Curli Formation Research Articles

Nucleator function of CsgB for the assembly of adhesive surface organelles in Escherichia coli.

Curli are surface organelles in Escherichia coli that assemble outside the bacterium through the precipitation of secreted soluble CsgA monomers, requiring the CsgB nucleator protein. Using immunoelectron microscopy and immunoblotting assays, CsgB is shown to be located on the bacterial surface and also as a minor component of wild-type curli. CsgB lacking its 20 N-terminal residues when fused to maltose-binding protein (MBP) can still trigger polymerization of CsgA monomers in vivo. However, the resulting surface organelles are only formed at one of the two bacterial poles and are morphologically distinct from wild-type curli. These Bfco organelles (CsgB-Free Curli-related Organelles) are highly regular structures reacting with anti-CsgA, but not anti-CsgB antibodies. The CsgB of the active MBP-CsgBII fusion is surface exposed but, unlike the native CsgB in wild-type curli, is not detectable in the Bfco organelles. Overexpression of csgB within a csgA mutant results in the formation of short CsgB polymers on the cell surface. It is suggested that in wild-type bacteria, both CsgA and CsgB are secreted proteins. Interaction between CsgA and CsgB triggers wild-type curli formation, resulting in CsgA-CsgB heteropolymers, while surface-anchored CsgB in MBP-CsgBII triggers morphologically distinct, CsgB-free/CsgA Bfco organelles. In the absence of CsgA, CsgB can self-assemble into polymers.

Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12.

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA 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 csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

Expression of two csg operons is required for production of fibronectin‐ and Congo red‐binding curli polymers in Escherichia coli K‐12

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor σs in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.

The Crl protein activates cryptic genes for curli formation and fibronectin binding in Escherichia coli HB101.

Curli are thin, coiled, temperature-regulated fibres on fibronectin-binding Escherichia coli. The subunit protein of curli was highly homologous at its amino terminus to SEF-17, the subunit protein of thin, aggregative fimbriae of Salmonella enteritidis 27655 strain 3b, suggesting that these fibres form a novel class of surface organelles on enterobacteria. E. coli HB101 is non-curliated and unable to bind soluble, iodinated fibronectin. The phenotypically cryptic curlin subunit gene, csgA, in HB101 is transcriptionally activated by expressing the cytoplasmic Crl on a multicopy plasmid. Transcriptional activation of csgA by Crl was observed after growth at 26 degrees C but not at 37 degrees C, even though crl transcription was not thermoregulated. A deletion of the 39 carboxy-terminal residues abolished Crl activity, whereas a deletion of 10 residues at the C-terminus did not, implying that a region between residue 93 and 122 in the 132-amino-acid-residue large Crl protein is required for activating curli expression in E. coli HB101. crl is a normal housekeeping gene in E. coli and it is suggested that its gene product may either be a DNA-binding protein affecting chromatin structure as has been suggested for histone-like protein H1 or interact with specific regulatory protein(s) controlling transcription of genes required for curli formation and fibronectin binding.