Nutrition and Metabolism of Marine Bacteria1

JOURNAL OF BACTERIOLOGY, July, 1966 Copyright 1966 American Society for Microbiology Vol. 92, No. I Printed in U.S.A. Nutrition and Metabolism of Marine Bacteria1 XV. Relation of Na+-Activated Transport to the Na+ Requirement of a Marine Pseudomonad for Growth GABRIEL R. DRAPEAU,2 TIBOR I. MATULA, AND ROBERT A. MAcLEOD Department of Microbiology, Macdonald College of McGill University, and Marine Sciences Center, McGill University, Montreal, Quebec, Canada Received for publication 16 April 1966 ABSTRACT 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-H3 into the cells. The same organism required 150 to 200 mm Na+ for the oxidation of L-alanine and for the transport of 4-aminoisobutyric acid-c'4 (AIB-C14) 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 whenl-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-C14 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-C14 by the cells. All marine bacteria investigated in detail have been found to have a highly specific requirement for Na+ for growth. In this respect, they differ from most terrestrial species examined [see MacLeod (8) for a review]. Previous efforts to determine the function of Na+ in the metabolism of a marine pseudomonad revealed that Na+ was required for the oxidation of exogenous substrates by resting-cell suspensions of the organism, but not by any of the enzymes tested in extracts derived from the cells (9, 10). Similarly, Pratt and Happold (14) observed a requirement for Na+ for indole production from tryptophan by whole cells but not by cell extracts of a marine Vibrio. Drapeau and MacLeod (4), using the nonmetabolizable substrates a-aminoisobutyric acid (AIB) and D-fucose to separate transport processes from metabolism, showed that there is a specific requirement for Na+ for the uptake of 1 Issued as Macdonald College Journal Series No. 543. 2Present address: Department of Biological Sciences, Stanford University, Stanford, Calif. these compounds by resting-cell suspensions of a marine pseudomonad. Since the quantitative requirement for Na+ for transport of the AIB was significantly greater than the Nat requirement for oxidation of the oxidizable substrate present, or for stimulation of the endogenous metabolism of the cells, it was evident that there was a role for Na+ in the uptake of the compound which was separate from any other possible role for Nat in the oxidative metabolism of the cells. In this investigation the relation of Na+-dependent transport to the Na+ requirements of the organism for oxidation and growth have been investigated. Some properties of a Nat-dependent amino acid and a sugar transport system in the marine pseudomonad have been documented, together with evidence for a Na+-dependent transport system, in the marine luminous bacterium Photobacterium fischeri. MATERIALS AND METHODS Cultures. The organism referred to as B-16 has been classified as a Pseudomonas species type IV and is maintained as culture NCMB 19 at the Torry Research Station, Aberdeen, Scotland. Studies on the 63

64 DRAPEAU, MATULA, AND MACLEOD J. BACTERIOL. nutrition and metabolism of this organism have been reported in some detail in previous communications. The culture of Photobacteriumfischeri used was kindly made available to us by W. D. McElroy of John Hopkins University. Media. The cultures of the two marine bacteria were maintained by monthly transfer on slants of a medium containing 1% Trypticase (BBL) and 1.5% agar in a salt solution consisting of 0.22 M NaCl, 0.026 M MgC2, 0.01 M KCl, and 0.1 mm FeSO4(NH4)2- SO4. Cells for study of the transport of AIB were grown in a liquid medium containing the same constituents with the agar omitted. For the study of D- fucose transport, 0.5% galactose was added to the liquid medium. The medium used to determine the Na+ requirement of marine pseudomonad B-16 for growth contained: 0.05 M MgSO4, 0.01 M KCI, 0.1 M tris(hydroxymethyl)- aminomethane (Tris) chloride buffer containing 2 mm H3PO4 (ph 7.2), 0.1% (NH4)2SO4, and either 1% D-galactose or 1% L-alanine. The methods used to determine the growth response to Na+ have been described (11, 12). Preparation of inocula. In the study of the Na+ requirement for growth, the inocula used were prepared in two ways. Inoculum A was obtained by transferring cells from a stock agar slant to Trypticase medium to which 1% L-alanine and 1% D-galactose had been added. After incubation of the medium for 16 to 18 hr at 25 C on a rotary shaker, the cells were washed three times by resuspension