# 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  
At 754 bases, the non-coding region between _csgD_ and _csgB_ is the fifth largest region between divergent operons in _E. coli_ K-12 and the thirteenth largest intergenic region overall (Table S6 and Figure S5). The intergenic region has strong inherent curvature and an AT content of 65.5% [108],which aids binding of nucleoid proteins such as IHF and H-NS which can induce sharp bending in DNA [34]. At least twelve proteins and five small RNAs have been shown to bind within the intergenic region between _csgD_ and _csgB_[34; 64; 109; 110] (Figure S5). Previously, most genes found affecting curli production did so through transcriptional changes at one or both curli operons [16] or by modifying cyclic-di-GMP metabolism [111] (Table S4). We identified many additional regulatory elements that also affect curli production. In fact, more than a fifth of the genes hit in the CR screen encode either signal transduction proteins or transcription factors (Table 1). The high number of regulatory proteins and the large intergenic region between _csg_ operons, are consistent with curli biogenesis being an intricately-regulated process. Indeed, 26 of the 32 mapped protein binding sites are within 200 bp of the transcription start of _csgDEFG_ operon, the highest such density in _E. coli_ (Figure S5 and Table S6). \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Function** & **COG Group** & **Number (\% Total)** & **Genes (Dark Mutants Are Bold)** \\ \hline
**Information Storage** & & & & \\ \hline Translation, ribosome structure, and biogenesis & J & 13 (4.2) & _efp_, _minA_, _pcnB_, _poxA_, _rbfA_, _rimK_, _rimM_, _rplA_, _rpsF_, _rpsT_, _rpsA_, _srmB_, _trtrB_ &  
Several mutants identified in our screen have been shown to directly or indirectly regulate \(\sigma^{\text{S}}\) levels or function, including _crp_, _clpP_, _clpX_, _dksA_, _dnaK_, _galU_, _lns_, _lftq_, _nlpD_, _nuoG_, _pgm_, and _mdoA_ (_mdoA_) [112; 113; 114; 115]. Similar to an _rpoS_ mutant, an _nlpD_ mutant completely lacks curli, probably due to loss of the major _rpoS_ promoter within _nlpD_[116]. Since DksA affects ppGpp-dependent induction of \(\sigma^{\text{S}}\)[117], we tested whether altering ppGpp production affected curli production. An MG1655 _relA spoT_ double mutant, which is defective in ppGpp synthesis [118], was light pink on CR plates and produced almost no curli proteins (Figure S6A). When the same mutants were made in the BW25113 strain, the CR binding phenotype was more severe and no CsgD or curli were detected by western blotting (Figure S6B). In addition to _rpoS_, we found that deletion of the sigma factor genes _rpoN_ and _rpoZ_ decreased curli production (Table S1), underscoring the complexity of curli regulation by different sigma factors. Several genes involved in quorum sensing and virulence were found to affect curli production including _qseB_, _qseC_, _aaeR_, _lsrF_, _ygiU_ (_mqsR_), _sidia_, and _flgM_. The _qseC_ strain produced more curli than WT; however, _qseB_ had mostly light pink colonies with a few dark red suppressor colonies (Tables S1 and S4). Previously, QseC but not QseB was found to be important for curli, type I pili, and flagella production in uropathogenic _E. coli_[119]. The Keio _flgM_ strain overproduced flagella and made less curli consistent with the antagonistic relationship between these two extracellular appendages [109; 120]. Similarly, the _hdfR_ strain, which lacks a repressor of the _flhCD_ operon, was defective for curli. However, _sidia_, which also overproduces flagella [121], had higher levels of curli production (Table S1). A mutation in the Na\({}^{+}\):H antiporter encoding gene _nhaA_ resulted in cells that stained light pink on CR indicator plates, and had much lower levels of CsgA and CsgG (Figure 5A,B,D and Table S1).