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Conditions optimum for the assay of alkaline phosphatase of marine pseudomonad B-16 (ATCC 19855) and for maintaining the activity of the enzyme have been determined. The pH for optimal activity of the cell-bound enzyme was 9.0, whereas that for the enzyme after its release from the cells exceeded 9.4. Release was effected by first washing the cells in 0.5 M NaCl and then suspending them in 0.5 M sucrose. In the absence of salts, the activity of the cell-bound enzyme decreased rapidly at 25 C and less rapidly at 4 C. This loss of activity could be arrested but not restored by adding Mg(2+). In the presence of Na(+), activity of the cell-bound enzyme dropped to about 50% of that prevailing initially, but in this case adding Mg(2+) restored enzyme activity completely. The activity of the enzyme after its release from the cells into 0.5 M sucrose was approximately 50% of that of the equivalent amount of enzyme in the original cells. This activity was relatively stable at both 25 and 4 C. Adding Mg(2+) to the released enzyme restored its activity to that of the cell-bound form. The synthesis of alkaline phosphatase by the cells was not affected by adding 50 mM inorganic phosphate to the growth medium. The K(m) of the released enzyme for p-nitrophenyl phosphate was found to be 6.1 x 10(-5) M.

When cells of Alteromonas haloplanktis 214 (ATCC 19855) were preloaded with alpha-[(14)C]aminoisobutyric acid or the K(+) in the cells was labeled with (42)K by incubation in a buffered salt solution containing 0.05 M MgSO(4), 0.01 M KCl, and 0.3 M NaCl, the cells retained their radioactivity when resuspended in the same salt solution. When NaCl was omitted from the solution, 80 to 90% of the radioactivity was lost from the cells. Cells suspended at intermediate concentrations of NaCl also lost radioactivity. New steady-state levels of the intracellular solutes were established within 15 s of suspending the cells; the percentage of radioactivity retained at each level decreased proportionately as the osmolality of the NaCl in the suspending solution decreased. With minor variations in effectiveness, MgCl(2), LiCl, and sucrose could substitute for NaCl on an equiosmolal basis for the retention of radioactivity by the cells. KCl, RbCl, and CsCl were appreciably less effective as replacements for NaCl, particularly when their osmolalities in the suspending solutions were low. The amount of alpha-[(14)C]aminoisobutyric acid taken up by the cells at the steady-state level increased to a maximum as the NaCl concentration in the suspending medium increased to 0.3 M. At suboptimal levels of NaCl, either LiCl or sucrose could substitute for NaCl in increasing the steady-state levels. The results obtained indicate that the porosity of the cytoplasmic membrane of this organism is determined by the difference between the osmotic pressure of the cytoplasm and the suspending medium. The lesser effectiveness of K(+), Rb(+), and Cs(+) than Na(+), Li, or Mg(2+) in permitting the retention of solutes by the cells is attributed to the greater penetrability of the hydrated ions of the former group through the dilated pores of a stretched cytoplasmic membrane.

Transport of K(+) by K(+)-depleted cells of marine pseudomonad B-16 (ATCC 19855) exhibited saturation kinetics. Rb(+) inhibited both K(+) transport and the K(+)-dependent transport of alpha-aminoisobutyric acid (AIB) into K(+)-depleted cells of the organism in proportion to the concentration of Rb(+) in the suspending medium. Inhibition of the K(+)-dependent uptake of AIB into K(+)-depleted cells by Rb(+) could be overcome by increasing the concentration of K(+) in the medium. When AIB and K(+) were added simultaneously to a suspension of K(+)-depleted cells, the uptake of K(+) occurred immediately and rapidly, whereas the accumulation of AIB occurred only after a lag. The initial uptake rate of AIB was directly proportional to the intracellular K(+) concentration. The intracellular concentration of K(+) and AIB at their steady-state levels increased to a maximum as the Na(+) concentration in the suspending medium was increased. At Na(+) concentrations between 0.2 and 0.3 M, the molar ratio of K(+) to AIB at their intracellular steady-state concentrations was constant at 1.6. At external Na(+) concentrations less than 0.2 M, the cells maintained a relatively higher K(+) intracellular steady-state level than AIB.