in and centrifugation from volumes of 0.05 M MgSO4 equal to those of the growth medium. One drop of the final suspension was added to each assay tube. Inoculum B was prepared by first transferring cells from the stock agar slant to slants of the chemically defined medium prepared with either D-galactose or L-alanine and solidified with 1.5% agar. When growth became visible on the slants (after 4 to 5 days), a further transfer was made to the corresponding chemically defined liquid medium. The resulting cells, appropriately adapted to growth on either D-galactose or L-alanine, were washed with 0.05 M MgSO4 for use as inocula. Preparation of cell suspensions. Cells of marine pseudomonad B-16 were harvested after 10 to 12 hr of incubation in the growth medium, and P. fischeri after 16 to 18 hr, by centrifugation at 12,000 X g at 4 C. Cells of organism B-16 were washed three times by resuspension in and centrifugation from volumes of 0.05 M MgSO4 equal to those of the growth medium. For the study of D-fucose-H3 uptake, the cells were washed four times. P. fischeri was washed with a solution containing 0.05 M MgSO4 and 0.2 M KCl, since cells washed in 0.05 M MgSO4 alone had little capacity to take up the substrate. The dry weight of cells in the washed suspensions was determined turbidimetrically by reference to a previously calibrated curve. Measurement of amino acid transport. Cells of the two organisms were suspended at a level of 200,ug/ml in a medium containing, unless otherwise indicated, 0.01 M KCI, 0.026 M MgSO4, 0.1 M Tris phosphate buffer (ph 7.2), 100,g/ml of chloramphenicol, 5 X 10-5 M (0.22,c/,umole) a-aminoisobutyric acid-i-c'4 (AIB-C14), and various concentrations of NaCl. The suspensions were contained in Erlenmeyer flasks incubated in a New Brunswick rotary water-bath shaker. All of the components except the AIB-C'4 were mixed and incubated at 25 C for 5 min before the labeled compound was added. At appropriate intervals after the addition of the AIB-C'4, 1.0-ml samples of the suspensions were filtered through Millipore HA filters. The cells on the filter were washed three times with 1-ml portions of the salt solution used in the growth medium. Measurement of sugar transport. Owing to the very rapid uptake of D-fucose-H' by the organism, the procedure for measuring AIB-C'4 transport was modified as follows. The cells were incubated in an Erlenmeyer flask for 5 min in the same salt solution used for AIB-C'4 uptake. A 0.95-ml portion of the suspension was then pipetted onto a Millipore filter already in place in a filter funnel, and 0.05 ml of a solution of D-fucose-H3 (0.083,umole, 0.112,uc) was added by use of a syringe. At the end of a period of incubation (usually 2 min), suction was applied to the filter apparatus. The cells were washed three times with a salt solution in which NaCl was replaced with LiCl. Measurement ofradioactivity. For C'4 measurement, the filters and their adhering cells were transferred to 20-ml screw-cap vials, dried slowly under an infrared lamp, and 5 ml of scintillation phosphor solution was added. For tritium measurement, the filters and their adhering cells were transferred to vials, and 0.1 ml of 5% trichloroacetic acid was added to disrupt the cells, followed by 2.5 ml of absolute ethyl alcohol and 10 ml of scintillation phosphor solution. The phosphor solution contained 5 g of 2,5-diphenyloxazole and 0.3 g ot 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene per liter of toluene solution. All radioactivity measurements were made in a Packard Tricarb liquid scintillation spectrometer. Manometric measurements. The usual techniques of Warburg manometry were used in measuring oxygen uptake by resting-cell suspensions. The suspending medium in the Warburg flasks contained 0.01 M KCI, 0.026 M MgSO4, 0.1 M Tris phosphate buffer (ph 7.2), various concentrations of NaCl, either 0.15 M D-galactose or 0.03 M L-alanine, and cells equivalent to 5 mg (dry weight), in a final volume of 3.0 ml. The gas phase was air, and the temperature of incubation was 25 C. RESULTS Requirement for Na+ for oxidation. Previous studies with this organism have shown that the quantitative requirements for Na+ for oxidation of substrates by the cells are not fixed, but vary with the substrate being oxidized. Monobasic acids required 50 to 100 mm Na+ for maximal rate of oxidation, whereas di- and tribasic acids required from 150 to 250 mm Na+, depending on the compound being oxidized (9). Similarly, when the quantitative requirements for Na+ for oxidation of D-galactose and L-alanine by cell

