Curli biogenesis in the absence of CsgE and CsgF. (A) Negative-stain EM micrographs of MHR592 (csgF−) bacteria grown on YESCA plates at 26°C for 48 hours. (B) CsgA visualized by Western analysis with anti-CsgA () and bacteria grown at 26°C on YESCA plates for 48 hours. Circular plugs of 8 mm, including cells and underlying agar (to collect soluble, unpolymerized and secreted CsgA), were collected and resuspended in 200 μl of 1.5× SDS loading buffer either with or without prior FA treatment. The etracts loaded in each lane are as follows: lanes 1 and 2, MC4100 (wild type); lanes 3 and 4, LSR10 (csgA−); lanes 5 and 6, MHR480 (csgE−); lanes 7 and 8, MHR261 (csgB−); and lanes 9 and 10 MHR592 (csgF−). (C) Negative-stain EM micrographs of MHR480 (csgE−) bacteria grown on YESCA plates at 26°C for 48 hours. These fibers often looped into imperfect circles (see inset). (D) Interbacterial complementation and CR binding in csgE− and csgF− mutants. The CsgA+ donor strain MHR261 (I) and the CsgB+ recipient strain LSR10 (II) were streaked from the top of the plate to the bottom. The horizontal cross-streaks were made from left to right with the following strains: MC4100 (wild type) (I-1 and II-1), csgA− (I-2), csgB− (II-2), csgE− (I-3 and II-3), csgEB− (I-4 and II-4), csgF− (I-5 and II-5), and csgFB− (I-6 and II-6). Bars in (A) and (C), 200 nm.