Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons.

Analysis of the bacterial genome sequences shows that many human and animal pathogens encode primary membrane Na+ pumps, Na+-transporting dicarboxylate decarboxylases or Na+ translocating NADH:ubiquinone oxidoreductase, and a number of Na+ -dependent permeases. This indicates that these bacteria can utilize Na+ as a coupling ion instead of or in addition to the H+ cycle. This capability to use a Na+ cycle might be an important virulence factor for such pathogens as Vibrio cholerae, Neisseria meningitidis, Salmonella enterica serovar Typhi, and Yersinia pestis. In Treponema pallidum, Chlamydia trachomatis, and Chlamydia pneumoniae, the Na+ gradient may well be the only energy source for secondary transport. A survey of preliminary genome sequences of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola indicates that these oral pathogens also rely on the Na+ cycle for at least part of their energy metabolism. The possible roles of the Na+ cycling in the energy metabolism and pathogenicity of these organisms are reviewed. The recent discovery of an effective natural antibiotic, korormicin, targeted against the Na+ -translocating NADH:ubiquinone oxidoreductase, suggests a potential use of Na+ pumps as drug targets and/or vaccine candidates. The antimicrobial potential of other inhibitors of the Na+ cycle, such as monensin, Li+ and Ag+ ions, and amiloride derivatives, is discussed.

Motility and chemotaxis in Bacillus sphaericus. Dependence upon stage of growth.

Chemotaxis and motility of B. sphaericus 2362 were monitored as the function of a batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemoeffectors peaked at the mid-logarithmic phase. A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified. The extent of label incorporation in this protein from L-[methyl-3H]methionine was maximal in mid- and late logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids. Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of the microbial population to survive.

Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362. The influence of L-ethionine, and of aminophenylboronic acid.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

Altered chemotaxis of a Bacillus sphaericus L-ethionine-resistant sporulation mutant. A probable link between chemotaxis and sporulation.

A UV irradiation-induced mutant of B. sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. The ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

Motility and chemotaxis in Bacillus sphaericus. Dependence upon stage of growth.

Chemotaxis and motility of Bacillus sphaericus 2362 were monitored as a function of the batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemo-effectors peaked at the mid-logarithmic phase. A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified. The extent of label incorporation in this protein from L-[methyl-3H]methionine was maximal in mid- and late-logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids. Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of microbial population to survive.

Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362. The influence of L-ethionine, and of aminophenylboronic acid.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

Altered chemotaxis of Bacillus sphaericus L-ethionine-resistant sporulation mutant. A probable link between chemotaxis and sporulation.

A UV irradiation-induced mutant of Bacillus sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. Ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

Mechanism of Na+/H+ exchange by Escherichia coli NhaA in reconstituted proteoliposomes.

Purified NhaA, a Na+/H+ antiporter from Escherichia coli, reconstituted into proteoliposomes was used to study partial reactions catalyzed by this protein. Homologous Na+/Na+ exchange as well as Na+/Li+ exchange via NhaA were detected by monitoring the effects of external Li+ and Na+ ions on the delta pH-driven sodium uptake into NH4 Cl-loaded vesicles. Furthermore, a sodium counterflow reaction was demonstrated in proteoliposomes preloaded with non-radioactive Na+ and placed into the experimental buffer containing low amounts of 22Na+ under experimental conditions when both components of protonmotive force generated by the antiporter. delta psi and delta pH, were dissipated by corresponding ionophores. The apparent Km for sodium counterflow is 1.1 mM, and Vmax is 80 mumol/min/mg of protein. External Na+ accelerates the downhill efflux of 22Na+ suggesting that the translocation of the Na(+)-loaded form of the carrier is faster than the rest of the catalytic cycle.

Calcium transport mediated by NhaA, a Na+/H+ antiporter from Escherichia coli.

In everted membrane vesicles of E. coli strain EP432/pGM42, which has only one Na+/H+ antiporter (NhaA), external CaCl2 inhibits dissipation of the respiration-dependent delta pH in response to the addition of NaCl at pH 7.5, and decreases equilibrium concentration of the intravesicular Na+. In the NhaA proteoliposomes, imposition of an artificial delta pH (acid inside) leads to the several-fold accumulation of calcium. The apparent Km for this delta pH-driven Ca2+ uptake at pH 8.5 is 2 mM, and the Vmax is 1.79 mumol/min/mg of protein. Dissipation of delta pH causes release of calcium from the vesicles. CaCl2 was found to inhibit the delta pH-driven Na+ uptake mediated by reconstituted NhaA, and vice versa. Further, heterological Ca2+/Na+ exchange has been demonstrated in proteoliposomes containing NhaA. Transmembrane electric potential difference proved to drive this process. All these data are consistent with the assumption that NhaA can also catalyze Ca2+/H+ exchange.

Adaptation of Bacillus FTU and Escherichia coli to alkaline conditions: the Na(+)-motive respiration.

