Continuous-Flow Capillary Assay for Measuring Bacterial Chemotaxis

Abstract

Bacterial chemotaxis may have a significant impact on the structure and function of bacterial communities. Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the bacterial transport parameters used in predictive models of chemotaxis. When the chemotactic bacteria consume the chemoeffector, the chemoeffector gradient to which the bacteria respond may be significantly perturbed by the consumption. Therefore, consumption of the chemoeffector can confound chemotaxis measurements if it is not accounted for. Current methods of quantifying chemotaxis use bacterial concentrations that are too high to preclude chemoeffector consumption or involve ill-defined conditions that make quantifying chemotaxis difficult. We developed a method of quantifying bacterial chemotaxis at low cell concentrations (ϳ10 5 CFU/ml), so metabolism of the chemoeffector is minimized. The method facilitates quantification of bacterial-transport parameters by providing well-defined boundary conditions and can be used with volatile and semivolatile chemoeffectors.Chemotaxis, the self-directed movement of an organism or cell toward or away from a chemical (chemoeffector) along a concentration gradient, is a well-known bacterial behavior (1,5,6,12). The interest in bacterial chemotaxis is broad. Knowledge is sought of how the bacteria sense chemicals (4, 21-23), how chemotaxis affects pathogenesis and symbiosis (10,29,57), the role of chemotaxis in the formation and structure of bacterial communities (70), the influence of chemotaxis on oceanic nutrient cycling (8,9,17,75), and the effect of chemotaxis on pollutant biodegradation (43,52,(54)(55)(56).The chemoeffectors to which bacteria are attracted (chemoattractants) are often nutrients that the bacteria consume. Many methods for quantifying chemotactic transport use bacterial concentrations that are high enough that significant consumption of the chemoattractant occurs, affecting the chemoattractant gradient that forms. For example, Marx and Aitken (49) reported diminished chemotaxis at bacterial concentrations of Ն10 6 CFU per ml, which was believed to be a result of significant consumption of the chemoattractant (naphthalene). Therefore, complexity is introduced when interpreting measurements of bacterial migration by chemotaxis because consumption of the chemoeffector must be accounted for. Furthermore, bacterial accumulation by chemotaxis and consumption of a chemoattractant may create a secondary gradient that the bacteria also sense. For example, aerobic biodegradation of a chemoattractant by bacteria that are also aerotactic (responsive to oxygen) could create an oxygen gradient that influences bacterial migration, confounding measurements of movement toward the primary chemoattractant. Methods that are used to quantify chemotaxis, therefore, should minimize metabolism of a biodegradable chemoeffector so that the observed bacterial motion would be in response only to diffusion of the primary chemoeffector.Methods in which metabolism can be minimized...

Respuesta de Physalis peruviana L. A la inoculación con bacterias solubilizadoras de fosfato

“…Additionally, the four strains responded similarly evaluated with the different carbon sources used in this study; which stimulated the greatest amount in the solubilization of phosphorus was malic acid, followed by mannitol, sucrose, glucose, and glutamic acid. Importantly, malic acid has been widely reported as one of the greatest microbial compounds generates attraction in the rhizosphere of various crops, including a minimum concentration of 4 mM (Law and Aitken, 2005;Bais et al, 2006). This result suggests that solubilization of phosphorus in the rhizosphere can be affected by numerous environmental factors, particularly the composition of the plant exudates.…”

The effect of oxygen on chemotaxis to naphthalene by Pseudomonas putida G7

“…Capillaries were modified 5-mL PCR pipets with plungers (Drummond Scientific, Broomall, PA). Details on the assembly by which the capillaries are inserted into the flow channel are provided elsewhere (Law and Aitken, 2005). The flowing bacterial suspension creates constant concentrations of bacteria and chemoattractant at the mouths of the capillaries, which facilitates quantification of bacterial transport.…”

“…After approximately 100 mL of buffer was passed through the flow channel, each capillary was rinsed twice by filling and expelling the buffer. The capillary was then filled with a 40-mm liquid plug, the capillary was withdrawn 10 mm so the protective sheath covered the mouth of the capillary (Law and Aitken, 2005), flow was stopped, and the exit tube of the flow channel was clamped. The dissolved oxygen concentration in the capillaries was assumed to be equal to the concentration in the buffer that flowed through the flow channel, which was measured by a fiber-optic oxygen sensor at the exit of the flow channel.…”

“…Quantification of bacterial chemotaxis toward naphthalene and oxygen was measured using a continuous-flow capillary assay (Law and Aitken, 2005). The continuous-flow capillary assay is a modification of the traditional capillary assay (Adler, 1969(Adler, , 1973.…”

“…Attraction of P. putida G7 toward oxygen and the influence of oxygen on random motility and chemotaxis was assessed using a modified capillary assay (Law and Aitken, 2005). The assay is useful for quantifying responses to attractants that are consumed because attractant gradients are not significantly affected by consumption and unintended secondary gradients in electron acceptor are minimized.…”