# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### Cell Envelope
(**A**) Silver stain of LPS from LPS mutants and BW25113 (WT) resolved on a 14% polyacrylamide gel electrophoresis (PAGE) Tricine gel (top panel), and the respective whole cell Western blots probed with antibodies to CsgA and CsgG are shown in the bottom part of panel A. (**B**) Addition of divalent salts MnSO\({}_{4}\) (0.5 mM), MgSO\({}_{4}\) (0.5 mM), ZnCl\({}_{2}\) (0.1 mM), or a mixture of all three divalent and CaCl\({}_{2}\) (0.5 mM) to CR indicator plates had varying abilities to suppress the CR phenotype of the indicated LPS mutants. (**C**) Whole cell Western blots of LPS mutants probed with anti-CsgG and anti-CsgA antibodies. All samples were grown on YESCA plates supplemented with the indicated salts at 26 \({}^{\circ}\)C for two days and treated with hexafluoroisopropanol (HFIP). (**D**) Transmission electron microscopy (TEM) images of _waF_ grown on YESCA and YESCA amended with a mixture of divalent cations. Scale bar equals 1 \(\upmu\)M. (**E**) The addition of 2 \(\upmu\)L of 0.1 M MgSO\({}_{4}\) to LPS mutants _waC_ and _waF_ resulted in their ability to bind CR only when they were grown on plates surrounded by BW25113. (**F**) Overexpression of _csgG_ from pMC1 did not rescue CsgA secretion in inner core LPS mutants. (**G**) The CsgG of WT (BW25113) and _waaC_ had similar mobility in a 13% sodium dodecyl sulfate (SDS)-PAGE gel after a 10 min treatment at the indicated temperatures as in previous studies [26] except samples were 10 \(\upmu\)L of 0.5 OD\({}_{600}\) resuspended cells grown on YESCA for 2 days at 26 \({}^{\circ}\)C. The Divalent mix was MnSO\({}_{4}\) (0.5 mM), MgSO\({}_{4}\) (0.5 mM), ZnCl\({}_{2}\) (0.1 mM), and CaCl\({}_{2}\) (0.5 mM). with altered curli production include _rffA_, _rffC_, _rffT_, _wzxE_, and _rfe_ (Figure S4B), which are involved in synthesis or addition of thymidine diphosphate 4N-acetyl-\(\alpha\)-D-fucosamine (TDP-Fuc4 NAc) to lipid II (Figure S4A) [71] and have increased DegP levels via \(\sigma^{\mathrm{E}}\) and Cpx induction [72]. These results suggest ECA was not required for curli production, as only mutants accumulating lipids II and III were curli deficient. Conversely, the _rfe_ strain, which accumulates undecaprenyl-P, produces more curli than WT cells (Figure S4B). Mutants in _rfe_ suppress the activation of _degP_ in the _rffA_, _rffH_, and _rffT_ strains suggesting undecaprenyl-P and lipid II accumulation have opposing roles in envelope stress responses and curli production [72]. The opposing curli phenotypes within ECA mutants are consistent with previous studies that demonstrated that increased \(\sigma^{\mathrm{E}}\) activity has a negative effect on curli production [73]. ECA mutants that induce the \(\sigma^{\mathrm{E}}\) or Cpx stress response make less curli than WT, while strains with lower induction produce more curli (Figure S4B). Similarly, mutations in _tolA_ and _pal_ have higher \(\sigma^{\mathrm{E}}\) levels [74] and were defective for curli (Table S1). The _rseA_ strain, which lacks the anti-sigma factor of \(\sigma^{\mathrm{E}}\)[75; 76; 77], produced less curli and was light pink on CR indicator plates (Figure S4C). Furthermore, ectopic expression of the anti-sigma factor _rseA_ in WT resulted in increased curli production (Figure S4D). The \(\sigma^{\mathrm{E}}\) stress response may function to limit the production of extracellular fibers during outer membrane stress. However, the _csg_ genes lack the \(\sigma^{\mathrm{E}}\) consensus sequence and overproduction of \(\sigma^{\mathrm{E}}\) did not significantly affect their transcript levels [78; 79]. Defects in LPS biosynthesis also result in \(\sigma^{\mathrm{E}}\) induction [80; 81]. Inner core LPS mutants such as _waaC_, _waaC_, _waaC_, _gaAl_D_, and _galU_ have drastically altered outer membrane protein profiles [53; 62], increased \(\sigma^{\mathrm{E}}\)-dependent transcription [82; 83; 84], and produce little or no curli (Figure 3C). The Cpx two-component system, which also negatively regulates curli specific genes [85; 86], is induced in many of these LPS mutants [72; 84].