Signal transduction in chemotaxis to oxygen in Escherichia coli and Salmonella typhimurium.

Pathways previously proposed for sensory transduction in chemotaxis to oxygen (aerotaxis) involved either (i) cytochrome o, the electron transport system, and proton motive force or (ii) enzyme IIGlucose and the phosphoenolpyruvate:carbohydrate phosphotransferase system for active transport. This investigation distinguished between these possibilities. Aerotaxis was absent in a cyo cyd strain of Escherichia coli that lacked both cytochrome o and cytochrome d, which are the terminal oxidases for the branched electron transport system in E. coli. Aerotaxis, measured by either a spatial or temporal assay, was normal in E. coli strains that had a cyo+ or cyd+ gene or both. The membrane potential of all oxidase-positive strains was approximately -170 mV in aerated medium at pH 7.5. Behavioral responses to changes in oxygen concentration correlated with changes in proton motive force. Aerotaxis was normal in ptsG and ptsI strains that lack enzyme IIGlucose and enzyme I, respectively, and are deficient in the phosphotransferase system. A cya strain that is deficient in adenylate cyclase also had normal aerotaxis. We concluded that aerotaxis was mediated by the electron transport system and that either the cytochrome d or the cytochrome o branch of the pathway could mediate aerotaxis.     

Oxygen taxis and proton motive force in Azospirillum brasilense.

The microaerophilic nitrogen-fixing bacterium Azospirillum brasilense formed a sharply defined band in a spatial gradient of oxygen. As a result of aerotaxis, the bacteria were attracted to a specific low concentration of oxygen (3 to 5 microM). Bacteria swimming away from the aerotactic band were repelled by the higher or lower concentration of oxygen that they encountered and returned to the band. This behavior was confirmed by using temporal gradients of oxygen. The cellular energy level in A. brasilense, monitored by measuring the proton motive force, was maximal at 3 to 5 microM oxygen. The proton motive force was lower at oxygen concentrations that were higher or lower than the preferred oxygen concentration. Bacteria swimming toward the aerotactic band would experience an increase in the proton motive force, and bacteria swimming away from the band would experience a decrease in the proton motive force. It is proposed that the change in the proton motive force is the signal that regulates positive and negative aerotaxis. The preferred oxygen concentration for aerotaxis was similar to the preferred oxygen concentration for nitrogen fixation. Aerotaxis is an important adaptive behavioral response that can guide these free-living diazotrophs to the optimal niche for nitrogen fixation in the rhizosphere.     

Aerotaxis in Salmonella typhimurium: role of electron transport

Sensory transduction in aerotaxis required electron transport, in contrast to chemotaxis, which is independent of electron transport. Assays for aerotaxis were developed by employing spatial and temporal oxygen gradients imposed independently of respiration. By varying the step increase in oxygen concentration in the temporal assay, the dose-response relationship was obtained for aerotaxis in Salmonella typhimurium. A half-maximal response at 0.4 microM oxygen and inhibition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochrome o. The response was independent of adenosine triphosphate formation via oxidative phosphorylation but did correlate with changes in membrane potential monitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate, which are alternative electron acceptors for the respiratory chain in S. typhimurium, inhibited aerotaxis when nitrate reductase and fumarate reductase were induced. These results support the hypothesis that taxis to oxygen, nitrate, and fumarate is mediated by the electron transport system and by changes in the proton motive force. Aerotaxis was normal in Escherichia coli mutants that were defective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulate methylation of the products of these genes. A cheC mutant which shows an inverse response to chemoattractants also gave an inverse response to oxygen. Therefore, aerotaxis is transduced by a distinct and unidentified signally protein but is focused into the common chemosensory pathway before the step involving the cheC product. When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis supported slow swimming but not tumbling, indicating that a minimum proton motive force was required for tumbling. The bacteria rapidly adapted to the anaerobic condition and resumed tumbling after about 3 min. The adaptation period was much shorter when the bacteria had been previously grown anaerobically.     

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T₃-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.     

Mechanism of loss of thermodynamic control in mitochondria due to hyperthyroidism and temperature.

Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.     

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

Role of Na+ cycle in cell volume regulation of Mycoplasma gallisepticum.

The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-s period and showed almost the same rate in the pH range of 6.5 to 8.0. 22Na+ efflux was markedly inhibited by dicyclohexylcarbodiimide (DCCD, 10 microM), but not by the proton-conducting ionophores SF6847 (0.5 microM) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) over the entire pH range tested. An ammonium diffusion potential and a pH gradient were created by diluting intact cells or sealed membrane vesicles of M. gallisepticum loaded with NH4Cl into a choline chloride solution. The imposed H+ gradient (inside acid) was not affected by the addition of either NaCl or KCl to the medium. Dissipation of the proton motive force by CCCP had no effect on the growth of M. gallisepticum in the pH range of 7.2 to 7.8 in an Na+-rich medium. Additionally, energized M. gallisepticum cells were stable in an isoosmotic NaCl solution, even in the presence of proton conductors, whereas nonenergized cells tended to swell and lyse. These results show that in M. gallisepticum Na+ movement was neither driven nor inhibited by the collapse of the electrochemical gradient of H+, suggesting that in this organism Na+ is extruded by an electrogenic primary Na+ pump rather than by an Na+-H+ exchange system energized by the proton motive force.     

