Availability of the fibre subunit CsgA and the nucleator protein CsgB during assembly of fibronectin‐binding curli is limited by the intracellular concentration of the novel lipoprotein CsgG

Abstract

SummaryCurli, an adhesive surface fibre produced by Escherichia coli and salmonellae, was proposed on the basis of genetic evidence to follow a distinct assembly pathway involving an extracellular intermediate of the fibre subunit CsgA, the polymerization of which can be induced at the cell surface by a 'nucleator' protein (CsgB). Here we show biochemically that CsgA is actively secreted to the extracellular milieu and that CsgB is surface located. We demonstrate that the putative curli assembly factor CsgG is an outer membrane-located lipoprotein. CsgG is highly resistant to protease digestion both in vivo and in vitro. During curli assembly, CsgG is required to maintain the stability of CsgA and CsgB. In line with this, it is possible to modulate the steady-state levels of CsgA and CsgB by varying intracellular levels of CsgG. This suggests that, in the absence of CsgG, CsgA and CsgB are proteolytically degraded. Moreover, curli production and steady-state levels of CsgA and CsgB can be increased above wild-type levels by overexpression of CsgG, meaning that the quantity of assembled curli fibres can be controlled by this lipoprotein.

The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization

“…WT csgB and csgB trunc were expressed in the ⌬csg strain LSR12 that contained a plasmid-encoding CsgG, the proposed curli outer membrane secretion protein (29,30). Western blot analysis revealed that WT CsgB was cell-associated, but CsgB trunc was secreted away from the cells in a CsgG-dependent manner (Fig.…”

“…The first strain overexpressed CsgG, proposed to be a central component of the curlin secretion apparatus (29). Overexpression of CsgG has been demonstrated to increase the secretion of CsgA (30). When grown under curli-expressing conditions on Congo red indicator plates, csgB cells containing pNH1 (csgB trunc ) and pMC1 (csgG) stained darker red than csgB cells containing pNH1 (csgB trunc ) or pMC1 (csgG) alone (SI Fig.…”

AgfC and AgfE facilitate extracellular thin aggregative fimbriae synthesis in Salmonella Enteritidis

“…agfD encodes a transcriptional regulator that positively regulates the expression of Tafi (Romling et al, 1998), cellulose (Romling et al, 2000), O-Ag capsule and serine hydroxymethyltransferase (Chirwa & Herrington, 2003), and negatively regulates factors that inhibit biofilm formation (Prigent-Combaret et al, 2001). The agfGFEC gene products are homologous to csgGFEC in E. coli, where csgG encodes an outer-membrane lipoprotein required for fimbrin secretion and stabilization (Chapman et al, 2002;Loferer et al, 1997), and assembles into an oligomeric complex that interacts with the products of csgEF (Robinson et al, 2006). CsgE and -F have uncharacterized roles in assembly, but CsgE at least has been called a chaperone (Chapman et al, 2002).…”

Escherichia coli tol and rcs genes participate in the complex network affecting curli synthesis

“…CsgA encodes the curlin subunit, CsgB is thought to nucleate CsgA curlin fibres (Bian & Normark, 1997), CsgD is a transcriptional activator of the csgBA operon, and CsgE, CsgF and CsgG are three putative curli assembly factors. The lipoprotein CsgG localizes to the inner leaflet of the outer membrane and may serve as a curli assembly platform (Loferer et al, 1997); CsgE and CsgF are thought to be chaperone-like proteins (Chapman et al, 2002). The csg genes appear to be expressed in environmental strains, clinical isolates and some laboratory strains of E. coli.…”