# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### Multiple Regulatory Networks Control Curli Gene Expression
NhaA is one of three sodium ion antiporters which uses the proton electrochemical gradient to expel sodium ions [64], and had been loosely linked to amyloid production in _Shewanella_[122]. The _nhaA_ mutant was more motile than WT (Figure 5C), had more FliC protein when measured by western blot (Figure 5B), and more flagella than wild type when examined by transmission electron microscopy (TEM) (Figure 5D). When bound to Na\({}^{+}\), NhaR activates _nhaA_ and other genes including _pgaABCD_ and _osmC_[64]. Since high levels of intracellular Na\({}^{+}\) might result in a constitutively active NhaR, we made a _nhaAR_ deletion, which did produce a small amount of curli (Figure 5D). Curli fibers are maximally produced at room temperature in low salt conditions [19]. High salt and osmolarity typically repress _csg_ transcription through the OmpR/EnvZ and Cpx systems [86], which respond to high osmolarity in the periplasm. However, YESCA is a low salt medium able to support curli production in _E. coli_. High Na\({}^{+}\) also inhibits the proper assembly of FtsZ in vitro [123]. When we examined _nhaA_ and _nhaAR_, we found many filamentous cells (Figure 5D). Similary, a _kdpD_ mutant displayed cell division defects and had reduced curli production (Figure 5B,D). KdpD regulates the influx of potassium, which promotes FtsZ assembly [64]. Because high levels of CpxR-P results in aberrant cell division [124], we examined the levels of Cpx and \(\sigma^{\text{E}}\) regulated genes in _nhaA_ and _nhaAR_. Double deletions of _nhaA cpxR_ and _nhaAR cpxR_ both produced more curli than _nhaA_ and _nhaAR_ (Figure 5F); however, over expression of _rseA_ or _rseAB_ resulted in lower curli production (Figure 5E). Collectively, these results suggest the high intracellular sodium in _nhaA_ strains is inhibiting cell division and results in Cpx induction which decreases curli production. Figure 5: Sodium antiporter gene _nhaA_ is required for curli production. (**A**) Strains were grown on CR indicator plates at 26 \({}^{\circ}\)C for two days. (**B**) Whole cell Western blots probed with antibodies to CsgA, CsgG, and FilC at 24 and 48 h. All samples were treated with HFIP; (**C**) Motility of WT, _csgA_, _csgG_, and _nhaA_ strains in 0.2% YESCA motility plates at 26 \({}^{\circ}\)C. (**D**) TEM images from cells grown for 26 \({}^{\circ}\)C for two days on YESCA plates. Black arrows indicate flagella. Red arrows indicate curli. Green arrows indicate filamentous cells. Scale bar equals 1 \(\mu\)m. (**E**) Expression of _rseA_ in trans using pRseA or pRseAB does not rescue curli expression in _nhaA_ or _nhaAR_ strains as detected by CR binding or Western blot probed with antibodies to CsgA or CsgG. (**F**) Expression of _rseA_ in trans from pRseA or pRseAB does not rescue curli production in _nhaA_ cpxR or _nhaAR_ cpxR_ double deletions as detected by CR binding or Western blot probed with antibodies to CsgA or CsgG. The master biofilm regulator, CsgD, has been shown to affect the transcription of genes outside the curli operon, including those promoting biofilm production, gluconeogenic metabolism, and peptide import (see Table S5). CsgD also plays an important role in decreasing flagella rotation and production, promoting the switch from single planktonic growth to community behavior, through the activity of AdrA, ci-di-GMP, and \(\sigma^{\text{S}}\) as well as direct repression of the _fliE_ and _fliFGHIJK_ operons [109; 120]. Here, _ffgM_, _flhc_, _fliI_, _fliG_, and _fliT_ had altered curli production (Tables S1 and S4). To further explore the intersection of flagella and curli we compared the results from our screen on CR indicator plates with other screens involving the Keio collection. [PERSON] et al. [125] screened the Keio collection for defects in swarming motility using Eiken Agar and subsequently checked the swarming mutants for reduced swimming motility [125; 126]. Using GeneVenn [127], motility genes identified by [PERSON] et al. were compared to the genes identified in our screen (see Figure 6).