# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 4 Conclusions  
More than 300 gene deletions that altered curli amyloid levels were identified in our CR screen. Several of the mutants focused on in this study are part of regulatory cascades, including stress response systems, which affect _csg_ transcription. General themes arose for two major stress response systems, \(\sigma^{\text{S}}\) and Cpx. Mutations that that result in elevated \(\sigma^{\text{S}}\) levels produced more curli, while, those that induced \(\sigma^{\text{E}}\) or the Cpx system, produced less curli. An intact cell envelope was required to support curli biogenesis, as mutations in LPS and OPG resulted in less extracellular amyloid. Curli amyloid production was tied to key metabolic pathways such as the TCA cycle, the nucleotide synthesis pathways, and the catabolite utilization pathways. Collectively, the number and diversity of mutations that result in altered CR binding demonstrate that amyloid biogenesis is a complex and highly regulated developmental pathway in _E. coli_.  
Supplementary Materials: The following are available online at www.mdpi.com/2218-273X/7/4/75/s1, Figure S1: Congo Red and hydrophobicity of different LPS mutant _E. coli_ strains, Figure S2: Differences in Congo Red phenotypes of Keio collection strains on YESCA and CFA plates and CFA plates without Coomassieie Brilliant Blue counterstain, Figure S3: qRT-PCR of inner core LPS and _nhaA_ mutants, Figure S4: Effects of eE induction on curli production, Figure S5: The intergenic region between _csgD_ and _csgB_ has many transcriptional binding sites, Figure S6: Low ppGpp strains and _dksA_ mutants produce less curli, Table S1: Congo red phenotypes and Western blot data from Keio strains with altered curli production, Table S2: Phenotypes of suppressor strains with more than one CR phenotype and the primers used, Table S3: Cellular localization of identified gene products, Table S4: Genes required for or known to affect curli production, Table S5: CsgD regulon excluding _csgBAC_ and _csgDEF_G, Table S6: Intergenic regions in _E. coli_ and the number of transcriptional binding sites in divergent intergenic regions, Table S7: Venn diagram gene List, Table S8: Congo red phenotypes of Keio strains on YESCA and CFA plates or CFA plates without Coomassieie Brilliant Blue, Table S9: CsgD protein and transcript levels relative to WT, Table S10: Strains, plasmids, and primers used.  
**Acknowledgments:** We would like to thank [PERSON] and [PERSON] for providing strains and plasmids. We would also like to thank [PERSON], [PERSON], [PERSON], [PERSON] and members of the [PERSON] lab for helpful discussions and critically reading the manuscript. We also want to thank the three reviewers who painstakingly vetted the expansive data in this work. Their comments helped us prepare a stronger manuscript. This work was funded by National Institutes of Health grant R01 GM118651 to MRC.  
**Author Contributions:** [PERSON] and [PERSON] conceived and designed the experiments; [PERSON] performed most of the experiments with [PERSON], [PERSON], [PERSON] and [PERSON] helping with the RT PCR, CsgD western blots, and the initial screening of the Keio collection on Congo Red plates. The data was analyzed by [PERSON], [PERSON], [PERSON] and [PERSON]. [PERSON] wrote the manuscript with help from [PERSON], [PERSON] and [PERSON].  
**Conflicts of Interest:** The authors declare no conflict of interest.