VOL. 92, 1966 Na+ REQUIREMENT AND TRANSPORT 65 suspensions of the marine pseudomonad were compared, it was found that both the Nat optima for maximal rate of oxidation of the two substrates and the shape of the Na+ response curves were different (Fig. 1). This confirmed previous findings with this organism (9, 19), and showed that galactose, like the monobasic acids, had a lower requirement for Na+ for oxidation than did alanine, which behaved like the di- and tribasic acids. Requirement for Na+ for transport. As reported previously, there is a requirement for Na+ for the uptake of AIB and D-fucose by resting-cell suspensions of marine pseudomonad B-16 (4). A comparison of the quantitative requirements for the uptake of these two compounds by cell suspensions of the organism is shown in Fig. 2. It is noteworthy that the requirement for Na+ for maximal rate of uptake of D-fuCose and the shape of the Na+ response curve were both similar to the corresponding parameters for galactose oxidation as seen in Fig. 1. In the same way, the Na+ requirements for uptake of AIB and for the oxidation of L-alanine were quantitatively similar. Specificity of the requirement for Na+. The previous report showed that NaCi could not be replaced by any one of a number of other salts or sucrose in the uptake of AIB-C14 by the cells. The possibility always exists that the failure of the other compounds to permit permease action was due to their own inhibitory effects. That this was not the case in this instance is evident from the results in Table 1, which show that in all. 160 140-120 w 100 IC 80 60 40 20 11 IO CURVE 1. D-GALAC 0 50 100 150 200 No (mm) as NoCI 2 400o / / z Z 2 z w/4 a. FIG. 2. Na+ requirement focurve t. D-FUCOSE-H CURVE 2. Ari-Cm4 FIG. 1. Na+ requirement for the oxida galactose and L-alanine by resting-cell sus pensions Dsuccinate and L-alanine at of a rapid rate. Similarly, marine pseudomonad B-16. Both curves riprnesents O the oxidation of D-fucose did not occur under uptake in the presence ofthe substrate, with endogenous conditions permitting D-galactose oxidation. oxygen uptake subtracted after 60 min of incubation. The possibility that the compounds were Na+ added as NaCl. metabolized in other ways than by oxidation was I- O0200 2-3 0 50 00o 150 200 250 300 Na' (mm) as NoCi FIG. 2. Na+ requirement for the transport Of D- fucose-h2l and AIB-C'4 into cells ofmarine pseudomonad B-16, as determined by measuring the uptake of radioactivity by the cells after incubation for 2 min in the presence of D-fucose-H3 and 70 min with AIB-CI4. TABLE 1. Effect of various salts on the uptake of AIB-C04 by cells of marine pseudomonad B-16 in the presence and absence of NaCI Compound added* Radioactivity (count/min) in the cells No additional NaCl In the presence of 0.2 ms NaCl None... 68 6,358 NaC1... 6,)358 7,540 RbCl... 4 6,102 NH4C1... 0 7,510 LiCl.... 6 4,242 KCl... 3 7,084 Sucrose... 18 1,544 * Each was tested at a level of 0.2 M, except sucrose which was added at 0.4 M. cases uptake of AIB-C14 occurred in the presence of all of the compounds when NaCl was added to the incubation medium, though some suppression of permease activity was apparent in the presence of sucrose. Assuming no inhibitory effects of ;TOSE the other compounds tested, the results in Table 2 CURVE 2. L-ALANIINE show that Na+ salts were required specifically for the uptake of D-fucose-IP by the cells. Identity of the accumulated material. Respirometer studies revealed that marine bacterium B-16 250 300 could not metabolize AIB with the uptake of ztion ofdoxygen under conditions enabling it to oxidize I

66 DRAPEAU, MATULA, AND MACLEOD J. BACTERIOL. TABLE 2. Effect of various salts anid sucrose on the uptake of D-fucose-H3 by cells of marine pseudomonad B-16 Salt added* Radioactivity in the cells cozntl/nin None... 95 NaNO3... 1,486 Na2SO4... 1,308 NaCl... 1,200 RbCl... 138 NH4Cl.72 LiCl... 111 KCl... 156 Sucrose... 