Two different aa3-type cytochromes can be purified from the bacterium Bacillus cereus.

Two aa3-type cytochromes were purified from membranes of sporulating Bacillus cereus. One of them, an aa3 complex, was found to be composed of two subunits (51 and 31 kDa), two a hemes and three copper atoms, thus being similar to the cytochrome aa3 previously purified from vegetative B. cereus [García-Horsman, J. A., Barquera, B., González-Halphen, D. & Escamilla, J. E. (1991) Mol. Microbiol. 5, 197-205]. The second isoform, a caa3 complex, was expressed in sporulating cells only, and was found to be composed of two subunits (51 and 37 kDa). The 37-kDa subunit (subunit II) is a heme-c-containing polypeptide as shown by its peroxidase activity in SDS/PAGE gels and by its spectral features. Both subunits of the caa3 complex immunologically cross-reacted with antiserum raised against B. cereus cytochrome aa3, suggesting homology between the two enzymes. Also, the heme-c-containing subunit of the caa3 complex was reactive with anti-(bovine cytochrome c) antiserum, but not with anti-(bovine cytochrome c1) antiserum. In addition to one heme c and two hemes a, the caa3 complex contained three copper atoms. Kinetic comparison of aa3 and caa3 complexes revealed that the latter is slightly more active (k = 150 s-1) and has a lower affinity to yeast cytochrome c (Km = 76 microM) and to oxygen (Km = 2 microM) as compared with cytochrome aa3 (100 s-1, 10 microM, and 5 microM, respectively).     

Generation of a membrane potential by Lactococcus lactis through aerobic electron transport

Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3',3'-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Delta psi). Wild-type cells, grown aerobically in the presence of heme, generated a Delta psi even in the presence of the F(1)-F(o) ATPase inhibitor N,N'-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydA Delta) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydA Delta cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force.

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.     

Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain

The Gram-positive bacterium Bacillus cereus is a facultative anaerobe that is still poorly characterized metabolically. In this study, the aerobic vegetative growth and anaerobic vegetative growth of the food-borne pathogen B. cereus F4430/73 strain were compared with those of the genome-sequenced ATCC14579 strain using glucose and glycerol as fermentative and nonfermentative carbon sources, respectively. Uncontrolled batch cultures on several defined media showed that B. cereus strains had high amino acid or pyruvate requirements for anaerobic fermentative growth. In addition, growth performance was considerably improved by maintaining the pH of the culture medium near neutrality. Spectra of fermentation by-products were typically (per mole of glucose) 0.2-0.4 acetate, 1.1-1.4 L-lactate, 0.3-0.4 formate, and 0.05-0.2 ethanol with only traces of succinate, pyruvate, and 2,3-butanediol. These spectra were drastically changed in the presence of 20 mmol nitrate x L(-1), which stimulated anaerobic growth. During anaerobic and aerobic respiration, the persistent production of acetate and other by-products indicated overflow metabolisms. This was especially true in glucose-grown cells for which respiratory complex III made only a minor contribution to growth. Surprisingly, oxygen uptake rates linked to the cytochrome c and quinol branches of the respiratory chain were maintained at high levels in anaerobic, respiring, or fermenting cells. Growth and metabolic features of B. cereus F4430/73 are discussed using biochemical and genomic data.     

Mitochondrial energy metabolism is regulated via nuclear-coded subunits of cytochrome c oxidase

A new mechanism on regulation of mitochondrial energy metabolism is proposed on the basis of reversible control of respiration by the intramitochondrial ATP/ADP ratio and slip of proton pumping (decreased H+/e- stoichiometry) in cytochrome c oxidase (COX) at high proton motive force delta p. cAMP-dependent phosphorylation of COX switches on and Ca2+-dependent dephosphorylation switches off the allosteric ATP-inhibition of COX (nucleotides bind to subunit IV). Control of respiration via phosphorylated COX by the ATP/ADP ratio keeps delta p (mainly delta psi(m)) low. Hormone induced Ca2+-dependent dephosphorylation results in loss of ATP-inhibition, increase of respiration and delta p with consequent slip in proton pumping. Slip in COX increases the free energy of reaction, resulting in increased rates of respiration, thermogenesis and ATP-synthesis. Increased delta psi(m) stimulates production of reactive oxygen species (ROS), mutations of mitochondrial DNA and accelerates aging. Slip of proton pumping without dephosphorylation and increase of delta p is found permanently in the liver-type isozyme of COX (subunit VIaL) and at high intramitochondrial ATP/ADP ratios in the heart-type isozyme (subunit VIaH). High substrate pressure (sigmoidal v/s kinetics), palmitate and 3,5-diiodothyronine (binding to subunit Va) increase also delta p, ROS production and slip but without dephosphorylation of COX.     