Mechanisms of Na+ transport into the inside-out subcellular vesicles of alkalo- and halotolerant Bacillus FTU and of Escherichia coli grown at different pH have been studied. Both microorganisms growing at pH 7.5 are shown to possess a system of the respiration-dependent Na+ transport which (i) is inhibited by protonophorous uncoupler, by delta pH-discharging agent diethylammonium (DEA) acetate, by micromolar cyanide arresting the H(+)-motive respiratory chain, and by amiloride, and (ii) is resistant to the Na+/H+ antiporter monensin and to Ag+, inhibitor of the Na(+)-motive respiratory chain. Growth at pH 8.6 strongly changes the activator and inhibitor pattern. Now (1) protonophore stimulates the Na+ transport, (2) DEA acetate is without effect in the absence of protonophore and is stimulating in its presence, (3) amiloride and low cyanide are ineffective, (4) monensin and Ag+ completely arrest the Na+ accumulation in the vesicles. Independent of pH of the growth medium, (a) valinomycin is stimulatory for the Na+ transport, (b) Na+ ionophore ETH 157 is inhibitory and, (c) Na+ transport can be supported by NADH----fumarate as well as by ascorbate (TMPD)----O2 electron transfers. Growth at alkaline pH results in the appearance of ascorbate (TMPD) oxidation resistant to low and sensitive to high cyanide concentrations. These relationships are in agreement with the concept (Skulachev, V.P. (1984) Trends Biochem. Sci. 9, 483-485) that adaptation to alkaline conditions in bacteria growing in the high [Na+] media causes substitution of Na+ for H+ as a coupling ion. The obtained data indicate that under alkaline conditions, Na+ can be pumped from the cell by the Na(+)-motive respiratory chain with neither H(+)-motive respiration nor the Na+/H+ antiporter involved. In the Na(+)-motive respiratory chain of Bac. FTU or E. coli, two Na+ pumps are localized, one in its initial and the other in its terminal spans.

A study on Na+ -coupled oxidative phosphorylation: ATP formation supported by artificially imposed delta pNa and delta pK in Vibrio alginolyticus cells.

Addition of Na+ to the K+-loaded Vibrio alginolyticus cells, creating a 250-fold Na+ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na+/H+ antiporter, monensin. The delta pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K+ gradient (high [K+] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na+ pulse-induced ATP formation. The data agree with the assumption that Na+ -ATP-synthase is involved in oxidative phosphorylation in V alginolyticus. Interrelation of H+ and Na+ cycles in bacteria is discussed.

The ATP-driven primary Na+ pump in subcellular vesicles of Vibrio alginolyticus.

Subcellular vesicles of Vibrio alginolyticus hydrolyze ATP and accumulate Na+ in an ATP-dependent fashion. The Na+ uptake is (i) strongly stimulated by delta psi-discharging agents, i.e., the protonophorous uncoupler CCCP or valinomycin + K+ and (ii) arrested by DCCD at a concentration strongly inhibiting ATP hydrolysis. Lower concentrations of DCCD stimulate the Na+ accumulation supported by ATP hydrolysis as well as by NADH oxidation. It is concluded that there is an electrogenic DCCD-sensitive Na+-ATPase in the cytoplasmic membrane of V. alginolyticus.

[The role of Na+ ions in the respiration, formation of the membrane potential and movement of the alkali-resistant marine bacterium Vibrio alginolyticus]. / Rol' ionov Na+ v dykhanii, obrazovanii membrannogo potentsiala i dvizhenii morskoi shchelocheustoichivoi bakterii Vibrio alginolyticus.

Subbacterial vesicles capable of generating delta psi during NADH oxidation were obtained. The oxidation of NADH was stimulated by Na+ and inhibited by 2-heptyl-4-oxyquinoline-N-oxide (HQNO) in submicromolar concentrations. The generation of delta psi was inhibited by HQNO in low concentrations, cyanide, gramicidine D and carbonyl cyanide-m-chlorophenylhydrazone (CCCP) in combination with monensine. At the same time, in the absence of monensine CCCP influenced the delta psi generation in a much lesser degree. In subbacterial vesicles delta psi generation coupled with NADH oxidation necessitated Na+. Experiments with intact cells of V. alginolyticus revealed that cell motility depends on Na+, is sensitive to CCCP + monensine as well as to arsenate + HQNO, cyanide or anaerobiosis. In the absence of arsenate, the inhibition of respiration partly decreased the rate of bacterial movement. In the presence of HQNO and arsenate, NaCl addition to K+-loaded cells led to the monensine preventing restoration of the cell motility during a few minutes. However, no stimulating effect was observed in the case of artificial delta pH formation as a result of acidification of the medium (from pH 8.6 to pH 6.5). The experimental results suggest that delta mu Na+ generated by the respiratory chain and by the arsenate-sensitive enzymatic system (presumably, glycolysis and Na+-ATPase) can be utilized by the Na+-driven molecular motor responsible for the motility of V. alginolyticus cells.

The sodium cycle. II. Na+-coupled oxidative phosphorylation in Vibrio alginolyticus cells.

The role of Na+ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na+-motive respiratory chain of V. alginolyticus. Na+ loaded cells incubated in a K+ or Li+ medium fail to synthesize ATP in response to lactate addition. The addition of Na+ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na+ level. Neither lactate oxidation nor delta psi generation coupled with this oxidation is increased by external Na+ in the Na+-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse delta pNa, i.e., [Na+]in greater than [Na+]out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K+-loaded cells incubated in a high Na+ medium, i.e., when delta pNa of the proper direction [( Na+]in less than [Na+]out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K+ medium (delta pNa is low) is decreased by CCCP even without monensin. Artificial formation of delta pNa by adding 0.25 M NaCl to the K+-loaded cells (Na+ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na+ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na+ instead of H+ as the coupling ion.

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus.

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.

[Dependence of monotrachial bacteria chemotaxis on the direction of flagella rotation]. / O zavisimosti khemotaksisa monotrakhial'nykh bakterii ot napravleniia vrashcheniia flagelly.

Monotrichous bacteria V. harveyi periodically reverse their direction when moving in a liquid environment. It is reported that in a uniform surrounding the motility pattern is composed of a repeat of two sequential runs: a short and a long one separated by a reversal. The main duration of the short run was 0.4 sec and of the long one - 1.3 sec. Upon the addition of attractants or repellents the long runs became prolonged or shortened correspondingly. The duration of the short runs was independent of attractant or repellent stimuli. It is concluded that the flagellum is unresponsive to the taxis signal when rotating clockwise, which corresponds to the short run.