Flow-Based Microffuidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients

Flow-Based Microffuidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients

8 Pages · 2009 · 1.34 MB · English

Attraction to AI-2 overcame repulsion by indole in equal, competing gradients. Our data suggest that concentration-dependent interactions between 

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY , July 2009, p 4557–4564Vol 75, No 13 00992240/09/$08000 doi:101128/AEM0295208 Copyright © 2009, American Society for Microbiology All Rights Reserved FlowBased Microuidic Device for Quantifying Bacterial Chemotaxis in Stable, Competing Gradients  Derek L Englert, 1Michael D Manson, 2and Arul Jayaraman 1,3 * Artie McFerrin Department of Chemical Engineering, 1Department of Biology, 2and Department of Biomedical Engineering, 3 Texas A&M University, College Station, Texas 77843 Received 28 December 2008/Accepted 27 April 2009 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 micro?uidic 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 colichemotaxis Laminar ?owbased diffusive mixing was used to generate gradients, and the chemotactic responses of cells expressing green ?uorescent protein were determined using ?uorescence microscopy Quanti?cation of the migration pro?les indicated that E coliwas attracted to the quorumsensing molecule autoinducer2 (AI2) but was repelled from the stationaryphase signal indole Cells also migrated toward higher concentrations of isatin (indole2,3dione), an oxidized derivative of indole Attraction to AI2 overcame repulsion by indole in equal, competing gradients Our data suggest that concentrationdependent 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 specic molecules, comparing the current concentration to the concen tration detected a few seconds earlier This comparison deter mines the net direction of movement (6, 22) Chemotaxis al lows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals Natural habitats for Esch erichia coli, such as the gastrointestinal (GI) tract, are typically heterogeneous and contain multiple chemoeffectors with po tentially 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 chemo taxis, such as the swim plate and capillary (1) assays, are not ideal for quantifying bacterial migration Chemotacticring for mation in semisolid agar requires metabolizable attractants and is subject to multiple factors, and both it and the tradi tional capillary assay are poorly designed to investigate repel lent taxis Mao et al (23) were the rst to investigate bacterial taxis in a microuidic ow 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 threechannel microu idic devices (7, 8) in which a linear gradient is generated in the absence of ow or a Tchannel device that monitors chemo taxis perpendicular to the direction of uid ow (18), were developed subsequently The Tchannel system has many of the limitations of the device developed by Mao et al (23), and nonow systems, like the capillary assay (1), suffer from a lack of temporal stability of the gradients Here, we report a owbased microuidic chemotaxis device that is coupled to a gradient generator Bacteria are exposed to precise and temporally stable concentration gradients of che moeffectors over the length of the microuidic channel This device was used to quantify E colichemotaxis in response to the canonical chemoeffectors Laspartate and Ni 2 The device was also used to investigate chemotaxis toward cellcell com munication signals such as autoinducer2 (AI2), indole, and isatin that are likely to be present in the in vivo microenviron ment in which E coliis present (eg, the human GI tract) The data obtained reinforce the idea that concentrationdependent interactions between different chemical signals could be im portant determinants of bacterial colonization in natural envi ronments MATERIALS AND METHODS Bacterial strains, materials, and growth media E coliRP437 (23) is wild type for chemotaxis, and strain RP437 eda tar(9) is blind to aspartate and Ni 2E coli TG1 was obtained

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