Aerotaxis in Halobacterium salinarium is methylation-dependent.

The behavioural response to a gradient of oxygen (aerotaxis) has been characterized in the archaeon, Halobacterium salinarium. When the gas surrounding a drop of H. salinarium strain S9-P culture was changed abruptly from 10% (v/v) O2 to 100% N2, the bacteria transiently increased the frequency of reversing before they adapted and resumed random swimming. When the gas was returned to 10% O2 the bacteria responded by swimming smoothly for approximately 45 s. Aerotaxis was strongest when respiration in H. salinarium was highest and when bacteriorhodopsin and halorhodopsin were not contributing to the proton motive force. Starvation for methionine of the auxotrophic H. salinarium essentially abolished the step-down aerotactic response. Methanol production from demethylation of methyl-accepting chemotaxis proteins was transiently increased in H. salinarium S9-P by a step down or step up in oxygen concentration, as observed in methylation-dependent chemotaxis in H. salinarium. The taxis-negative and methyltransferase-deficient mutant, H. salinarium strain Pho72 did not exhibit changes in methanol release in response to aerotaxis or chemotaxis stimuli. This is the first report of an aerotactic response that is dependent on methylation of methyl-accepting chemotaxis proteins. Aerotaxis in Escherichia coli and Salmonella typhimurium is independent of transducer methylation.

Lucigenin chemiluminescence as a probe for measuring reactive oxygen species production in Escherichia coli.

Addition of oxygen to whole cells of Escherichia coli suspended in the presence of the chemiluminescent probe bis-N-methylacridinium nitrate (lucigenin) resulted in a light emission increase of 200% of control. Addition of air to cells showed a chemiluminescent response far less than the response to oxygen. The redox cycling agents paraquat and menadione, which are known to increase intracellular production of O2- and H2O2, were also found to cause a measurable increase in lucigenin chemiluminescence in E. coli cells when added at concentrations of 1 and 0.1 mM, respectively. The oxygen-induced chemiluminescent response was not suppressed by extracellularly added superoxide dismutase or catalase. Further, the lucigenin-dependent chemiluminescent response of aerobically grown E. coli to oxygen was significantly greater than that of cells grown anaerobically. Heat-killed cells showed no increase in chemiluminescence on the addition of either oxygen, paraquat, or menadione. These results show that lucigenin may be used as a chemiluminescent probe to demonstrate continuous intracellular production of reactive oxygen metabolites in E. coli.

Inhibition of Spirochaeta aurantia chemotaxis by neurotoxins.

The effects of neurotoxic compounds on the chemotactic response of Spirochaeta aurantia were investigated. In the presence of neurotoxins that affect action potential generation and transmission in excitable eucaryotic cells, D-xylose taxis was inhibited by 69 to 93%. Inhibition of chemotaxis was not due to decreased viability or motility. This study supports the hypothesis that the molecular basis for sensory signal transduction in S. aurantia involves ion fluxes across the cytoplasmic membrane.

A voltage clamp inhibits chemotaxis of Spirochaeta aurantia.

Anaerobic conditions were employed to study the relationship between membrane potential and chemotaxis in Spirochaeta aurantia. When cells were grown anaerobically and suspended in anaerobic potassium phosphate buffer (pH 5.5), membranes did not appear to be polarized. Nevertheless, motility was supported by a transmembrane pH gradient, and the anaerobic cells exhibited D-xylose taxis. Introduction of trace amounts of air into anaerobic cell suspensions resulted in a transient membrane polarization. The addition of valinomycin to cells suspended under anaerobic conditions did not alter the steady-state value of membrane potential appreciably but served to clamp membrane potential at the preset level. Although there was no detectable effect of valinomycin on the motility of anaerobic cells in potassium phosphate buffer, D-xylose taxis was completely inhibited by this treatment. These data indicate the the action of valinomycin as a voltage clamp serves to inhibit the chemotaxis of S. aurantia and provide evidence to support the suggestion that the mechanism of chemotaxis in this organism involves the transduction of sensory signals in the form of membrane potential fluctuations.

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.

Relationship between proton motive force and motility in Spirochaeta aurantia.

The effects of various metabolic inhibitors on the motility of Spirochaeta aurantia were investigated. After 15 min in sodium arsenate buffer, 90% of cells remained motile even though adenosine triphosphate levels dropped from 5.6 to 0.1 nmol/mg (dry weight) of cells. After 70 min in sodium arsenate, 5% of cells were motile. Addition of phenazine methosulfate plus ascorbate at this time resulted in motility of 95% of cells, but adenosine triphosphate levels remained at 0.1 nmol/mg of cell dry weight. Carbonyl cyanide-m-chlorophenyl hydrazone rapidly (within 1 min) and completely inhibited motility of metabolizing cells in potassium phosphate buffer. However, after 15 min in the presence of carbonyl cyanide m-chlorophenyl hydrazone the cellular adenosine triphosphate level was 3.4 nmol/mg (dry weight) of cells, and the rate of oxygen uptake was 44% of the rate measured in the absence of carbonyl cyanide m-chlorophenyl hydrazone. Cells remained motile under conditions where either the electrical potential or the pH gradient across the membrane of S. aurantia was dissipated. However, if both gradients were simultaneously dissipated, motility was rapidly inhibited. This study indicates that a proton motive force, in the form of either a transmembrane electrical potential or a transmembrane pH gradient, is required for motility in S. aurantia. Adenosine triphosphate does not appear to directly activate the motility system in this spirochete.