Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.     

The role of osmotic effects in haloadaptation of Vibrio costicola

Growth rates of Vibrio costicola showed a broad optimum between 0.8 and 1.5 M-NaCl, and there was no growth above 3.3 M-NaCl in a peptone-based medium. The minimum requirement of 0.5 M-NaCl for growth in NaCl alone was reduced to 0.3 M-NaCl when the total solute concentration was raised to 0.5 to 1.0 M equivalent with sucrose or glycerol. Compared with equivalent NaCl concentrations, higher concentrations of sucrose were more inhibitory to growth, whereas glycerol had less effect. Increasing the medium NaCl concentration suddenly by 2- or 3-fold with either a constant starting, or final, salt concentration showed that, after the shift-up, the lag in growth, the rate of growth, and the inhibition of phospholipid synthesis depended both on the final NaCl concentration and the magnitude of the shift in salinity. The time-courses of phospholipid synthesis following a 2- or 3-fold shift-up in NaCl or sucrose media were very similar and exhibited a relative increase in phosphatidylglycerol synthesis over that of phosphatidylethanolamine. This 'switch-over' was not seen following shift-up in glycerol media when there was also a stimulation, rather than inhibition, of phospholipid synthesis. It is concluded that during phenotypic haloadaptation of V. costicola, osmotic effects play a significant part in the sensing of and response to raised external salinity.     

Preparation and properties of highly-purified Vibrio costicola polynucleotide phosphorylase

Vibrio costicola polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) has been purified to electrophoretic homogeneity. It has an approximate molecular weight of 220 000 and consists of identical subunits with an approximate molecular weight of 72 000. The enzyme appears to be a fairly typical polynucleotide phosphorylase with respect to its pH optima, substrate specificity and requirement for a divalent cation cofactor. However, the effect of salt concentration on its physiologically important phosphorolysis activity suggests that it is a moderately halophilic enzyme, able to function at the intracellular ionic strength of the bacterium. In addition, its ADP polymerization activity is remarkably stimulated by polylysine.     

Effects of penicillin on a moderately halophilic bacterium,Vibrio costicola

Penicillin inhibited growth and killed growing cells of the moderate halophileVibrio costicola. Cells were more susceptible to penicillin at lower than at higher NaCl concentrations. Inhibition of growth by KCN prevented the lethal action of penicillin. Envelopes of treated cells showed structural damage. Chains of cells separated under the action of the drug. Treated cells swelled rapidly, an unexpected reaction in a bacterium whose internal and external osmolarities are thought to be the same.     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

The susceptibility of the moderate halophile Vibrio costicola to heavy metals

Fifty-eight strains of the moderate halophile Vibrio costicola , including both culture collection strains and freshly isolated strains from solar salterns, were examined for their susceptibility to 10 heavy metal ions by using an agar dilution technique. All strains were sensitive to cadmium, copper, silver, zinc and mercury. This latter ion showed the highest activity even at 0·05 mmol/1 metal concentration. On the other hand, all strains were similarly tolerant to lead, and a great proportion of them were also tolerant to nickel (91%) and chromium (88%). Only 44% and 14% of them showed tolerance to arsenate and cobalt, respectively. The majority of strains (96·4%) were multiply metal-tolerant, with three different metal ion tolerances as the major pattern.     

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola.

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.     

Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola.

Vibrio costicola grown in the presence of different NaCl concentrations contains cell-associated Na+ and K+ ions whose sum is equal to or greater than the external Na+ concentration. In the presence of 0.5 M NaCl, virtually no in vitro protein is synthesized in extracts of cells grown in 1.0 M NaCl. However, we report here that active in vitro protein synthesis occurred in 0.6 M or higher concentrations of Na2SO4, sodium formate, sodium acetate, sodium aspartate, or sodium glutamate, whereas 0.6 M NaF, NaCl, or NaBr completely inhibited protein synthesis as measured by polyuridylic acid-directed incorporation of [14C]phenylalanine. Sodium glutamate, sodium aspartate, and betaine (0.3 M) counteracted the inhibitory action of 0.6 M NaCl. The cell-associated Cl- concentration was 0.22 mol/kg in cells grown in 1.0 M NaCl. Of this, the free intracellular Cl- concentration was only 0.02 mol/kg. Cells contained 0.11 mol of glutamate per kg and small concentrations of other amino acids. All of the negative counterions for cell-associated Na+ and K+ have not yet been determined. In vitro protein synthesis by Escherichia coli was inhibited by sodium glutamate. Hybridization experiments with ribosomes and the soluble (S-100) fractions from extracts of E. coli and V. costicola showed that the glutamate-sensitive fraction was found in the soluble, not the ribosomal, part of the system. The phenylalanyl-tRNA synthetase of V. costicola was not inhibited by 0.5 M or higher concentrations of NaCl; it was slightly more sensitive to high concentrations of sodium glutamate. Therefore, this enzyme was not responsible for the salt response of the V. costicola in vitro protein-synthesizing system.     