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide.     

The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation

In previous studies the allosteric inhibition of cytochrome c oxidase at high intramitochondrial ATP/ADP-ratios via binding of the nucleotides to the matrix domain of subunit IV was demonstrated. Here we show that the allosteric ATP-inhibition of the isolated bovine heart enzyme is switched on by cAMP-dependent phosphorylation with protein kinase A of subunits II (and/or III) and Vb, and switched off by subsequent incubation with protein phosphatase 1. It is suggested that after cAMP-dependent phosphorylation of cytochrome c oxidase mitochondrial respiration is controlled by the ATP/ADP-ratio keeping the proton motive force Deltap low, and the efficiency of energy transduction high. After Ca(2+)-induced dephosphorylation this control is lost, accompanied by increase of Deltap, slip of proton pumping (decreased H(+)/e(-) stoichiometry), and increase of the rate of respiration and ATP-synthesis at a decreased efficiency of energy transduction.     

The relative proton stoichiometries of the mitochondrial proton pumps are independent of the proton motive force

Several different proton pumps were used to generate a proton motive force (delta p, proton motive force across the mitochondrial inner membrane) in isolated rat liver mitochondria, and the relationship between delta p and pump rate was investigated by titrating with various inhibitors of the pumps. It was found that this relationship was the same for mitochondria respiring on succinate irrespective of whether respiration was inhibited with malonate, antimycin or cyanide, indicating that the relationship was independent of the redox state of the respiratory chain. When delta p was generated by either the cytochrome bc1 complex, cytochrome oxidase, both together, or ATP hydrolysis (and transport), the reaction rates (in moles of electrons or ATP) were in the ratio of close to 3:1.5:1:1, respectively, at all accessible values of delta p. This suggests that the proton stoichiometries (H+/e and H+/ATP, where H+/e is the number of protons translocated vectorially across the inner membrane per electron transferred by the respiratory chain and H+/ATP is the number of protons translocated vectorially per ATP molecule hydrolyzed externally) were in the ratio of close to 1:2:3:3, respectively, at all values of delta p. Possible reasons for previous contradictory results are suggested.     

Proton motive force is not obligatory for growth of Escherichia coli.

When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5.     

Quantitative measurements of proton motive force and motility in Bacillus subtilis.

The protein motive force of metabolizing Bacillus subtilis cells was only slightly affected by changes in the external pH between 5 and 8, although the electrical component and the chemical component of the proton motive force contributed differently at different external pH. The electrical component of the proton motive force was very small at pH 5, and the chemical component was almost negligible at pH 7.5. At external pH values between 6 and 7.7, swimming speed of the cells stayed constant. Thus, either the electrical component or the chemical component of the proton motive force could drive the flagellar motor. When the proton motive force of valinomycin-treated cells was quantitatively decreased by increasing the external K+ concentration, the swimming speed of the cells changed in a unique way: the swimming speed was not affected until about--100 mV, then decreased linearly with further decrease in the proton motive force, and was almost zero at about--30 mV. The rotation rate of a flagellum, measured by a tethered cell, showed essentially the same characteristics. Thus, there are a threshold proton motive force and a saturating proton motive force for the rotation of the B. subtilis flagellar motor.     

Comparative study of energy-transducing properties of cytoplasmic membranes from mesophilic and thermophilic Bacillus species.

The properties of enzymes involved in energy transduction from a mesophilic (Bacillus subtilis) and a thermophilic (B. stearothermophilus) bacterium were compared. Membrane preparations of the two organisms contained dehydrogenases for NADH, succinate, L-alpha-glycerophosphate, and L-lactate. Maximum NADH and cytochrome c oxidation rates were obtained at the respective growth temperatures of the two bacteria. The enzymes involved in the oxidation reactions in membranes of the thermophilic species were more thermostable than those of the mesophilic species. The apparent microviscosities of the two membrane preparations were studied at different temperatures. At the respective optimal growth temperatures, the apparent microviscosities of the membranes of the two organisms were remarkably similar. The transition from the gel to the liquid-crystalline state occurred at different temperatures in the two species. In the two species, the oxidation of physiological (NADH) and nonphysiological (N,N,N',N'-tetramethyl-p-phenylenediamine or phenazine methosulfate) electron donors led to generation of a proton motive force which varied strongly with temperature. At increasing temperatures, the efficiency of energy transduction declined because of increasing H+ permeability. At the growth temperature, the efficiency of energy transduction was lower in B. stearothermophilus than in the mesophilic species. Extremely high respiratory activities enabled B. stearothermophilus to maintain a high proton motive force at elevated temperatures. The pH dependence of proton motive force generation appeared to be similar in the two membrane preparations. The highest proton motive forces were generated at low external pH, mainly because of a high pH gradient. At increasing external pH, the proton motive force declined.