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.

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization ofcurlin to fimbriae-like structures (curli) on the surface ofEscherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleatorpresenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.

Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy. Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype). Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C)and csgDEFG, from S. typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli. The divergence of the curli region between S. typhimurium and E. coli at the nucleotide level is above average (22.4%). However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products. Consequently, S. typhimurium genes on low-copy-number plasmids were able to complement respective E. coli mutants, although not always to wild-type levels. rpoS and ompR are required for transcriptional activation of (at least) the csgDpromoter. The high degree of conservation at the protein level and the identical regulation patterns in E. coli and S. typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species.

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The selective autophagy substrate p62 serves as a molecular link between autophagy and cancer. Suppression of autophagy causes p62 accumulation and thereby contributes to tumorigenesis. Here we demonstrate that autophagy deficiency promotes cell proliferation and migration through p62-dependent stabilization of the oncogenic transcription factor Twist1. p62 binds to Twist1 and inhibits degradation of Twist1. In mice, p62 up-regulation promotes tumor cell growth and metastasis in a Twist1-dependent manner. Our findings demonstrate that Twist1 is a key downstream effector of p62 in regulation of cell proliferation and migration and suggest that targeting p62-mediated Twist1 stabilization is a promising therapeutic strategy for prevention and treatment of cancer.acroautophagy (hereafter autophagy) is a catabolic process by which cellular proteins, cytoplasm, and organelles are captured and targeted for proteolytic degradation in lysosomes (1, 2). Autophagy dysfunction is associated with multiple human diseases, such as neurodegeneration, microbial infection, metabolic diseases, cardiovascular diseases, aging, and cancer (2-4). The multidomain protein p62/A170/SQSTM1 (hereafter referred to as "p62") has been shown to be both a selective autophagy substrate and an autophagy adaptor protein that acts as a link between ubiquitination and autophagy (5, 6). Several studies have demonstrated the oncogenic role of p62 in tumor formation and/ or progression (7, 8) through regulating NF-kappaB (9, 10) and NRF2 (11-13). Furthermore, Ras induces p62 expression in tumorigenesis (10). However, much remains to be elucidated with regard to its function and interaction with other critical cellular pathways.The transcription factor Twist1 is a core regulator in both early embryonic morphogenesis and cancer development and metastasis (14-17). It induces loss of epithelial (E)-cadherin-mediated cellcell adhesion and facilitates the epithelial-mesenchymal transition (EMT) (16), and it promotes cell proliferation (17). Twist1 is a basic helix-loop-helix (bHLH) protein and is structurally unrelated to other EMT factors including Slug and Snail. Recent studies have shown that Twist1 is a labile protein regulated by the ubiquitin-proteasome system through the F-box protein and E3 ubiquitin ligase Ppa (18). Despite these advances, the regulatory and functional role of Twist1 remains poorly understood. Here we demonstrate that p62 stabilizes Twist1 protein to increase cell proliferation and migration in vitro and in mice. Results Autophagy Deficiency Decreases E-Cadherin Expression and PromotesCell Migration, Invasion, and Proliferation. Two distinctive hallmarks of autophagy are the conversion of light chain 3-I (LC3-I) to LC3-II and the selective degradation of p62 (19,20). Multiple mammalian homologs of products of the autophagy-related genes (Atg) originally identified in yeast have been identified (3). Compared with wild-type (WT) mouse embryonic fibroblast (MEF) cells, cells with Atg3, Atg5, Atg9, and Atg12 knockout (KO) blocked the co...