# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 2 Results and Discussion  
### Forward Genetic Screen to Identify Genes Involved in Curli Production  
To better understand curli production and regulation, we screened the Keio collection for mutants with altered curli production using Congo red (CR) indicator plates (Figure 1A,B). Curliated colonies turn red when grown on CR plates, while strains that do not produce curli remain white [41]. The Keio collection consists of 3985 nonessential gene mutants comprising more than 90% of the 4390 open reading frames of _E. coli_ K-12 (named wild type, WT, in the remaining manuscript) [40]. Following growth in conditions that favor curli production (26 \({}^{\circ}\)C for 48 h), strains with changes in CR phenotypes were isolated and scored according to their color, which varied from the white color of _csgA_ to the darker red of _cpxR_ (Figure 1B). Genes known to affect curli production, such as curli specific genes (_csgABEFG_) and previously described transcription factors (_cpxR_ and _ompF_) were identified, validating our screening technique. Strains such as _thyA_, _glnA_, _ubiE_ and _ubiF_, did not grow well under the conditions tested and were not further analyzed (see _ubiF_ Figure 1A and Table S1 strains marked with '). Strains with an altered phenotype were regrown on CR indicator plates for verification and scored numerically according to color from lightest to darkest (Figure 1B and Table S1). We isolated 332 Keio mutants with reproducible CR phenotypes. Of these, 64 strains had two or more phenotypes upon isolation of single colonies. Each genotype from these gene deletion strains was verified by polymerase chain reaction (PCR), revealing that most strains had the correct Kan' insert for each phenotype (Table S2). We excluded the 64 mixed phenotype strains from further study as they likely have second site suppressors. However, we included the strains _cmk_, _fabF_, _mdoG_, _pgm_, _trpD_, _trpE_, _yiaK_, and _ymgE_ because both of their CR phenotypes were defective (lighter red than WT cells). The resulting 276 mutants were 14.3% white, 16.5% light pink, 27.2% pink, 18.6% light red, and 23.3% dark red when grown on CR indicator plates (Figure 1C). To better quantify curli production in the identified mutants, CsgG and CsgA protein levels were measured in whole cell extracts by Western blotting (sample of color groups Figure 1D and Table S1). Protein levels were generally consistent with CR phenotypes (Figure 1D). In some light pink mutants, the curli secretion protein CsgG [23; 24; 25] was present; however, no CsgA was detectable (Table S1). We divided the gene mutations with altered CR binding into clusters of orthologous groups (COGs) [40; 42; 43; 44; 45] and used Echobase [46] to assign a cellular location to each protein product  
Figure 1: Overview of screen for mutants affecting curli production. (**A**) The Keio collection was screened on CR indicator plates after inoculation with a bolt replicator and incubation for two days at 26 \({}^{\circ}\)C. The collection was screened three times. (**B**) Associated phenotype scoring: 1 or white = _csgA_; 2 or light pink = _fbfq_; 3 or pink = _nagA_; 4 or light red = _greA_; 5 or wild type (WT) = BW25113; and 6 or dark red = _cpxR_. (**C**) Distribution of Congo red (CR) phenotypes based on scoring from white to dark red. (**D**) Whole cell Western blots of strains probed with anti-CsgG and anti-CsgA antibodies. (Table 1 and Table S3, Figure 2). Collectively, the genes identified in the CR screen are predicted to encode proteins that are primarily localized to the cytoplasm. Genes in the transcription COG (K) are likely to have collateral effects as regulators often control multiple gene products, such as the case with cAMP receptor protein (CRP) which has been shown to have 70 unique targets [47]. Because of this, of the 43 transcription related genes, only those with previously reported relationships or verified binding sites on the _csg_ intragenic region were studied.