During infection in the gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) O157:H7 is exposed to a wide range of signaling molecules, including the eukaryotic hormones epinephrine and norepinephrine, and bacterial signal molecules such as indole. Since these signaling molecules have been shown to be involved in the regulation of phenotypes such as motility and virulence that are crucial for EHEC infections, we hypothesized that these molecules also govern the initial recognition of the large intestine environment and attachment to the host cell surface. Here, we report that, compared to indole, epinephrine and norepinephrine exert divergent effects on EHEC chemotaxis, motility, biofilm formation, gene expression, and colonization of HeLa cells. Using a novel two-fluorophore chemotaxis assay, it was found that EHEC is attracted to epinephrine and norepinephrine while it is repelled by indole. In addition, epinephrine and norepinephrine also increased EHEC motility and biofilm formation while indole attenuated these phenotypes. DNA microarray analysis of surface-associated EHEC indicated that epinephrine/norepinephrine up-regulated the expression of genes involved in surface colonization and virulence while exposure to indole decreased their expression. The gene expression data also suggested that autoinducer 2 uptake was repressed upon exposure to epinephrine/ norepinephrine but not indole. In vitro adherence experiments confirmed that epinephrine and norepinephrine increased attachment to epithelial cells while indole decreased adherence. Taken together, these results suggest that epinephrine and norepinephrine increase EHEC infection while indole attenuates the process. The intestinal tract is colonized by approximately 1012 commensal bacteria consisting of hundreds of bacterial species, including the genus Escherichia (9, 10, 19). The introduction of pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157:H7 into the human gastrointestinal (GI) tract results in colonization of host cells and leads to the onset of bloody diarrhea and hemolytic uremic syndrome (24, 25). EHEC infections progress through a three-step mechanism, the first of which involves adhesion of bacteria to host cells and the formation of microcolonies (23,46). EHEC infections pose a serious clinical problem as they are often associated with complications and permanent disabilities, including neurological defects, hypertension, and renal insufficiency (35). Understanding the mechanisms underlying EHEC pathogenicity could lead to better approaches for attenuating the deleterious consequences associated with GI tract infections (52).E. coli O157:H7 is exposed to a wide range of signaling molecules in the GI tract. These include bacterial quorum-sensing molecules such as autoinducer 2 and 3 (AI-2 and AI-3, respectively) that are involved in the regulation of phenotypes crucial for virulence and infection (44, 49). For example, Sperandio et al. (43,44) have shown that the adhesion of pathogenic E. coli to host c...

Chemotaxis is the migration of cells in gradients of chemoeffector molecules. Although multiple, competing gradients must often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to gradients of multiple signals. In this work, we developed a microfluidic device for generating precise and stable gradients of signaling molecules. We used the device to investigate the effects of individual and combined chemoeffector gradients on Escherichia coli chemotaxis. Laminar flow-based diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green fluorescent protein were determined using fluorescence microscopy. Quantification of the migration profiles indicated that E. coli was attracted to the quorum-sensing molecule autoinducer-2 (AI-2) but was repelled from the stationary-phase signal indole. Cells also migrated toward higher concentrations of isatin (indole-2,3-dione), an oxidized derivative of indole. Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between attractant and repellent signals may be important determinants of bacterial colonization of the gut.Bacteria sense chemoeffectors using cell surface receptors (13,29). Cells constantly monitor the concentration of specific molecules, comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement (6,22). Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Natural habitats for Escherichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with potentially opposing effects. The integrated chemotactic response in such environments is thus likely to be an important factor in bacterial colonization.Conventional approaches for investigating bacterial chemotaxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration. Chemotactic-ring formation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the traditional capillary assay are poorly designed to investigate repellent taxis. Mao et al. (23) were the first to investigate bacterial taxis in a microfluidic flow cell. In their device, a concentration gradient is formed by the diffusive mixing of two inlet streams. However, the exposure to a fully developed gradient in this device is limited because it takes time for the gradient to develop.Variations of this technique, such as three-channel microfluidic devices (7,8) in which a linear gradient is generated in the absence of flow or a T-channel device that monitors chemotaxis perpendicular to the direction of fluid flow (18), were developed subsequently. The T-channel system has many of the limitations of the device developed by Mao et al. (23), and nonflow systems,...