Role of membrane-bound 5''-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).     

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions.

In vitro protein synthesis in Vibrio costicola [poly(U)-directed incorporation of phenylalanine] was studied. The extent of protein synthesis was limited by the number of ribosomes present. Density gradient centrifugation experiments suggested that, after runoff of ribosomes from the artificial messenger, the 50S subunit was unable to attach to the 30S-messenger complex. As shown previously (M. Kamekura and D. J. Kushner, J. Bacteriol. 160:385-390, 1984), Cl- ions inhibited protein synthesis; indeed, the highest rate of synthesis took place in the lowest attainable Cl- concentration (37 mM). The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. The strongest reversal was seen when both glutamate and betaine were present. Cl- ions can prevent binding of ribosomes to poly(U) and displace ribosomes already bound to this artificial messenger. The effects of Cl- ions on binding were also reversed by glutamate and betaine. Cl- ions did not affect accuracy of translation; they were shown previously (Kamekura and Kushner, J. Bacteriol. 160:385-390, 1984) not to affect phenylalanyl-tRNA synthetase. It was also found that washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. On the contrary, these ribosomes were more active than before they were washed. We conclude that the main site of action of Cl- in the system studied is on the binding of ribosomes to the mRNA.     

Phosphoenolpyruvate carboxykinase from the moderate halophile Vibrio costicola. Purification, physicochemical properties and the effect of univalent-cation salts.

Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophilic bacterium Vibrio costicola. The enzyme is monomeric, with an Mr of 62,000, as determined by the Svedberg equation, by using values of s0(20,w) 4.4 x 10(-13) s, D20,w 6.13 x 10(-7) cm2.s-1 and v 0.719 cm3.g-1. Compared with other, non-halophilic, PEPCKs, the enzyme from V. costicola had a significantly lower total content of hydrophobic amino acids. The contents of glycine and serine were higher in the V. costicola enzyme (16.7 and 10.22% respectively) than in the non-halophilic PEPCKs (6.8-9.6% and 4.67-6.28% respectively). These results resemble those obtained by De Médicis & Rossignol [(1979) Experientia 35, 1546-1547] with the pyruvate kinase from V. costicola, and agree with the proposal by Lanyi [(1974) Bacteriol. Rev. 38, 272-290] of partial replacement of hydrophobic amino acids by glycine and serine to maintain the balance between hydrophobic and hydrophilic forces in halophilic enzymes. In agreement with this 'halophilic' characteristic, the PEPCK was somewhat stabilized by 1 M-KCl or -NaCl and by 20% (v/v) glycerol, and its oxaloacetate-decarboxylation and 14CO2-oxaloacetate-exchange reactions were activated by KCl and NaCl up to 1 M, whereas the fixation of CO2 on PEP had a maximum at 0.025-0.05 M salt. These facts suggest that the salts, at concentrations probably physiological for the bacterium, increase the formation of the complex of oxaloacetate and ATP with the enzyme, and the liberation of the products, PEP and ADP, thus favouring PEP synthesis.     

Interactive effects of salt concentration and temperature on growth and lipid composition in the moderately halophilic bacterium Vibrio costicola.

The interactive effects of NaCl concentration and growth temperature on the growth and lipid composition of the moderately halophilic eubacterium Vibrio costicola have been investigated. Vibrio costicola was shown to be capable of growth over the temperature range 4-37 degrees C. Maximum growth yields were obtained at 30 degrees C when the optimum NaCl concentration was 1.0 M NaCl. In contrast with some previous studies, at higher or lower growth temperatures both the optimum and lower limit of NaCl concentration were higher, but there was no change in the upper limit of NaCl concentration for growth. There were no differences between the lipid compositions of cultures grown in 1 M NaCl at 30 or 37 degrees C, but as the growth temperature was lowered from 30 to 10 or 4 degrees C, the ratio of phosphatidylethanolamine to phosphatidylglycerol increased significantly as a result of the conversion of phosphatidylglycerol to diphosphatidylglycerol; in addition, at the lower growth temperatures the phospholipid fatty acyl composition became more unsaturated and the mean acyl chain length was shorter. It is suggested that the altered salt dependence of V. costicola at temperatures below the optimum for growth is due to a modification in membrane lipid phase behavior and stability brought about by changes in lipid composition, whereas a different mechanism operates above the growth temperature optimum.     