The transport of alpha-aminoisobutyric acid and K(+) into K(+)-depleted cells of a marine pseudomonad (ATCC 19855) was stimulated strongly by ethanol, reduced nicotinamide adenine dinucleotide (NADH), and ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine. In the presence of the quinone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide, only ascorbate-reduced N, N, N', N'-tetramethyl-p-phenylenediamine was active. Primary and secondary, but not tertiary, alcohols from ethanol to n-amyl alcohol stimulated both alpha-aminoisobutyric acid and K(+) transport and were oxidized by the cells. Malate and succinate, which were oxidized rapidly by the cells, had little or no capacity to energize the transport of alpha-aminoisobutyric acid into K(+)-depleted cells but were partially effective in promoting K(+) uptake. Ethanol stimulated the transport of alpha-aminoisobutyric acid into K(+)-preloaded cells. The transport of both alpha-aminoisobutyric acid and K(+) was inhibited 20% by iodoacetate, 85% by N-ethylmaleimide, and 90 to 100% by both NaCN and p-chloromercuribenzoate. The addition of Na(3)Fe(CN)(6) permitted the ethanol-induced transport of alpha-aminoisobutyric acid into K(+)-preloaded cells in the presence of NaCN, but little or no uptake of alpha-aminoisobutyric acid or of K(+) into K(+)-depleted cells under the same conditions. The transport of alpha-aminoisobutyric acid into K(+)-depleted cells required both K(+) and an electron donor. The oxidation of NADH and ethanol by K(+)-depleted cells was stimulated strongly by K(+). Parallels between these studies and those with membrane vesicles show that results with membrane vesicles of the marine pseudomonad have physiological significance for the intact cells. The results support the conclusion that the energy for the active transport of both alpha-aminoisobutyric acid and K(+) into cells of this organism is provided by electron flow through a region of the respiratory chain lying between cytochrome c and O(2).

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.

Lipopolysaccharide (LPS) extracted from Alteromonas haloplanktis 214, variants 1 and 3, separated into three fractions when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions appeared in the gels as bands which stained for carbohydrate with the periodate-Schiff reagent. Variant 1, a smooth variant of the organism, and variant 3, a rough colonial variant, produced identical banding patterns. Under similar conditions, LPS from Neisseria meningitidis SDIC, Escherichia coli O111:B4, and Salmonella typhimurium LT2 gave rise to one, two, and three bands, respectively. LPS from Pseudomonas aeruginosa (ATCC 9027) failed to stain clearly with the reagent used. The banding pattern obtained with A. haloplanktis LPS was found not to be due to artifacts produced by the extraction or solubilization procedures employed or to the amount of protein associated with the LPS. When Triton X-100 replaced sodium dodecyl sulfate in the electrophoresis system, LPS failed to migrate into the gel. The lipid A but not the degraded polysaccharide fraction obtained by mild acid hydrolysis of the LPS migrated into the gel on electrophoresis. The three carbohydrate-staining bands obtained with A. haloplanktis LPS and referred to as LPS I, II, and III, in order of increasing electrophoretic mobility, were detected in each of the three outer layers of the cell wall of the organism. Estimations from densitometer scans indicated that 17% of the total LPS in the cell was present in the outer membrane, with the remainder divided almost equally between the loosely bound outer layer and the periplasmic space. Of the three fractions, LPS II was present in each of the layers in greatest amounts. Less LPS I and more LPS III were present in the outer membrane than in the periplasmic space. Pulse-labeling studies indicated that LPS I and II may be synthesized independently, whereas LPS III, which appeared only in cells in the stationary phase of growth, may be a degradation product of LPS I.

Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N', N'-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.