44 * At a level of 125 mm, except for Na2SO4 which was added at 62.5 mm. tested as follows. The cells were allowed to accumulate the radioactive compounds; they were then suspended in a small volume of distilled water and heated in a boiling-water bath. Portions of the boiled suspensions, including all insoluble components, were placed on paper for chromatography. In the case of each compound, only one radioactive spot was revealed which corresponded in RF to authentic samples of the radioactive compounds run separately. No trace of radioactivity was detected in the insoluble material remaining at the origin. For AIB-C'4 detection, the two solvent systems used were n-butanolacetic acid-water (50:25:25) and water-saturated phenol. For D-fucose-H3, n-butanol-pyridinewater (6:4:3) and water-saturated phenol were employed. Evidence for the identity ofthe AIB and L-alanine transport systems. The effect of nonradioactive amino acids on AIB-C14 uptake was tested to determine the specificity of the transport system. The results (Table 3) show that, of 12 amino acids and glycinamide tested, 7 inhibited the uptake completely whereas 6 had at most only a small inhibitory effect. Both L-alanine and AIB prevented the uptake of the radioactive AIB; such a result is indicative of competition for a common carrier, though kinetic studies would be required to establish this point beyond question [see Brock and Moo-Penn (1)]. Evidence for the identity of the D-fucose and D-galactose transport systems. More direct evidence was obtained of a common carrier for D-fucose and D-galactose. Cells of this organism require preliminary adaptation to oxidize galactose. If they are grown in a medium containing galactose, they oxidize the compound, but if they are grown in the absence of galactose they do not. The results in Fig. 3 show that significant amounts TABLE 3. Effect ofnonradioactive amino acids on the uptake of AIB-C14 by cells of marine pseudomonad B-16 Amino acid added Inhibition of uptake L-Alanine. 99 D-Alanine... 97 AIB (non-radioactive)... 98 Glycine... 97 L-Serine... 99 DL-Cysteine... 99 DL-Threonine... 98 DL-Valine. 23 L-Leucine... 33 L-Aspartic acid... 17 DL-Phenylalanine... 20 L-Proline... 32 Glycinamide. 8 * At 100 times the concentration of AIB-C14. I z 5 1200 cr w o- 800 r z 0 4001.I- - 1600 k D-FUCOSE UPTAKE BY 1. GALACTOSE ADAPTED CELLS o 2. UNADAPTED CELLS 0 0 2 4 6 TIME (MINUTES) FIG. 3. Effect of adapting cells to the oxidation oj galactose on the capacity of the cells to become radioactive wheni incubated with D-fucose-H3. Cells were adapted to galactose oxidation by growth in the Trypticase medium to which 0.5%0 D-galactose was added. NaCl concentration, 125 mai. of radioactivity were taken up by cells incubated with D-fucose only if the cells had been grown in a medium which had contained D-galactose. It would thus appear that adaptation of this organism to the metabolism of galactose involves the induction of a permease which can transport both D-galactose and D-fucose into the cells. Further evidence that D-galactose and D-fucose share a common carrier was obtained from competition experiments (Table 4). The results show that D-fucose, D-glucose, D-galactose, and a- 0 x x AX~~~Ix 8 10

VOL. 92, 1966 Na+ REQUIREMENT AND TRANSPORT 67 methyl glucoside all prevented the uptake of D-fucose-H3 by the cells. L-Fucose and the other compounds tested had either much less or no effect. Requirement for Na+ for growth. If the Na+ requirement of this organism for growth represents a requirement for Na+ for transport, and if transport of the oxidizable substrate is the rate-limiting step in growth, the optimal Nat requirement for growth should depend on the carbon and energy source in the medium and its requirement for Na+ for transport. To determine whether this was so, the Na+ requirement for growth was determined with either D-galactose or L-alanine as the sole source of carbon and energy in the medium. Those with inoculum A show that the Na+ requirement for growth with L-alanine as substrate was indeed similar to the Na+ requirement for the oxidation of L-alanine and for the transport of AIB (Fig. 4). The results with D-galactose, however, show that the Na+ requirement for growth with this compound was higher than for its oxidation or for the transport of D-fucose, and was essentially identical to the Nat requirement for growth with L-alanine as substrate. When an inoculum (B) was used which was specially adapted to more rapid growth in the chemically defined media, somewhat different results were obtained with D-galactose (Fig. 4). Here the growth response to Na+ with D-galactose as substrate shows less of a lag at low Na+ concentrations than occurred with inoculum A. This led to an initial conclusion that there was a close similarity between the Na+ requirements for growth, oxidation, and transport in the case of D-galactose, as there more clearly was in the case of L-alanine. Effect of inhibitors on Na+-dependent transport. When various metabolic inhibitors were tested, dinitrophenol and KCN were found to prevent TABLE 4. Effect of nonradioactive sugars and sugar derivatives on the uptake ofd-fucose-h3 by cells of marine pseudomonad B-16 Compound added* Inhibition of uptake D-Fucose... 