# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### Cell Envelope  
Curli assembly occurs at the cell surface, therefore, it is not surprising that genes involved in cell envelope and outer membrane biogenesis have defects in curli assembly (COG family group M) [49]. The cell envelope includes the inner membrane, the periplasm, the outer membrane, and extracellular structures including fimbiae and extracellular polysaccharides (EPS) [50]. Multiple lipopolysaccharide-accharide (LPS) biosynthesis gene mutants, especially those involved in assembling the inner core region of LPS, were defective for curli production (Figure 3A,B). These genes encode hexose and heptose transferases, enzymes that produce the inner core sugar building block adenosine diphosphate (ADP)-L-glycero-d-manno-heptose, the LPS kinase _waaP_, and the transcriptional antiterminator for the _waaQGPSBIJYZK_ operon _waaH_[51; 52; 53; 54]. Western blot analysis showed that many LPS mutants  
Figure 2: Genes affecting curli production perform diverse roles. Genes identified for altered curli production sorted by clusters of orthologous groups (COGs) showed involvement of regulation, environmental sensing, metabolism, cell envelope biogenesis, transport, and protein turnover in curli production. with CR defects had corresponding decreases in CsgA and CsgG levels (Figure 3A,C compare WT to _lpxA_, _gmlB_, and _waa_ mutants). Mutants in the inner core sugar transferases _waaC_, _waaF_, _waaG_, and the heptose biosynthesis genes _lpCA_ and _waaE_ were the most defective in CR binding and these strains had lower CsgG levels compared to WT and no CsgA was detected (Figure 3C). Previous studies have implicated the LPS biosynthesis genes _waaG_, _ddhc_ (_rfbH_), and _lpxM_ (_msbB_) as being important for curli production [55; 56]. In _Salmonella enterica_, both _waaG_ and _ddhc_ strains produce less curli than WT [55]. Similarly, the _waaG_ strain in K-12 _E. coli_ has reduced curli production. [57; 58]. We also looked at other lipid A modifying enzymes to see if they affected curli production. The _lpxL_, _lpxM_ (_msbB_), _lpxP_, _pageP_, and _arnT_ mutant strains all produced curli at WT levels suggesting the modification state of the lipid A core does not necessarily affect curli production in K-12 strains of _E. coli_ (Figure S2C, left plate). Analysis of mutations in other LPS synthesis genes revealed that curli production is likely affected by genes that synthesize or transfer the ADP-l-glycero-D-manno-heptose to Lipid A-KDOII [52] (Figure 3A,B). Mutations in _waaD_ lead to an accumulation of heptose free LPS [59], and _waaD_ mutants had decreased curli production compared to WT (Figures 3C and 4A). Mutations in _gmlB_ also accumulate heptose-free LPS and, like the _waaD_ mutants, the _gmlB_ strain had decreased curli production (Figure 3A) [60]. The composition of the LPS in the _waaD_ and _gmlB_ strains may explain their intermediate curli phenotype. Unlike the _waaG_ mutant strain and other inner core mutants, both _waaD_ and _gmlB_ strains have a small amount of glucose I and further modified LPS [60; 61] (Figure 4A). Accordingly, a mutation in _waaI_, which has fully glucose I modified LPS [52], is unaffected for curli production. Similarly, _waaP_, which lacks phosphorylation of heptose I [52], had less curli production  
Figure 3: Lipopolysaccharidescharide (LPS) mutants are defective for curli production. (**A**) LPS mutant strains and BW25113 (WT) grown on CR indicator plates at 26 \({}^{\circ}\)C for two days. (**B**) A schematic of LPS structure. Blue lines represent curli defective LPS mutants. Red lines represent LPS genes not in the Keio collection. (**C**) Whole cell Western blots of LPS mutants probed with anti-CsgG and anti-CsgA antibodies. All samples were grown on YESCA plates at 26 \({}^{\circ}\)C for two days and treated with formic acid. and partially substituted LPS (Figure 4A). Thus, it appears that even a small amount of glucose I modified LPS is capable of supporting curli subunit secretion and assembly into an amyloid fiber.