When cells of Pseudomonas aeruginosa were suspended in solutions containing increasing concentrations of NaCl, the optical density (OD) of the suspensions measured within 30 sec was found to increase in proportion to the increase in salt concentration. Measurement of intracellular fluid volumes indicated that the volume of the cells decreased roughly in proportion to the increase in salt concentration. After the initial increase in optical density, there was a slow decrease at all concentrations of NaCl tested except the highest, 500 mm. Metabolic inhibitors such as sodium azide and 2,4-dinitrophenol prevented the decrease. Direct analysis showed that the Na(+) and Cl(-) concentrations in the cells were 86 and 77%, respectively, of the concentrations of the ions in the suspending medium after 1 hr. Measurement of the (22)Na space in packed cells showed that Na(+) penetrated the total fluid space in the packed cells. The penetration of (22)Na was not prevented by the presence of metabolic inhibitors or by 500 mm NaCl in the suspending medium. The results indicate that the OD increases produced in suspensions of P. aeruginosa by NaCl are not due to the osmotic action of the salt. The subsequent optical density decreases observed are under metabolic control.

Colonies of Mycoplasma pneumoniae and Mycoplasma salivarium grown in PPLO agar were examined by light and electron microscopy. The main objective of the investigation was to attempt in situ fixation and minimize tonic changes in the organisms. Microscopy revealed that both organisms grew both in and upon the agar. The agar and surface growths of M. pneumoniae exhibited similar profiles, whereas those of M. salivarium differed strikingly. Both organisms are highly pleomorphic, but their matrix was denser and appeared more intact than in previously reported profiles. Cells which resemble the commonly reported mycoplasma were occasionally observed. The significance of these discrepant profiles remains unanswered. It is suggested that they may represent aged or osmotically damaged cells.

The cell wall of the gram-negative marine pseudomonad (American Type Culture Collection 19855) consists of three layers: the loosely bound outer layer, the outer double-track layer, and the underlying layer. These three layers constitute 4.7, 7.9, and 6.1%, respectively, of the dry weight of the whole cells. All three layers contained protein, lipid, and carbohydrate. The loosely bound outer layer and underlying layer were lower in protein and lipid and higher in amino and nonamino carbohydrate than the outer double-track layer. All three layers contained proteins with similar amino acid compositions. Minicell-like forms attached to the ends of cells were separated with and fractionated from the units of loosely bound outer layer. Examination of negatively stained preparations by electron microscopy revealed the loosely bound outer layer to be composed largely of units ranging from 400 to 1000 nm in diameter. The outer double-track layer, by the same technique, appeared as large, usually rounded sheets, each with a distinct rim. Washing this layer changed the gross chemical composition but did not affect the bimolecular leaflet appearance in thin sections. The underlying layer, when negatively stained, appeared to be composed of a heterogeneous mixture of particles differing in size and shape. It was separated by gel filtration into a large fraction with a molecular weight range in excess of 20 x 10(6) to 40 x 10(6) and a small fraction with a lower range of molecular weight. The larger fraction contained both protein and hexosamine, whereas the smaller one contained protein and only traces of hexosamine. A cytochrome-like pigment separated with this latter fraction.

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.

Drapeau, Gabriel R., (Macdonald College of McGill University, Quebec, Canada) and Robert A. MacLeod. Nutrition and metabolism of marine bacteria. XII. Ion activation of adenosine triphosphatase in membranes of marine bacterial cells. J. Bacteriol. 85:1413-1419. 1963.-Isolated membranes of two species of marine bacteria, a Pseudomonas and a Cytophaga, have been shown to possess adenosine triphosphatase activity. The optimal pH for enzyme action of both organisms was 8.8. The enzyme system was found to be capable of splitting inorganic o-phosphate from adenosine triphosphate (ATP), adenosine diphosphate, adenosine monophosphate, and inosine triphosphate but not from inorganic pyrophosphate. Mg(++) was required for enzyme activity; with the Pseudomonas species, the optimal Mg(++) to ATP ratio was 1:1. Ca(++) could not replace Mg(++). In the presence of the optimal concentration of Mg(++), the enzyme system was further stimulated, nonspecifically, by a number of different salts. Maximal activation was achieved at an ionic strength of 0.3 to 0.4. No evidence of an adenosine triphosphatase specifically activated by a combination of Na(+) and K(+) was obtained with either organism. No effect of ouabain on either the membrane adenosine triphosphatase activity or Na(+) transport by whole cells could be detected. The results suggest that the mechanism of ion regulation in marine bacterial cells is different from that in animal cells.

