# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion
### CsgD Transcript and Protein Levels Are Altered in Several Strains with CR Phenotypes  
CsgD is considered the \"master\" biofilm transcription factor [19; 130]. We selected a total of 38 mutant strains, sampling each CR phenotype and representing each COG, to further investigate the regulation of _csgD_ at both transcript and protein level. _csgD_ transcript levels were measured in all 38 strains using quantitative real time polymerase chain reaction (RT-qPCR) (Table 2 and Table S9) and CsgD protein levels were measured by Western blot analysis (Table S9 and representative blot Figure 7). Twenty-one of the 38 strains had _csgD_ transcript levels that were significantly different from WT (Table 3). There was a correlation of CR phenotypes with _csgD_ transcript and CsgD protein levels. For example, of twelve mutants tested that had increased CR binding, eight had increased levels of _csgD_ transcripts and ten had increased levels of CspD protein (Table 2 and Table S9). Mutants _mdoC_, _perR_, and _cusB_, which presented increased _csgD_ transcripts, did not have a detectable change in CsgD protein levels while _truB_ and _qseC_ exhibited no change in _csgD_ transcripts to account for the increase of CsgD levels (Tables 2 and S9). Of 19 mutants that were white, light pink, or pink on CR plates, 57% of them also had significantly decreased _csgD_ transcript levels (Table 2) and all but _nagA_, _dkSA_, _aneR_, _glvG_, _cmr_, and _pltp_ had decreased CgsD levels consistent with their CR coloring and qPCR data (Table S9). There were also notable cases where the _csgD_ transcript or protein levels did not predictably correlate with CR binding. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**CR Phenotype** & **Strain** & **Average Relative to BW25113 (WT)** & **Standard Deviation** & **Outcome of \(t\)-Test** \\ \hline Red & BW25113 & 1 & N/A & N/A \\ White & _csgD_ & N.D. & N/A & N/A \\ White & _nuA_ & 0.018 & \(\pm\)0.0068 & _p_-value \textless{} 0.001 \\ White & _pltp_ & 0.841 & \(\pm\)0.130 & Not significant \\ White & _purD_ & 0.036 & \(\pm\)0.053 & _p_-value \textless{} 0.001 \\ White & _lon_ & 0.392 & \(\pm\)0.187 & _p_-value \textless{} 0.05 \\ Light Pink & _nuaC_ & 1.118 & \(\pm\)0.363 & Not significant \\ Light Pink & _duaK_ & 0.221 & \(\pm\)0.034 & _p_-value \textless{} 0.001 \\ Light Pink & _speB_ & 0.578 & \(\pm\)0.114 & _p_-value \textless{} 0.05 \\ Light Pink & _hfg_ & 0.06 & \(\pm\)0.080 & _p_-value \textless{} 0.001 \\ Light Pink & _aroA_ & 0.223 & \(\pm\)0.057 & _p_-value \textless{} 0.05 \\ Pink & _fabH_ & 1.171 & \(\pm\)0.315 & Not significant \\ Pink & _f IgM_ & 0.86 & \(\pm\)0.254 & Not significant \\ Pink & _ddpD_ & 0.766 & \(\pm\)0.337 & Not significant \\ Pink & _pyrC_ & 0.045 & \(\pm\)0.020 & _p_-value \textless{} 0.001 \\ Pink & _nagA_ * & 3.38 & \(\pm\)1.946 & _p_-value \textless{} 0.05 \\ Pink & _fihA_ & 0.695 & \(\pm\)0.132 & _p_-value \textless{} 0.05 \\ Pink & _dksA_ * & 5.115 & \(\pm\)1.076 & _p_-value \textless{} 0.001 \\ Pink & _lngbD_ & 0.278 & \(\pm\)0.043 & _p_-value \textless{} 0.05 \\ Pink & _rstA_ & 0.492 & \(\pm\)0.187 & _p_-value \textless{} 0.05  
\end{table} 
Table 2: Selected mutant _csgD_ levels as compared to WT. Figure 7: CsgD protein levels are changed in several mutants. A representative western blot is shown for the data presented in Table S9. CsgD protein levels are shown in green and \(\sigma\)70 levels are shown in red as a loading control. CsgD levels were normalized to \(\sigma\)70, and then each mutant was normalized to WT to yield the relative CsgD protein levels for each mutant list of genes. \(\sigma\)70 migrates father in the _hybD_ line consistently over multiple trials on different days. Two mutants that were pink on CR plates, _nagA_ and _dksA_, had significantly increased _csgD_ transcript levels (Table 2). However, it appears that the 16s ribosomal RNA (rRNA) levels were changed in these mutants and when we normalized by total RNA, the _csgD_ transcript levels were similar to WT (Table 4).