88 D-Glucose... 90 D-Galactose... 86 a-methyl glucoside... 94 3-0-methylglucose... 33 L-Fucose... 0.6 D-Lyxose... 0.8 D-Mannose...... 1.0 Maltose.. 0.8 * At 100 times the concentration of D-fucose-H3. Incubation time: 4 min. The cells used were grown on a medium containing D-galactose. the uptake of AIB-C14 by the cells (Table 5), providing further support to a previous conclusion (4) that the uptake of this compound is energydependent. The two mercaptide forming agents tested, p-chloromercuribenzoic acid (PCMB) and iodoacetate had opposite effects on AIB-C14 accumulation. PCMB prevented uptake completely, whereas iodoacetate stimulated it. The latter effect has been observed in other transport systems (7), and has been ascribed to a preferential effect of certain inhibitors on the exit reaction. Ouabain, an inhibitor of Na+-dependent transport in animal cells, had a slight stimulatory effect in the bacterial system. Effect of other ions on transport. When other ions than Na+ were removed from the suspending medium, a requirement for K+ for the transport of AIB-C'4 was detected (Fig. 5). In this experiment, the Tris buffer concentration was reduced from 0.1 to 0.01 M to lower the residual uptake of AIB-C'4 which occurred in the absence of added K+ Ḋeletion of Mg++ lowered the rate of uptake of AIB-C'4, though an absolute requirement for this ion could not be demonstrated. Cells for this study were washed with 0.5 M NaCl. The extent of the Mg++ effect varied considerably from one experiment to another. Previous experiments have shown that the requirement for Na+ for transport could be satisfied by any one of a number of Na+ salts. Since C1- was supplied by other salts in the incubation medium, these results threw no light on the requirement for Cl- for transport. Although halide ion is not required for growth by marine pseudomonad B-16, both Cl- and Brstrongly stimulated early growth of the organism (12). Since the amount of halide required for maximal stimulation was similar to the optimal Na+ requirement for growth, it was suggested that the function of the two ions might be closely related in the metabolism of the organism. It was, therefore, of considerable interest to know whether halide ions had any effect on Na+dependent transport. When all the chloride salts in the incubation medium were replaced by sulfates, the rate of incorporation of AIB-C'4 into the cells was reduced as compared with the same medium with chloride ion added (Table 6). Part A of Table 6 shows that the rate of AIB-C14 uptake increased as the Cl- concentration was increased to 200 mm. In this experiment, the Na+ concentration was maintained constant at 200 mm by the addition of appropriate amounts of Na2SO4. That the response obtained was due to stimulation by chloride rather than to inhibition by sulfate is evident from the results in part

68 DRAPEAU, MATULA, AND MACLEOD J. BACTERIOL. Z 40 I W 50-0-~~~~~~~~~~~ 0~~~~~~~~~~~ 70 0 z < 80 /.90 100 907 -I 50 100 150 200 250 50 too ISO 200 250 Na* (mm) as NaCI FIG. 4. Effect of carbon source and method of preparing the inoculum on the Na+ requirement of marine pseudomonad B-16 for growth in a chemically defined medium. Curve 1, D-galactose as sole carbon source; curve 2, L-alanine. B. When Na2SO4 at a concentration sufficient to provide 200 mm Na+ was combined with 0.2 M NaCl, the rate of uptake of AIB-C14 was not appreciably less than that which occurred in 0.4 M NaCI. The results also show that Br- was appreciably more active than Cl- in stimulating transport, though for growth the two ions were essentially interchangeable. Iodide was only slightly inhibitory for transport, though it was quite toxic for growth. Na+-dependent transport in P. fischeri. The marine luminous bacterium P. fischeri was found also to require Na+ specifically for the uptake of AIB-C14 by the cells. The optimal level of Na+ for maximal rate of uptake of the compound was somewhat lower than the requirement of marine pseudomonad B-16 for this purpose (Fig. 6). Competition studies showed that, as in the case of marine pseudomonad B-16, L-alanine prevented the uptake of AIB-C14 by the cells. DIscussIoN The results reported here provide strong support for the conclusion that in marine pseudomonad B-16 the Nat requirement for oxidation of substrates reflects a need for Na+ for the TABLE 5. Effect of inhibitors on the uptake of AIB-C14 by cells of a marine pseudomonad Inhibitor Concn Radioactivity of cells M count/min None... 3,300 KCN... 10-2 355 NaN3. 