EXPRESSION OF 2 CSG OPERONS IS REQUIRED FOR PRODUCTION OF FIBRONECTIN-BINDING AND CONGO-RED-BINDING CURLI POLYMERS IN ESCHERICHIA-COLI K-12

Two divergently transcribed operons in Escherichia coli required for t he expression of fibronectin- and Congo red-binding curli polymers wer e identified and characterized by transposon mutagenesis, sequencing a nd transcriptional analyses, as well as for their ability to produce t he curli subunit protein, The csgBA operon encodes CsgA, the major sub unit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of Cs gA, but the subunit protein is not assembled into insoluble fibre poly mers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in thi s putative gene abolishes the autoagglutinating ability typical of cur liated cells without affecting the production of the fibre. The promot er for the oppositely transcribed csgDEFG operon was identified by pri mer extension and shown, like the csgSA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma(s) in wild type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csg SA promoter, Therefore, any regulatory effect on the csgBA promoter mi ght be secondary to events controlling the csgDEFG promoter and/or act ivation of CsgD. Insertions in csgE, csgF and csgG abolish curli forma tion but allow CsgA expression suggesting that one or more of these ge ne products are involved in secretion/assembly of the CsgA subunit pro tein. No amino acid sequence homologies were found between the CsgE, C sgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a nov el pathway.