curli amyloid biogenesis.