When distilled water from a tin-lined still served as the plating diluent, cells of Aerobacter aerogenes developed symptoms of metabolic injury as evidenced by increased counts on supplemented, as compared with minimal, plating medium. Cysteine was as effective as yeast extract as a supplement to the minimal medium in increasing the viable count. Mg(++) and, to a lesser extent, phosphate buffer at the concentrations tested protected unfrozen cells, but not cells which had been frozen and stored, against the loss of capacity to grow on minimal medium. When the plating diluent consisted of distilled water redistilled in an all-glass still, the symptoms of metabolic injury did not appear. Spectrographic analysis revealed the presence of 10(-7)m Cu(++) in the distilled water, and Cu(++) added to redistilled water serving as the plating diluent reproduced the metabolic injury effects induced by distilled water. It was concluded that freezing and storage damaged the cell membrane, rendering it more penetrable by toxic elements which were thereby enabled to act at sites in the cell where Mg(++) and other solutes in the plating diluent could not serve as effective antagonists. Increased recovery of cells on supplemented medium could be ascribed to the capacity of the supplements to remove toxic elements which had become bound to the cells during suspension in the plating diluent.

With procedures developed previously in this laboratory, the various layers of the cell wall of a gram-negative bacterium, a marine pseudomonad (ATCC 19855), were removed completely giving rise to true protoplasts. Membranes were isolated from the protoplasts formed. After treatment with ribonuclease, deoxyribonuclease, and washing, the membranes isolated were shown by electron microscopy and chemical analysis to be essentially free from both wall material and cytoplasmic constituents. The membranes gave rise to a single compact band in a sucrose density gradient. All of the lipid and protein were found to be associated in the membrane band. Analysis showed the membranes to contain 30.5% lipid (78% of which was phospholipid), 62.8% protein, and 2% carbohydrate. The predominant phospholipid present was phosphatidylethanolamine with a lesser amount of diphosphatidylglycerol and traces of unidentified compounds.

The peptidoglycan layer of a marine pseudomonad was observed by electron microscopy in thin sections of plasmolyzed intact cells and mureinoplasts but not in untreated intact cells. Only fragments of this layer could be isolated by sodium lauryl sulfate (SLS) treatment of mureinoplast envelopes. Sacculus-like peptidoglycan structures were obtained from growing cells by immediate heat inactivation of cellular autolytic enzymes and subsequent SLS, trypsin, and nuclease treatments. Recently, similar peptidoglycan sacculus-like structures have been obtained by adding SLS to the growing culture and treating the isolated particulate material with nucleases. Thin-sectioned and negatively stained preparations of whole cell peptidoglycan showed compressed profiles of cell-shaped sacculi. Peptidoglycan prepared by SLS treatment of mureinoplast envelopes had a similar composition to that prepared from whole cells. The major amino sugars and amino acids in the peptidoglycan component were glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in the molar ratios 1.18:1.24:1.77:1.00:0.79. Forty-five per cent of the epsilon-amino groups of diaminopimelic acid were cross-linked. The peptidoglycan was estimated to account for about 1% of the cell dry weight.