102 2,379 Dinitrophenol... 5 X 10-4 704 Iodoacetate. 3 X 10-3 5,300 p-chloromercuribenzoate.. 10-3 163 Ouabain... 103 3,612 transport of the substrates into the cells from the medium. This conclusion is based on the similarity between the Na+ requirement for oxidation of substrates and for the transport of their corresponding nonmetabolizable analogues in the case of two substrates having quite different quantitative requirements for Na+ for metabolism. It is further supported by the evidence that each substrate shares the same permease pathway with its corresponding analogue. It would therefore appear that the differences in Na+ require-

VOL. 92, 1966 Na+ REQUIREMENT AND TRANSPORT 69 TIME (MINUTES) FIG. 5. Evidence for the requirement for both Na+ and K+ for the uptake of AIB-C'4 by cells of marine pseudomonad B-16. TABLE 6. Effect of halide ions on the rate of transport of AIB-C14 into cells of marine pseudomonad B-16 Treatmenta Incubation timeb 20 min 40 min 60 min 120 min Part A NaC1 0 mm... 2,281 3,101 3,428 3,431 50 mm... 3,183 4,278 4,771 4,587 100 mm... 3,778 4,951 4,950 5,214 150 mm... 3,993 5,557 6,314 5,881 200 mm... 4,020 4,896 5,888 5,427 Part B NaC1, 0.4 M..3,880 5,571 6,360 6,831 Na2SO4, 0.1 M..2,281 3,101 3,428 3,431 NaSO4, 0.1 M + NaCl, 0.2 M.. 3,634 5,005 5,794 6,160 Na2SO4, 0.1 M + NaBr, 0.2 M..4,257 5,964 6,644 7,203 Na2SO4, 0.1 M + NaI, 0.2 M..1699 3,061 3,991 5,160 a In part A, the Na+ concentration was maintained constant at 200 mm by the addition of appropriate amounts of Na2SO4 - b Results expressed as counts per minute. ment for oxidation by cells of this organism, depending on the substrate being oxidized (9), are due to differences in the requirement for Na+ for the transport of the different substrates into the cells from the medium. The parallelism in the Na+ requirement for transport and oxidation extends to growth when l00 150 2c Na (mm) as NaoI FIG. 6. Quantitative requirement of Photobacterium fischeri for Na+ for the uptake ofaib-c14 by the cells. Radioactivity ofcells measured after 60 min of incubation in the presence ofaib-cq. L-alanine but not when D-galactose is the substrate. Less Na+ is needed for the oxidation of D-galactose and for the transport of D-fucose than is needed for growth when D-galactose serves as sole carbon source in the medium. The extra Na+ required for growth must be serving in some other capacity than transport. Rhodes and Payne (15) suggested that there is a specific requirement for Na+ for the induction of permeases in the marine bacterium Pseudomonas natriegens. They observed, however, that permease induction occurred in the presence of sufficient K+ if Mg++ was omitted from the medium. In marine pseudomonad B-16, the amount of Na+ required for growth with D-galactose as substrate could be reduced to the level required for transport if 0.2 M K+ was added to the medium (MacLeod, Matula, and Wong, unpublished data). Thus, the need for extra salt for growth with D-galactose over the specific requirement for Na+ for transport of this substrate would seem to reflect an additional requirement for a medium of appropriate ionic strength, and not an additional specific requirement for Na+. Earlier studies indicated a stimulation of endogenous metabolism by low concentrations of Na+ (19). It is necessary to consider whether this stimulation indicates one or more other functions of Na+ in these cells. Recent observations suggest a possible explanation for this phenomenon. In the absence of Na+, intracellular solutes are lost from cells of this organism and can be transported back in again when the Na+ concentration is restored (5). Such a loss and restoration of metabolizable intracellular solutes could account

70 DRAPEAU, MATULA, AND MACLEOD J. BACTERIOL. for the apparent stimulation of the endogenous metabolism by Nat. Evidence for a different mechanism of Na+ stimulation of endogenous metabolism has been obtained in the case of the moderate halophile Micrococcus halodenitrificans. Sierra and Gibbons (17) showed that cells of this organism oxidize poly-f-hydroxybutyric acid endogenously, and that the depolymerase necessary for the first step in the metabolism of this compound requires Na+ or Li+ for activity. There are small quantitative differences in the Na+ requirements for optimal rate of oxidation of the substrates and for the transport of the corresponding analogues. These differences may arise from the manner in which Na+ functions in the transport