Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coliχ7122 conferred mannose‐resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR‐like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain χ7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA–lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild‐type S. typhimurium SL1344 or SR‐11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low‐copy‐number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature‐ and RpoS‐independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS‐dependent curli and extracellular matrix production by E. coli and S. typhimurium.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3- While important advances have been made toward understanding how Salmonella interacts with the intestinal epithelium to initiate disease (reviewed in references 6 and 44), investigations into the interaction of Salmonella with the human intestinal epithelium have been limited by the lack of in vitro and in vivo models which faithfully replicate the in vivo condition. In particular, it is well documented that important differences exist between the pathogenesis of Salmonella enterica serovar Typhimurium in human infections and that in widely used cell culture and animal models (34,40,47). In vitro assays using cultured mammalian epithelial cells have long been used as a model for investigating the interaction between Salmonella and the intestinal mucosa. However, there are inherent limitations associated with the use of these cultured cell lines (34), as they are not exact models of the conditions faced in vivo by Salmonella. Several characteristics of conventional tissue culture models have raised concerns regarding their overall efficacy as models for microbial infectivity in general (34) due to the dedifferentiation of these cells during conventional cell culture. Indeed, many of the physiological differences between cultured cells and their in vivo counterparts are believed to be the result of the dissociation of cells from their native three-dimensional (3-D) geometry in vivo to their propagation on a two-dimensional substrate in vitro (10). Likewise, many characteristics of animal models fail to mimic the human disease, and animal models present a complex system in which many variables cannot be controlled. A high-fidelity enteric cell culture model could provide new insights into studies of Salmonella infectivity by bridging the gap between the inherent limitations of cultured mammalian cells and intact animals.DFor humans, S. enterica serovar Typhimurium is among the most common Salmonella serotypes isolated from individuals suffering from infectious gastroenteritis and has long been recognized as a major public health problem (23). Gastroenteritis results from infection of the small intestine after ingestion with Salmonella. Indeed, the ability to colonize the intestinal epithelium is an essential feature in the pathogenicity of Salmonella infection. Moreover, the initial interactions between Salmonella and the host intestinal epithelium are believed to play a key role in mediating the intense inflammatory and secretory response which is a hallmark of serovar Typhimurium infections in humans (reviewed in reference 6). Studies with cultured intestinal epithelial cells have shown that,