AI-2 is an autoinducer made by many bacteria. LsrB binds AI-2 in the periplasm, and Tsr is the L-serine chemoreceptor. We show that AI-2 strongly attracts Escherichia coli. Both LsrB and Tsr are necessary for sensing AI-2, but AI-2 uptake is not, suggesting that LsrB and Tsr interact directly in the periplasm.Many functions in bacteria are regulated by population density, including formation of biofilms and production of virulence factors (5). Assessment of population density, known as quorum sensing, relies on the ability of cells to determine the concentrations of compounds known as autoinducers (AIs). As the cell density increases, an AI accumulates to a concentration that triggers a quorum-sensing response. Autoinducers activate some genes and repress others. Induced genes typically include those responsible for production of the autoinducer, resulting in a positive feedback loop. Cell densities required to accumulate enough AI for good induction are 10 8 per ml or higher.AIs are of two basic types: species specific and general (22). Species-specific AI-1s are acyl homoserine lactones in Gramnegative bacteria and modified peptides in Gram-positive bacteria. Full induction of bioluminescence in the marine bacterium Vibrio harveyi, which colonizes dead organic matter, requires both a specific AI-1 inducer and a general autoinducer, called AI-2 (6). AI-2 is derived from spontaneous cyclization of 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is made from S-ribosylhomocysteine by the enzyme LuxS (25). S-Ribosylhomocysteine is an intermediate in the breakdown of S-adenosylhomocysteine, the product remaining after methyl group donation by S-adenosylmethionine.AI-2 is produced by a wide range of Gram-negative and Gram-positive bacteria and exists in multiple forms that are in equilibrium with each other (5). The form that is active in V. harveyi is (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate (S-THMF borate) (8), which binds to the periplasmic protein LuxP. In S. enterica serovar Typhimurium, a boron-free isomer of AI-2 [(2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetra-hydrofuran (R-THMF)] binds to the periplasmic LsrB protein (21). LsrB is the recognition component of an ABC transporter for AI-2. LsrACD are the membrane-bound components of the ABC transporter for AI-2. AI-2 is generated by the enzyme LuxS, and the YdgG (TqsA) protein has been implicated in AI-2 export from the cytoplasm (14).AI-2 is a known chemoattractant for Escherichia coli (4, 10), but the receptor(s) involved in AI-2 sensing has not been identified. This work was initiated to characterize the proteins involved in AI-2 recognition by E. coli strain CV1, which is equivalent to the standard wild-type chemotaxis strain RP437. The strains used in this study are shown in Table 1.The microplug (Plug) assay (9), a modified plug-in-pond assay, provides a qualitative but highly visual representation of chemotaxis. Cells containing the green fluorescent protein (GFP)-encoding plasmid pCM18 were grown overnight at 32°C in tryptone broth (TB) (20) co...

The plug-in-pond and capillary assays are convenient methods for measuring attractant and repellent bacterial chemotaxis. However, these assays do not provide quantitative information on the extent of migration and are not well-suited for investigating repellent taxis. Here, we describe a protocol for a flow-based microfluidic system (microFlow) to quantitatively investigate chemotaxis in response to concentration gradients of attractants and repellents. The microFlow device uses diffusive mixing to generate concentration gradients that are stable throughout the chemotaxis chamber and for the duration of the experiment. The gradients may be of any desired absolute concentration and gradient strength. GFP-expressing bacteria immediately encounter a stable concentration gradient when they enter the chemotaxis chamber, and the migration in response to the gradient is monitored by microscopy. The effects of different parameters that influence the extent of migration in the microFlow device-preparation of the motile bacterial population preparation, strength of the concentration gradient and duration of exposure to the gradient-are discussed in the context of repellent taxis of chemotactically wild-type Escherichia coli cells in a gradient of NiSO(4). Fabrication of the microfluidic device takes 1 d while preparing motile cells and carrying out the chemotaxis experiment takes 4-6 h to complete.

Biofilms are highly organized structures coordinately formed by multiple species of bacteria. Quorum sensing (QS) is one cell-cell communication mechanism that is used by bacteria during biofilm formation. Biofilm formation is widely acknowledged to occur through a sequence of spatially and temporally regulated colonization events. While several mathematical models exist for describing biofilm development, these have been developed for open systems and are not applicable to closed systems where biofilm development and hydrodynamics are interlinked. Here, we report the development of a mathematical model describing QS and biofilm formation in a closed system such as a microfluidic channel. The model takes into account the effect of the external environment viz the mass and momentum transport in the microfluidic channel on QS and biofilm development. Model predictions of biofilm thickness were verified experimentally by developing Pseudomonas aeruginosa PA14 biofilms in microfluidic chambers and reflect the interplay between the dynamics of biofilm community development, mass transport, and hydrodynamics. Our QS model is expected to guide the design of experiments in closed systems to address spatio-temporal aspects of QS in biofilm development and can lead to novel approaches for controlling biofilm formation through disruption of QS spatio-temporal dynamics.