# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### Carbohydrate Metabolism, Energy Production, and Gluconeogenesis  
Strains with mutations in several genes involved in global carbohydrate flux and sugar import were found to be defective for curli production: _cyaA_, _crp_, _fruR_ (_cra_), _ptsH_, _ptsI_, _aceE_, _fbp_, _gnd_, _tkA_, _tpiA_ (Table S1). The metabolic flux changes of the Keio _cyaA_, _crp_, and _fruR_ strains have been examined under different growth conditions and shown to produce less phosphoenolpyruvate (PEP) from oxaloacetate in glucose limiting conditions [90; 91]. PEP is used by the phosphotransferase system (PTS) to transport and phosphorylate many different sugars. FruR increases PEP production from pyruvate through a combination of _pykF_ repression and _ppsA_ activation [92]. The cAMP-CRP complex also activates _ptsHI-crr_[93], and _cyaA_ and _crp_ mutants, both of which are defective in cAMP-CRP mediated gene activation, and have low levels of glucose uptake due to a PTS defect [94]. Combined with the curli phenotype for the PTS genes, _ptsH_ (Enzyme I) and _ptsI_ (HPr), these results suggest that a defect in the PTS system leads to lower curli production. However, many of these gene products have global effects on gene transcription. The cAMP-CRP complex regulates multiple genes including direct activation of _csgDEFG_[95; 93]. Enzymes for central metabolism, energy production, and their coenzymes also play an important role in curli production. Citric acid cycle (TCA) mutants with reduced curli production include genes encoding enzymes for the complete conversion of \(\alpha\)-ketoglutate to fumarate: _sucA_,\(B\),\(C\),\(D\) and _sdhA_,\(B\) (Tables S1 and S5). The restriction of curli defective mutants to the upper TCA cycle and the curli defects in _fruR_, _fbp_, _tpiA_, and _sfCA_ (_maeA_) indicate that gluconeogenesis is required for curli production. FruR tightly regulates gluconeogenesis by increasing gluconeogenic enzymes and decreasing glycolytic enzymes to prevent a futile cycle [96]. In _S. typhimurium_, a curli producing WT strain had higher levels of gluconeogenic end products including glucose, glycogen, and trehalose as well as lower levels of succinate, fumarate, malate, and polyamines relative to a _csgD_ strain (Table S5) [97]. Gluconeogenic-specific genes including _pckA_, _maeB_, _ppsA_, and _fbp_ were also upregulated in the WT strain, and a _ppsA pckA_ double mutant in _S. typhimurium_ was defective for curli and glycogen production [97]. Thus, gluconeogenesis appears to not only be coregulated with but also required for curli production. Gluconeogenic metabolism is important for pathogenesis in ureopathogenic _E. coli_ (UPEC) [98]. Like urine, the media used to express curli including YESCA and colonization factor antigen (CFA) are mostly composed of amino acids and small peptides. When used as a carbon source, many amino acids are broken down into pyruvate or compounds of the upper TCA cycle such as \(\alpha\)-ketoglutarate, succinate, and formate. Glucose produced by gluconeogenesis is used in LPS, glycogen, trehalose, osmoregulated periplasmic glucans (OPG), or various EPS including cellulose, ECA, and colonic acid. Mutants in most of the pathways utilizing glucose were not defective for curli production. Interestingly, outer core LPS biosynthesis requires UDP-d-glucose which is converted from glucose-6P through the action of Pgm and GalU (Table S1) and added to the heptose II of inner LPS core by WaaG [52]. The _waaG_, _pgm_, and _galU_ strains were all defective for curli production, suggesting gluconeogenic mutants may lack curli due to glucose I defective LPS. Mutants that lack glucose-I LPS modifications can be partially suppressed by the addition of divalent ions (Figure 4B). Indeed, the addition of divalent ions to _sdhA_ and _sdhB_ resulted in increased CR binding, suggesting that the lack of the glucose-I LPS modification is at least partially responsible for the curli defect in gluconeogenesis mutants (Figure S4E). Anaerobic respiration is vital for _E. coli_ to persist as a pathogen [99].