Proton circulation during the photocycle of sensory rhodopsin II.

Sensory rhodopsin II (SRII) in Halobacterium salinarum membranes is a phototaxis receptor that signals through its bound transducer HtrII for avoidance of blue-green light. In the present study we investigated the proton movements during the photocycle of SRII in the HtrII-free and HtrII-complexed form. We monitored sustained light-induced pH changes with a pH electrode, and laser flash-induced pH changes with the pH indicator pyranine using sealed membrane vesicles and open sheets containing the free or the complexed receptor. The results demonstrated that SRII takes up a proton in M-to-O conversion and releases it during O-decay. The uptake and release are from and to the extracellular side, and therefore SRII does not transport the proton across the membrane. The pH dependence of the SRII photocycle indicated the presence of a protonatable group (pK(a) approximately 7.5) in the extracellular proton-conducting path, which plays a role in proton uptake by the Schiff base in the M-to-O conversion. The extracellular proton circulation produced by SRII was not blocked by HtrII complexation, unlike the cytoplasmic proton conduction in SRI that was found in the same series of measurements to be blocked by its transducer, HtrI. The implications of this finding for current models of SRI and SRII signaling are discussed.     

Respiration-driven Na+ pump and Na+ circulation in Vibrio parahaemolyticus.

Sodium circulation in Vibrio parahaemolyticus was investigated. We observed respiration-driven Na+ extrusion from cells by using a Na+ electrode. The Na+ extrusion was insensitive to a proton conductor, carbonyl cyanide m-chlorophenylhydrazone, and sensitive to a respiratory inhibitor, CN-. These results support the idea of the existence of a respiratory Na+ pump in V. parahaemolyticus. The respiration-driven Na+ extrusion was observed only under alkaline conditions.     

Growth of a marine Vibrio alginolyticus and moderately halophilic V. costicola becomes uncoupler resistant when the respiration-dependent Na+ pump functions

The growth of Vibrio alginolyticus and V. costicola, which possess respiration-dependent Na+ pumps, was highly resistant to the proton conductor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), in alkaline growth media, even though the membrane was rendered permeable to H+. The pH dependence of CCCP-resistant growth was similar to that of the Na+ pump. In contrast, Escherichia coli ML308-225 showed neither Na+ pump activity nor CCCP-resistant growth, even when grown in alkaline, Na+-rich media. These results suggest that certain bacteria possess the Na+ pump and are thus able to grow under the conditions where H+ circulation across the membrane does not take place. Moreover, V. alginolyticus growing in the presence of CCCP maintains normal levels of internal K+, Na+, and H+. The Na+ pump, therefore, makes the growth of these organisms resistant to CCCP by maintaining the intracellular cation environments.     

Respiration-Dependent Proton and Sodium Pumps in a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

Respiration-dependent proton and sodium flows in a psychrophilicbacterium, Vibrio sp. strain ABE-1, were examined. At alkalinepH, this bacterium grew without being affected by a proton conductor,carbonylcyanide m-chlorophenylhydrazone (CCCP). O₂-pulse intoanaerobic cell suspensions prepared with Na^$-free buffers inducedtransient alkalization in the presence of CCCP and acidificationat pH 8.5 and 6.5, respectively. However, using cells preparedwith Na^$-containing buffer, the transient pH changes of thecell suspension could be simultanously detected at both pHs.Several inhibitory experiments suggested that the acidificationand alkalization should be attributed to a respiration-dependentprimary H^$ pump and Na^$ pump, respectively, and that the latterwas similar to that first reported in a marine bacterium, Vibrioalginolyticus. This Na^$ pump may have supported the CCCP-resistantgrowth at alkaline pH. The H^$ and Na^$ pumps operated very actively at low temperatures,such as 5?C, and should markedly help sustain bacterial growthat low temperatures. (Received May 30, 1987; Accepted November 13, 1987)