process. Schultz and Zalusky (16) found that the Na+-dependent transport of sugars and amino acids in the isolated rabbit ileum not only requires Na+ but also brings about an increased rate of transmural Na+ transport. They suggest that the role of Na+ is not simply that of an activator of these transport processes, but instead that the active transport of both sugars and amino acids is mediated by a ternary sodium-sugar or amino acid-carrier complex. The formation of such complexes could account for the differences in Na+ requirement for the transport of different substrates reported here. Since the amount of Na+ required for the formation of the ternary complex would vary with the molecular constitution both of the substrate being transported and of the carrier used for transport, considerable differences in the Na+ requirement for transport could be expected from one carrier system to another. Even with compounds using the same carrier, however, small differences in the sodium requirement for complex formation could be anticipated, depending on the molecular constitution of the compound being transported. Three other ions, K+, Mg++, and halide, have been shown to affect transport into cells of this marine bacterium. Removal of K+ but not Mg++ from the incubation medium reduced the rate of amino acid transport into rat kidney cortex slices (6). K+ has also been shown to be required for transport in another marine pseudomonad (Payne and Rhodes, personal communication). How directly K+ and Mg++ may be involved in the transport processes has not been established in any of these systems. Since concentration data and inhibitor studies indicate that the uptake of the compound by these systems is energy-dependent, K+ and Mg++ may be nonspecifically involved in transport through their known influence on the energy-generating mechanisms in the cell. This possibility seems particularly likely in the case of K+, since separate experiments have shown (Srivastava and MacLeod, unpublished data) that K+, like other solutes (5), is lost from the cells of marine pseudomonad B-16 when this organism is washed with 0.05 M MgSO4 solution. That K+ probably plays a less direct role than Na+ in transport is indicated also by the observation that the requirement of cells of this organism for K+ for oxidation, unlike that for Na+, does not vary with the substrate being oxidized (9). Also, until quantitative data on the requirement for chloride or bromide for the transport of different substrates are available, it is not possible to come to any conclusions regarding the site of action of the halide ions. The lack of evidence for the direct participation of K+, Mg++, and halide ions in transport is in contrast to the situation with Nat. In the case of Na+, the quantitative differences in the requirements for Na+ depending on the substrate being transported point to a direct and specific role for Na+ in the transport process. There is a considerable body of evidence to suggest that Na+-dependent transport in animal cells requires the maintenance of a Na+ gradient between the inside and the outside of the cells (2). In marine pseudomonad B-16, no such gradient appears to exist. Direct analysis has shown that intracellular Na+ rapidly assumes the concentration prevailing in the medium (18). Furthermore, no evidence has been found for a Na+, K+ activated ATPase (4), an enzyme which in animal cells is believed to be responsible for maintaining the Nat gradient (13). There would thus seem to be a fundamental difference in the mechanism of Na+-dependent transport in animal cells and in this marine pseudomonad. ACKNOWLEDGMENTS We are indebted to R. K. Crane, Chicago Medical School, for generously making available the D-fucose-H3 used in this study. We thank W. J. Payne and M. E. Rhodes for a preprint of their manuscript. This investigation was supported by a grant from the National Research Council of Canada. LITERATURE CITED 1. BROCK, T. D., AND G. MOO-PENN. 1962. An amino acid transport system in Streptococcus faecium. Arch. Biochem. Biophys. 98:138-190. 2. CRANE, R. K. 1965. Na+-dependent transport in the intestine and other animal tissues. Federation Proc. 24:1000-1006. 3. DRAPEAU, G. R., AND R. A. MACLEOD. 1963. Nutrition and metabolism of marine bacteria. XII. Ion activation of adenosine triphosphatase in membranes of marine bacterial cells. J. Bacteriol. 85:1413-1419. 4. DRAPEAU, G. R., AND R. A. MACLEOD. 1963. Na+ dependent active transport of a-amino-

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