Many bacteria assemble extracellular amyloid fibers on their cell surface. controlled and ordered amyloid assembly. Here we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. and species (Fig. 1A) [10-13]. Curli are the major proteinaceous component of Chloroprocaine HCl biofilms Chloroprocaine HCl and are important for surface colonization and interacting with host factors and the host immune system [10 14 Curli production is usually easily scored in the lab by plating cells on agar plates made up of the amyloid binding dye Congo reddish [17 22 Like all amyloids the major curli subunit CsgA is usually capable of self-polymerizing into β-sheet-rich amyloid fibers that bind to the amyloid specific dye Thioflavin T (ThT) and can be visualized using transmission electron microscopy (TEM) [4 23 24 put together CsgA amyloid fibers are indistinguishable from fibers formed [23]. Physique 1 Curli production contributes to biofilms. A. k-12 strain BW25113 produced on Chloroprocaine HCl a low salt agar plate at 26°C produce cell surface associated curli fibers that are visible by transmission electron microscopy. Level bar is usually 500nm. B. The … The ability of curli to Chloroprocaine HCl act as a strong scaffolding agent in KCNRG biofilm formation stems from properties inherent to all amyloids. Curli are highly stable 6-12 nm wide non-branching fibers (Fig. 1A) that are resistant to degradation by proteases and denaturation by detergents [4]. Pretreatment with a strong denaturant such as formic acid or hexafluoroisopropanol is required to depolymerize curli fibers so that monomers of the major subunit protein CsgA can be resolved on an SDS-PAGE gel [4]. CsgA amyloids like other amyloids are β-sheet rich and assemble into a highly stable cross-β structure stabilized in large part by tight “steric zipper” interactions between side-chains on adjacent β-linens [4 25 The amyloid characteristics of curli fibers are clearly important for their biological function and afford many useful techniques for quick and study of curli biogenesis biofilm formation and integrity and amyloid assembly [28 29 2 Curli expression is usually highly coordinated Curli gene expression is usually a highly regulated and coordinated event both around the cellular level and within a biofilm community. Curli are the major protein component of biofilms yet the production of curli within a biofilms is restricted to a distinct subpopulation [14 30 Expression is usually controlled by several environmental signals and chemical gradients including heat osmolarity and oxygen [34-36]. Within a rugose or rough colony biofilm (Fig. 1B) curliated cells localize to the air-colony interface [30 33 This bimodal populace development can be triggered by oxidative stress and in turn rugose biofilms are guarded from oxidative damage resulting from exposure to hydrogen peroxide [30]. Bacteria within curli- Chloroprocaine HCl and cellulose-dependent rugose colonies are also guarded from desiccation and are more resistant to sodium hypochlorite treatment (or bleach) when compared to non-curliated colonies or planktonic cells respectively [37]. Successful biofilm formation requires spatial and temporal regulation of curli assembly which relies on sophisticated coordination of gene expression and protein production (Table 1). Table 1 Direct Regulators of the Curli Operons. The curli specific genes (are found in two divergently transcribed operons the intergenic region of which is the 7th largest in and subject to extensive regulation [17 38 39 The promoter is recognized as one of the most complexly regulated promoters in [40]. Curli are primarily expressed during stationary phase and at low heat (below 30°C) although some clinical isolates can express curli at 37°C [10 11 35 41 Both curli promoters are regulated by the stationary phase alternate sigma factor σS which is usually assisted by the thermo-sensitive protein Crl (Table 1) [10 41 42 Stationary phase expression of the promoter is usually likewise positively regulated by the stationary phase transcription factor MlrA (Table 1) [43 44 Curli expression is also internally regulated by the first gene product of the operon: CsgD (Table 1). CsgD is usually a member of the FixJ/LuxR family of transcriptional regulators that coordinates the expression of multiple biofilm components including curli and cellulose while repressing expression of flagellar genes [17 45 CsgD activity and stability is usually modulated by phosphorylation of an N-terminal aspartic acid residue (DNA binding is usually decreased by Chloroprocaine HCl phosphorylation) [48]. Several additional transcriptional regulators.