The outer layers of the cell envelope of a pseudomonad of marine origin were removed by washing the cells in 0.5 m NaCl followed by suspension in 0.5 m sucrose. The term mureinoplast has been suggested for the rod-shaped forms which resulted from this treatment. As previously established, these forms lacked the outer cell wall layers but still retained a rigid peptidoglycan structure. Mureinoplasts remained stable if suspended in a balanced salt solution containing 0.3 m NaCl, 0.05 m MgSO(4), and 0.01 m KCl but, unlike whole cells, lost ultraviolet (UV)-absorbing material if suspended in 0.5 m NaCl or 0.05 m MgCl(2). Sucrose added to the balanced salt solution also enhanced the loss of UV-absorbing material. Addition of lysozyme to suspensions of mureinoplasts in the balanced salt solution produced spherical forms which, by electron microscopy and the analysis of residual cell wall material, appeared to be true protoplasts. Only undamaged mureinoplasts, as judged by their capacity to fully retain alpha-aminoisobutyric acid, were capable of being converted to protoplasts. Protoplasts and undamaged mureinoplasts retained 100% transport capacity when compared to an equal number of whole cells. The Na(+) requirement for transport of alpha-aminoisobutyric acid and the sparing action of Li(+) on this Na(+) requirement were the same for both protoplasts and whole cells. These observations indicate that, in this gram-negative bacterium, the cell wall does not participate in the transport process though it does stabilize the cytoplasmic membrane against changes in porosity produced by unbalanced salt solutions. The results also indicate that the requirements for Na(+) for transport and for the retention of intracellular solutes are manifested at the level of the cytoplasmic membrane.

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.

Takacs, Frank P. (McGill University, Montreal, Quebec, Canada), Tibor I. Matula, and Robert A. MacLeod. Nutrition and metabolism of marine bacteria. XIII. Intracellular concentrations of sodium and potassium ions in a marine pseudomonad. J. Bacteriol. 87:510-518. 1964.-Washed cells of a marine pseudomonad were suspended in buffered salt solutions containing, in addition to MgSO(4), NaCl and KCl at various concentrations. The cells were centrifuged from the medium and analyzed for Na(+) and K(+). Inulin and C(14)-carboxypolyglucose were employed to estimate the volume of extracellular fluid associated with the packed cells. Intracellular Na(+) and K(+) concentrations were determined by correcting for the amount of Na(+) and K(+) in the extracellular fluid. At all levels of Na(+) in the suspending medium (0 to 1 m), the intracellular and extracellular Na(+) concentrations within the limits of experimental error were the same. The intracellular K(+) concentrations were approximately double the extracellular concentrations at the two levels of K(+) tested (0.01 and 0.15 m) and were not influenced by the amount of Na(+) present. Intracellular and extracellular Cl(-) concentrations were the same at the one level of Cl(-) examined. The intracellular fluid volume varied with the NaCl or KCl concentration of the suspending medium, being greatest in the absence of added salts, decreasing to a minimum at 0.3 m salt, and then increasing slightly at higher salt concentrations. Most of the intracellular Na(+) could be removed by washing with solutions of MgSO(4) or sucrose, but a small amount [10 to 15 mumoles/g (dry weight)] remained bound to the cells.

Suspensions of cells of a marine pseudomonad washed with 0.05 m MgSO(4) showed an immediate increase in optical density (first-phase optical change) when the salt concentration of the suspending medium was increased; a subsequent slow decrease in optical density (second-phase optical change) occurred if K(+) was present. The rate of the second-phase change was similar to the rate of uptake of (42)K(+) by the cells. Glutamate increased the rate and extent of the second-phase change and produced a parallel increase in the rate and extent of uptake of (42)K(+). Citrate increased the extent of the second-phase change in cells adapted to oxidize citrate but not in unadapted cells. Adapted, but not unadapted, cells accumulated (14)C-citrate. The nonmetabolizable alpha-aminoisobutyric acid (AIB) also increased the extent of the second-phase change under conditions leading to the uptake of (14)C-AIB by the cells. Cells maintained in a salt solution optimal for the retention of intracellular solutes were found to contain 0.184 m K(+). In the same salt solution, cells preloaded with (42)K(+) retained the isotope, but they lost it rapidly when suspended in 0.05 m MgSO(4). The second-phase changes can be accounted for by the energy-dependent accumulation in an osmotically active form of K(+) and other metabolites by cells depleted of intracellular solutes.

