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We have previously reported the cloning of the Salmonella enterica serovar Typhimurium SPI-1 secretion system and the use of this clone to functionally complement a ΔSPI-1 strain for type III secretion activity. In the current study, we discovered that S. Typhimurium cultures containing cloned SPI-1 display an adherent biofilm and cell clumps in the media. This phenotype was associated with hyper-expression of SPI-1 type III secretion functions. The biofilm and cell clumps were associated with copious amounts of secreted SPI-1 protein substrates SipA, SipB, SipC, SopB, SopE, and SptP. We used a C-terminally FLAG-tagged SipA protein to further demonstrate SPI-1 substrate association with the cell aggregates using fluorescence microscopy and immunogold electron microscopy. Different S. Typhimurium backgrounds and both flagellated and nonflagellated strains displayed the biofilm phenotype. Mutations in genes essential for known bacterial biofilm pathways (bcsA, csgBA, bapA) did not affect the biofilms formed here indicating that this phenomenon is independent of established biofilm mechanisms. The SPI-1-mediated biofilm was able to massively recruit heterologous non-biofilm forming bacteria into the adherent cell community. The results indicate a bacterial aggregation phenotype mediated by elevated SPI-1 type III secretion activity with applications for engineered biofilm formation, protein purification strategies, and antigen display.

Salmonella enterica serovar Typhimurium possesses a stimulon of genes that are differentially regulated in response to conditions of low fluid shear force that increase bacterial virulence and alter other phenotypes. In this study, we show that a previously uncharacterized member of this stimulon, ydcI or STM1625, encodes a highly conserved DNA binding protein with related homologs present in a range of Gram-negative bacterial genera. Gene expression analysis shows that ydcI is expressed in different bacterial genera and is involved in its autoregulation in S. Typhimurium. We demonstrate that purified YdcI protein specifically binds a DNA probe consisting of its own promoter sequence. We constructed an S. Typhimurium ⌬ydcI mutant strain and show that this strain is more sensitive to both organic and inorganic acid stress than is an isogenic WT strain, and this defect is complemented in trans. Moreover, our data indicate that ydcI is part of the rpoS regulon related to stress resistance. The S. Typhimurium ⌬ydcI mutant was able to invade cultured cells to the same degree as the WT strain, but a strain in which ydcI expression is induced invaded cells at a level 2.8 times higher than that of the WT. In addition, induction of ydcI expression in S. Typhimurium resulted in the formation of a biofilm in stationary-phase cultures. These data indicate the ydcI gene encodes a conserved DNA binding protein involved with aspects of prokaryotic biology related to stress resistance and possibly virulence.Bacterial growth environments characterized by low fluid shear force have been shown to induce a multitude of phenotypic responses, including altered acid, oxidative, thermal, and osmotic stress resistance (7,33,36,41,(52)(53)(54)(55), increased biofilm formation (6, 33, 52), altered protein secretion (14, 15), altered cell surface lipid and polysaccharide profiles (6, 7, 55), and increased survival in cellular and animal hosts (39,(52)(53)(54). Notably, the virulence of Salmonella enterica serovar Typhimurium is increased by low fluid shear growth conditions as measured using murine infection assays and tissue culture models (39,(52)(53)(54). Low fluid shear force (defined here as approximately Ͻ0.01 to 0.2 dynes/cm 2 ) is characterized by a lowturbulence, low-agitation environment, as opposed to high fluid shear (defined here as approximately from 5 to Ͼ50 dynes/cm 2 ) where liquid moves with higher velocity over the cellular surface (3-5, 21, 23, 25, 36). Low fluid shear growth environments include spaceflight, ground-based suspension culture models such as the rotating-wall vessel (RWV) bioreactor, and the spaces between cellular microvilli, the last of which is encountered by numerous pathogens during the natural course of infection (21,23,25,30,40,41). Previous work has shown that bacteria grown in low fluid shear environments induce a molecular response which includes genome-wide changes in gene expression (the low fluid shear stimulon) (6,7,39,48,52,53,55). Since growth under low fluid shear conditions is able to ind...

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