# The Production of Curli Amyloid Fibers Is Deeply Integrated into the Biology of _Escherichia coli_
## 1 Introduction  
Curli are thin aggregative fimbiae produced by many Enterobacteriaceae including _Escherichia coli_ and _Salmonella typhimurium_[1; 2]. As the major protein component of the extracellular matrix, curli are associated with biofilm development [3]. Furthermore, curlated bacteria adhere to and colonize a variety of surfaces [4; 5; 6; 7; 8] and are resistant to damage from predation, desiccation, oxidative stress, and other antimicrobial agents [4; 5; 9; 10; 11]. Curli fibers have also been implicated in pathogenesis and aid in host cell adhesion, invasion, and immune system activation [12; 13; 14]. Curli fibers are the product of a dedicated and highly regulated assembly system encoded by curli specific genes (_csg_), which are arranged on two divergent operons _csgDEF_ and _csgBAC_[15; 16; 17]. Found primarily in the genomes of Enterobacteriaceae, _csg_ genes are also present in other Gammaproteobacteria including members of the Pseudomonasaceae, Shewanellaceae, and Vibrionaceae families, as well as the more distantly related Bradyrhizobiologyaceae, Burkholderiaceae, andFlavobacteriaceae families [18]. CsgD is the master biofilm regulator, and is required for transcription of the _csgBAC_ operon [15; 19]. At the cell surface, the major curli fiber subunit CsgA is nucleated into an extracellular amyloid fiber by the minor fiber subunit CsgB [1; 20; 21; 22]. Secretion of both curli fiber subunits requires the lipoprotein CsgG, which forms a pore within the outer membrane [1; 23; 24; 25; 26]. CsgE acts as the cap for CsgG, blocking the unfolded CsgA from returning into the periplasm [27]. CsgF has been shown to interact with CsgG and may aid in its function [25; 26; 28], as well as keeping CsgB associated with the bacterial outer membrane [22; 29]. CsgC is dispensable for curli formation; however, it may influence curli fiber morphology, and it potentially discourages CsgA amyloid formation within the cell [15; 30; 31; 32]. As the first bacterial protein identified as an amyloid [1], curli are part of a growing class of functional amyloid proteins that have evolved to fulfill physiological roles [33]. Regulation of _csgDEFG_ operon is complex, and involves several proteins [16]. Transcription of the _csgDEFG_ operon is under the control of RpoS (\(\sigma^{\text{S}}\)), Crl, MlrA, H-NS, IHF, and a number of bacterial two component systems [16; 34] (see Table S4). The intervening region between the curli operons is the fifth largest divergent operon in _E. coli_, yet previous screens to find additional accessory factors and regulators have identified only a handful of genes including _nagA_, _ompR_, _dksA_, and _rssB_[15; 35; 36]. The current cohort of regulators does not sufficiently account for the diverse conditions that induce _E. coli_ expression of curli and other biofilm components [37; 38; 39]. Therefore, we performed a comprehensive screen for genes involved in curli production using the Keio collection [40]. The Keio collection is a near complete set of single gene knockouts in K-12 _E. coli_. By growing the collection on plates containing the amyloid specific dye Congo red (CR), we identified more than 300 strains that had changes in curli production. These strains lack genes involved in a variety of cellular processes including regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. Many of these genes are known to affect RpoS (\(\sigma^{\text{S}}\)) levels or induce the Cpx or RpoE (\(\sigma^{\text{E}}\)) stress responses. Induction of either the Cpx or \(\sigma^{\text{E}}\) stress response system resulted in less curli production, while stimulation of \(\sigma^{\text{S}}\) levels increased curli expression. Few of the genes identified here are involved in motility, underlying the fact that motility and biofilm formation are distinct microbial behaviors. We propose that curli fiber formation is part of a unique lifestyle in _E.