The addition of 2% Trypticase to a minimal salts-glucose plating medium increased the bacterial count of frozen and thawed suspensions of Escherichia coli 451B cells, even when precautions were taken to remove toxic trace elements from the plating diluent. Hydrolysis of the Trypticase with HCl or H(2)SO(4) reduced its count-increasing activity. Treatment of the H(2)SO(4) hydrolysate with a cation-exchange resin greatly improved its capacity to replace Trypticase. Addition of a mixture of amino acids approximating the composition of casein also increased the plate count when added at a level equivalent to 0.1% casein, but at 2% it depressed the count. Tests of amino acids in the mixture revealed that aspartic acid could replace Trypticase completely as a supplement to the basal medium. When added at a level of 2.5 mm, aspartic acid doubled and occasionally tripled the plate count of a suspension of frozen and thawed cells. Glutamic acid, alanine, and to a lesser extent certain other amino acids also showed a capacity to increase the count. Cysteine was without significant effect. Serine and other amino acids depressed the count. None of the amino acids or other supplements affected the count of suspensions of cells that had not been frozen. The effect of adding aspartic acid, cysteine, or Trypticase to the basal medium on the bacterial count of suspensions of various strains of E. coli, Aerobacter aerogenes, Serratia marcescens, and two species of Pseudomonas after freezing was examined. The response to the supplements was unique for each organism.

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.

Drapeau, Gabriel R., (McGill University, Montreal, Quebec, Canada), Tibor I. Matula, and Robert A. MacLeod. Nutrition and metabolism of marine bacteria. XV. Relation of Na(+)-activated transport to the Na(+) requirement of a marine pseudomonad for growth. J. Bacteriol. 92:63-71. 1966.-A marine pseudomonad was found to require 50 to 100 mm Na(+) for maximal rate of oxidation of d-galactose and for the transport of d-fucose-H(3) into the cells. The same organism required 150 to 200 mm Na(+) for the oxidation of l-alanine and for the transport of phi-aminoisobutyric acid-C(14) (AIB-C(14)) into the cells. Competition studies indicated that d-galactose and d-fucose on the one hand and l-alanine and AIB on the other shared common carriers for transporting the compounds into the cells. This parallelism in Na(+) response for oxidation and transport extended to growth when l-alanine was the sole carbon source in the medium. When d-galactose was the sole carbon source, an amount of Na(+) equal to that with l-alanine was needed. KCN and dinitrophenol but not ouabain inhibited the uptake of AIB-C(14) by the cells. K(+) in addition to Na(+) was required for transport, and both Mg(++) and either Cl(-) or Br(-) were stimulatory. Photobacterium fischeri was also found to require Na(+) specifically for the uptake of AIB-C(14) by the cells.

When cells of a marine pseudomonad were washed and suspended in 0.5 m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 m NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up alpha-aminoisobutyric acid by a Na(+)-dependent process.

When cells of a marine pseudomonad washed free of medium components with 0.05 m MgSO(4) were suspended in solutions containing 200-mm concentrations of various salts, there was an immediate increase in optical density (OD), followed by a slow decrease. The decrease following the initial increase, but not the increase itself, could be prevented by omitting K(+) from or by adding metabolic inhibitors to the suspending solution. With NaCl, the initial increase in OD rose to a maximum as the salt concentration was increased to 200 mm and then declined at 500 mm. There was a corresponding decrease in intracellular fluid volume to a minimum at 200-mm NaCl and then a rise. When the increased OD produced by NaCl was maintained, the internal Na(+) and Cl(-) could be shown to have reached essentially the same concentration in the cells as in the medium. Thus, the OD changes could not have been due to osmotic effects. No evidence was obtained of a salt-induced aggregation of nuclear material. The OD of suspensions of isolated cell envelopes increased in response to increases in NaCl concentration in the absence but not in the presence of 0.05 m MgSO(4). The data was interpreted to indicate that the salt-induced increases in OD occurring in suspensions of the cells resulted from an interaction of salts with components of the cell envelope, causing contraction of the envelopes and shrinkage of the cells.