# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### Cell Envelope
Several LPS mutant strains had a notably dry and crumbly texture when dragged across the plate (Figure S1A) [17; 53; 62], including _galU_ and _waaG_ and to a lesser degree _waF_. GalU is needed for growth on galactose and trehalose and is required for the production of uridine diphosphate (UDP)-D-glucose, trehalose, the LPS outer core, colonic acid, and periplasmic glucans [63; 64; 65; 66]. Since WaaG adds UDP-D-glucose to the inner core of LPS, the _galU_ and _waaG_ mutants should be functionally similar. Both strains displayed a light pink phenotype and had similarly low levels of CsgG by Western blotting. These mutants were also visibly drier than WT, crumbled when scraped from plates [62], autoagglutined when resuspended in PBS [53], and displayed identical LPS profiles on silver stained polyacrylamide gel electrophoresis (PAGE) gels [66] (Table S1, Figure 4A, Figure S1 compare _galU_ and _waaG_). Because of the dry colony phenotype of the _waaG_ and _galU_ strains (Figure S1A), we tested the cell hydrophobicity of the inner core sugar transferase mutants using the bacteria adhesion to hydrocarbons (BATH) method [67]. Mutant strains that had less developed LPS cores were more hydrophilic as measured using the BATH method (compare _waaC,F,G_ in Figure S1B and Figure 4A). As expected, both _galU_ and _waaG_ had similar cell hydrophobicity profiles. Because of the significant changes made to the membrane in the _galU_ and _waaG_ mutants, we looked at the integrity of the CsgG protein present within the membrane. Because CsgG is required for the stability and secretion of CsgA and CsgB [23; 24; 25], and several of the inner core LPS mutants had decreased levels of CsgG protein (Figure 4A), we asked if ectopic overexpression of CsgG could rescue the curli assembly defect in the inner core LPS mutants. Expression of _csgG_ from plasmid pMC1 increased the amount of CsgG protein in the _waaC,__waaF_ and _waaG_ strains, but was unable to restore CsgA protein levels or curli production (Figure 4F). Interestingly, the transcript levels of _csgD,__csgA_, and _rpoS_ were near WT levels in the _waaF_ strain (Figure S3). Taken together, these data suggested that the CsgG present in inner core LPS mutants was not able to properly fold or function in the outer membrane. To test this idea, we supplemented CR indicator plates with divalent cations. Divalent cations interact with LPS molecules in the outer membrane (OM) [52; 68], and their presence can stabilize outer membrane porins [69; 70]. When added to CR indicator plates, the divalents Mn\({}^{2+}\), Mg\({}^{2+}\), and Zn\({}^{2+}\) all increased CsgG levels and partially complemented CsgA secretion and curli production in inner core LPS mutants (Figure 4B-D). Addition of Mn\({}^{2+}\) near inner core mutants also resulted in increased CR binding (Figure 4E). Many LPS mutants displayed an aberrant CR phenotype: a rim of darker stained cells near the edge of the plate (Figure 3A and Figure S1A) where there is less competition for limiting divalent cations. The mechanism of complementation by addition of divalent cations is unclear. The divalent cations may be interacting with phosporhyl moieties to reshape the OM and decrease the higher phospholipid content of the OM outer leaflet in inner core LPS mutants [68]. Alternatively, they may stabilize the interactions between LPS and CsgG or a partner OM protein; however, addition of divalent cations did not alter the heat modified mobility of CsgG from _waaF_ relative to WT (Figure 4G), suggesting divalent cation rescue of CsgA levels is not due to direct interaction with CsgG. Other components of the cell envelope, including the enteric common antigen (ECA) and the periplasmic glucans, were found to affect curli production (Figure S4). Mutants in ECA biosynthesis  
Figure 4: Effects of inner core LPS mutants on curli production.