Non-chemotactic Strain Research Articles

Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae.

The role of chemotaxis in the virulence of gastrointestinal pathogens is ill defined. Counterintuitively, nonchemotactic mutants of the polarly flagellated pathogen Vibrio cholerae greatly out-compete the wild-type strain during infection of the small intestine. We show that the out-competition phenotype is dependent on the direction of flagellar rotation and independent of Toxin Co-regulated Pilus function. Specifically, the out-competition associated with the loss of chemotaxis required the presence of counterclockwise-biased flagellar rotation and smooth straight runs by the bacteria. In contrast, a nonchemotactic strain with clockwise-biased flagellar rotation was confined to small-scale net movement and was attenuated for infection. The significance of the out-competition phenotype was examined and was shown to correlate with a true increase in infectivity. Counterclockwise-biased mutants are aberrantly distributed throughout the infant mouse small intestine and we find that the expression of virulence factors occurs normally in all segments. Thus, alteration of the chemotactic properties of V. cholerae allows it to exploit additional niches in the host intestine.

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 626–634, 2002.

Quantitative analysis of experiments on bacterial chemotaxis to naphthalene.

A mathematical model was developed to quantify chemotaxis to naphthalene by Pseudomonas putida G7 (PpG7) and its influence on naphthalene degradation. The model was first used to estimate the three transport parameters (coefficients for naphthalene diffusion, random motility, and chemotactic sensitivity) by fitting it to experimental data on naphthalene removal from a discrete source in an aqueous system. The best-fit value of naphthalene diffusivity was close to the value estimated from molecular properties with the Wilke-Chang equation. Simulations applied to a non-chemotactic mutant strain only fit the experimental data well if random motility was negligible, suggesting that motility may be lost rapidly in the absence of substrate or that gravity may influence net random motion in a vertically oriented experimental system. For the chemotactic wild-type strain, random motility and gravity were predicted to have a negligible impact on naphthalene removal relative to the impact of chemotaxis. Based on simulations using the best-fit value of the chemotactic sensitivity coefficient, initial cell concentrations for a non-chemotactic strain would have to be several orders of magnitude higher than for a chemotactic strain to achieve similar rates of naphthalene removal under the experimental conditions we evaluated. The model was also applied to an experimental system representing an adaptation of the conventional capillary assay to evaluate chemotaxis in porous media. Our analysis suggests that it may be possible to quantify chemotaxis in porous media systems by simply adjusting the model's transport parameters to account for tortuosity, as has been suggested by others.

Isolation and Characterisation of Chemotactic Mutants of Chlamydomonas reinhardtii obtained by Insertional Mutagenesis

The swimming behaviour of the green flagellated protist Chlamydomonas reinhardtii is influenced by several different external stimuli including light and chemical attractants. Common components are involved in both the photo- and chemo-sensory transduction pathways, although the nature and organisation of these pathways are poorly understood. To learn more about the mechanism of chemotaxis in Chlamydomonas, we have generated nonchemotactic strains by insertional mutagenesis. The arginine-requiring strain arg7-8 was transformed with DNA carrying the wild-type ARG7 gene. Of the 8630 arginine-independenttransformants obtained, five are defective in their chemotaxis towards various sugars. Two of the mutants (CTX2 and CTX3) are blocked only in their response to xylose. Mutant CTX1 is blocked in its response to xylose, maltose and mannitol, but displays normal taxis to sucrose. Mutants CTX4 and CTX5 lack chemotactic responses to all sugars tested. CTX1, CTX4 and CTX5 represent novel chemotactic phenotypes not previously obtained using ultra-violet or chemical mutagenesis. Genetic analysis confirms that each mutation maps to a single nuclear locus that is unlinked to the mating-type locus. Further analysis of CTX4 indicates that the mutant allele is tagged by the transforming ARG7 DNA. CTX4 appears to be defective in a component specific for chemotactic signal transduction since it exhibits wild-type photobehavioural responses (phototaxis and photoshock) as well as the wild-type responses of EGTA-induced trans-flagellum inactivation and acid-induced deflagellation. Insertional mutagenesis has thus permitted the generation of novel chemotactic mutants that will be of value in the molecular dissection of the signalling machinery.

Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin.

Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli. However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established. In order to monitor internal free Ca2+ in E. coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain [BL21(DE3)] of E. coli. Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+. These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling. The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin. The resting level of free Ca2+ in E. coli (AB1157) was found to be 100 nM, which agrees with previous data [Gangola and Rosen (1987) J. Biol. Chem. 262, 12570-12574]. As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E. coli.

Effect of temperature on motility and chemotaxis of Escherichia coli.

The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature. The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C. The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature. When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed. A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling. These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition.