Vibrio Costicola Research Articles

Phospholipid Biosynthesis in the Moderately Halophilic Bacterium Vibvio costicola During Adaptation to Changing Salt Concentrations

The metabolism of phospholipids in Vibrio costicola, a moderately halophilic bacterium, has been investigated in relation to sudden changes in salinity. Both the absolute and relative rates of biosynthesis of phosphatidylglycerol and phosphatidylethanolamine depend on the salt concentration of the medium; a sudden rise in salt concentration has an instantaneous inhibitory effect on phospholipid biosynthesis, but this inhibition lessens as. the bacteria adapt to the higher salinity. There is no turnover of phospholipids during isotonic growth, nor when the salt concentration is suddenly altered. The alterations in biosynthetic rates of phosphatidylglycerol and phosphatidylethanolamine that occur after sudden changes in salt concentration are consistent with the known compositional changes. We conclude that the mechanisms of changes in phospholipid composition during adaptation to raised or lowered salt concentrations are not necessarily the same.

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 220000 and consists of identical subunits with an approximate molecular weight of 72000. 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.

Proton circulation in Vibrio costicola.

The importance of proton movements was assessed in the moderate halophile Vibrio costicola. When anaerobic cells in acidic buffer (pH 6.5) were given an O2 pulse, protons were extruded regardless of the presence of Na+. At pH 8.5, however, V. costicola produced an acidic response to an O2 pulse in the absence of Na+ and an alkaline response when Na+ was present. An Na+/H+ antiport activity was confirmed at pH 8.5. All of these effects were prevented by protonophores or butanol treatment. Growth in complex medium at pH 8.5 was prevented by a high concentration (50 microM) of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or a low concentration (5 microM) of another protonophore, 3,3',4',5-tetrachlorosalicylanilide (TCS). The relative ineffectiveness of the former protonophore was caused by the proteose peptone and tryptone ingredients of the complex medium, since 5 microM completely prevented growth in their absence. The results are explained by a primary respiratory-linked proton efflux coupled to a secondary Na+/H+ antiport operating at alkaline pH. Evidence was seen for a role of Na+ in stimulating proton influx at alkaline pH, presumably via the pH homeostasis mechanism.

Study of microflora in Malaysian dried fishes and their decontamination by gamma-irradiation.

The distribution of microorganisms in 10 samples of salted dried fish and the effects of irradiation of them were studied. The total aerobic bacteria in commercial dried fish were determined to be from 2 × 104 to 3 × 106 per gram. Mold counts were 1 × 102 to 7 × 103 per gram with a lower amount of yeasts. In spoiled dried fish, total aerobic bacteria were determined to be 4× 106 or 1 × 107 per gram with a few yeasts. Coliforms were not isolated on MacConkey agar plates from any of the samples. The predominant bacteria occurring in spoiled dried fish were Pediococcus halophilus, Vibrio costicola and Planococcus sp. More than 50% of the molds consisted of the Aspergillus niger group, whereas lower amounts of the A. flavus, A. fumigatus and A. ochraceus groups, Penicillium chrysogenum series, etc. were also isolated from many samples of dried fish. All kinds of putrefactive microorganisms were radiation sensitive, and a dose of ca. 500 krad appears to be sufficient for extension of the shelf-life of dried fi...

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

Active transport of α-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.

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.

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na + ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O 2-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na + to Na +-deficient cells, showing that Na + is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N, N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na + chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na + gradient is also required.

The effect of salt concentration on the phospholipid and fatty acid composition of the moderate halophile Vibrio costicola

The major lipids of Vibrio costicola membranes and whole cells are phosphatidyl glycerol and phosphatidyl ethanolamine. Lesser amounts of lysophosphatidyl ethanolamine, cardiolipin, a glycolipid, and two other phospholipids, tentatively identified as lysocardiolipin and lysophosphatidyl glycerol, are also present. Phosphatidyl glycerol content markedly increased, whereas that of phosphatidyl ethanolamine decreased, with increasing salt concentration (0.5 to 3 M NaCl) in the growth medium. These changes result in an increase in the mole ratio of negatively charged polar lipids to neutral polar lipids. Little effect of growth phase on the proportions of the lipid components or their fatty acids was observed in cells grown in 1 M salt. The major fatty acids found were 16:0, 16:1, and 18:1; minor amounts of 14:0, 17:1, and 18:0, and traces of 12:0, 12:1, 14:1, 15:0, 15:1, and 17:0 were also found. The proportions of certain fatty acids also changed with changing salt concentration in the medium.

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.

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.

Distribution of Isoprenoid Quinones in Halophilic Bacteria

Menaquinones were the sole isoprenoid quinones found in 28 of the 34 halophilic organisms examined. Unsaturated and dihydrogenated menaquinones with eight isoprene units were found in some alkalophilic halophiles and representatives of the genera Halobacterium and Halococcus. Brevibacterium halotolerans and Micrococcus halobius possessed major amounts of unsaturated menaquinones with seven and eight isoprene units, respectively. Actinopolyspora halophila possessed complex mixtures of partially hydrogenated menaquinones with tetrahydrogenated menaquinones with nine isoprene units predominating. Vibrio costicola possessed both menaquinones and ubiquinones with eight isoprene units. Ectothiorhodospira halophila, Flavobacterium halmephilum, Paracoccus halodenitrificans and Pseudomonas beijerinkii contained ubiquinones as their sole respiratory quinones. Ectothiorhodospira halophila possessed major amounts of ubiquinones with eight isoprene units whereas ubiquinones with nine isoprene units predominated in Flavobacterium halmephilum, Paracoccus halodenitrificans and Pseudomonas beijerinkii.

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).

Some properties of the NADP-specific malic enzyme from the moderate halophile Vibrio costicola.

NADP-specific malic enzyme (EC 1.1.1.40) has been purified about 160-fold from the moderate halophile Vibrio costicola. The enzyme has a molecular weight of about 120,000. The purified enzyme was unstable in dilute solutions but could be stabilised by NaCl or glycerol. NH4Cl or KCI caused maximal activation at 0.1M, but higher concentrations were inhibitory. NaCl did not activate and was instead a mixed-type inhibitor towards NH4Cl or KCI. The salt concentration affected the kinetic parameters of the reaction. The apparent Km for L-malate reached a minimal value at about 0.1 M salt; the value for NADP, on the other hand, increased continuosly with the Co2+ or Mg2+. NADH was a mixed-type inhibitor towards both substrates, whereas oxaloacetate was strictly competitive towards L-malate and non-competitive towards NADP. The inhibition kinetics were sigmoidal for NADH and hyperbolic for oxaloacetate. The malic enzyme form V. costicola was similar to those of a marine Pseudomonas and Halobacterium cutirubrum in kinetic and regulatory properties but showed a response to salts intermediate between those of the latter enzymes.

The N-terminal sequence of the ribosomal ‘A’ protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 11227)

The ‘A’ protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The ‘A’ protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the ‘A’ proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the ‘A’ protein from both moderate halophiles showed substantial homology to EL 12 (75–80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli we were also isolated and their amino acid compositions determined.

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K + with Na + effectively prevented the lysis by SCN −. K + salts of NO 3 −, Br −, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN − than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN − is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na + observed here was considered to be manifested by specific interactions of Na + with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.

Salt-sensitive in vitro protein synthesis by a moderately halophilic bacterium.

EXTREMELY halophilic bacteria, such as Halobacterium cutirubrum, grow only in high NaCl concentrations (2.5–5 M) and have very high (5 M or higher) internal concentrations of salts, mainly KCl1,2. The ribosomes and protein-synthesising systems of these organisms seem especially adapted to function in such concentrations3. In contrast, moderately halophilic bacteria, such as Vibrio costicola, grow over a much wider range of NaCl concentrations (at least 0.5–3.5 M)4. In V. costicola, though not necessarily in all moderate halophiles, the cell-associated monovalent cations are at least as concentrated as those of the external medium. For example, cells growing exponentially in medium containing 1 M NaCl and 0.008 M KC1 can have internal Na+, K+, and NH4+ concentrations of about 0.6, 0.7 and 0.4 M respectively, as well as 40 mM Mg2+ (ref. 5 and our unpublished results). Ribosomes from V. costicola differ from those of both extremely halophilic and non-halophilic bacteria in their ability to maintain a ‘standard’ sedimentation pattern (30, 50 and 70S) over a wide range of salt concentrations. This pattern is not changed by the NaCl concentration in which the cells are grown6. Such properties, however, do not really tell us how well the ribosomes function at different salt concentrations. Studies of in vitro protein synthesis, reported here, suggest that ribosomes may function at much lower salt concentrations than measurements of total cell-associated ions indicated to be present in the cell.

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 effect of some gram-negative bacteria on the ripening and quality of dry sausage

The possible use of gram-negative bacteria as starter cultures in dry sausage was studied by inoculating the following gram-negative bacteria into dry sausage both alone and with Lactobacillus plantarum: Aeromonas x, Aeromonas 19. Vibrio costicolus, Achromobacter 22, Achromobacter guttatus, Achromobacter X, Escherichia coli, or Proteus vulgaris. Sausage without any inoculation and sausage containing Micrococcus sp. + Lactobacillus plantarum were used as references. Aeromonas x and 19 strains had a very favourable effect on the quality of dry sausage when inoculated together with lactobacilli. Both strains reduced nitrate, and the colour formed during the first 3 days. The pH value of Aeromonas + Lactobacillus sausages decreased so quickly that the consistency of these sausages developed within one week and was at least as good as, and often firmer than in Micrococcus + Lactobacillus sausages. The consistency of Aeromonas 19 + Lactobacillus sausages was better at the 0.05 significance level than that of Micrococcus + Lactobacillus samples. The aroma and flavour of Aeromonas + Lactobacillus sausages were as good as or better than those of Micrococcus 4- Lactobacillus sausages. The aroma and flavour of Aeromonas 19 + Lactobacillus sausages were significantly better (significance level 0.001) than Micrococcus-f Lactobacillus sausages. The flavour of Aeromonas x + Lactobacillus sausages was better at the 0.05 level than Micrococcus + Lactobacillus sausages. The number of bacteria, roughly the number of lactobacilli. was often significantly higher in Aeromonas + Lactobacillus sausages (range 2—4 x 108/g between 3 and 21 days of ripening) than in Micrococcus + Lactobacillus sausages (range 8 X 107 1.5 X 108/g between 3 and 21 days of ripening). For this reason the former sausages ripened more quickly than the latter. Vibrio 21 and three Achromobacter strains did not thrive in dry sausage and disappeared during the first 7 days of ripening. When inoculated together with lactobacilli, Escherichia coli had almost as favourable an effect as micrococci on the quality of dry sausage. Inoculations of E. coli alone and Proteus vulgaris, both alone and with lactobacilli, did not improve the quality of dry sausage. P. vulgaris actually had a detrimental effect. Conclusion The investigation has proved that gram-negative bacterial strains Aeromonas x and 19 inoculated with lactobacilli improve the quality of dry sausage and that, especially with the Aeromonas 19 + Lactobacillus inoculum, better dry sausage was obtained than with micrococci and lactobacilli.

Nutrition and distribution of salt response in populations of moderately halophilic bacteria.

Cultures of Micrococcus halodenitrificans and Vibrio costicolus can grow in the presence of 0.4–3.5 M NaCl. Three lines of investigation: attempts to select for more or less salt-tolerant cells; colony counts at different salt concentrations; and replica plating experiments, suggested that populations of these organisms were genetically homogeneous in their salt response. That is, each cell in a population could grow over the whole range of salt concentration in which the culture grew.The nutritional requirements of V. costicolus were studied. This organism can grow in a minimal (salts–glucose) medium at pH values above 6.0 and in a salt concentration range 0.5–2.2 M. Increasing the concentration of phosphate buffer greatly increased cell yield. The range of salt concentration in which V. costicolus could grow depended on the nutrient supply, being widest in complex media. A wider range was possible in the presence of amino acids than in a salts–glucose medium. All growth factors permitting growth to occur at high salt concentrations were not identified.

THE NUTRITION OF VIBRIO COSTICOLUS. II. CYST(E)INE UTILIZATION.

The effect of cyst(e)ine and sodium chloride concentration on oxygen uptake, and hydrogen sulphide, ammonia, and pyruvic acid production by Vibrio costicolus is described. Added pyridoxal was unnecessary for maximum desulphydrase activity. Generally, oxidation curves of cyst(e)ine concentrations 4 μM or less were the customary type whereas curves which resulted from the oxidation of 6.25 μM cyst(e}ine and greater were of a type indicating accumulation of a product toxic to the system. This occurred in the presence of 1 M NaCl and the suppressed type of oxidation could be returned to normal by increasing the concentration of NaCl. Pyruvic acid accumulated in the flasks showing the suppressed type of oxidation curves, but did not accumulate in flasks showing customary oxidation curves. Pyruvic acid did not accumulate in flasks containing 6.25 μM cyst(e)ine if the sodium chloride concentration was increased to 1.5 M or greater. Pyruvic acid did accumulate with 3.125 μM cyst(e)ine if a citric acid cycle inhibitor (sodium fluoroacetate) was added to the flask contents. These studies suggest that the most likely pathway for the utilization of cyst(e)ine, an essential nutrient for V. costicolus, is its conversion to pyruvate to enter the citric acid cycle.