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JOURNAL OF IACTEIOLOGY, Dec. 1975, p. 1177-1190 Copyright 0 1975 American Society for Microbiology

Vol. 124, No. 3 Printed in U.S.A.

Characterization of Neutral Amino Acid Transport in a Marine Pseudomonad JARED E. FEIN' AND ROBERT A. MAcLEOD* Department of Microbiology, Macdonald Campus, McGill University and Marine Sciences Centre, McGill University, Montreal, Quebec, Canada Received for publication 3 July 1975

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and a-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (IXV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, i-valine, and L-alanine. This system exhibited low affinity for a-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified a-amino or a-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine,

K, values of 4.6 x 10- 6 and 1.9 x 10-4 M and Vmax

values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems, respectively. a-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and K, values of 2.8 x 10-' and 1.4 x 10-' M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.

In recent years, a considerable body of information has been accumulated on the nature of amino acid transport systems in a wide variety of cells and tissues. In general, the systems found in bacteria tend to exhibit high substrate specificities and, consequently, serve the uptakes of only single or closely related amino acids (for recent reviews, see references 14 and 28). In a few cases, however, bacterial transport systems capable of recognizing a wider range of amino acids have been reported (2, 17, 33). Eukaryotic amino acid transport systems, on the other hand, tend to be less specific than those of bacteria, although systems having narrow substrate specificities have been described (16, 39). In both groups of organisms, the transport of amino acids by multiple transport systems with overlapping affinities has been well documented (12, 14, 16, 28, 34). Various approaches to the problem of distinguishing the

different amino acid transport systems of a cell have been suggested (4, 29). The transport systems mediating amino acid uptake in marine bacteria have not been identified previously. Unlike most prokaryotic transport systems, those of marine bacteria are distinguished by their requirements for Na+ for activity (10, 25). Recent communications (15, 37, 38, 42, 43) from this laboratory have considered the roles of inorganic cations and the mechanism of energy coupling in the active transport of neutral amino acids in the marine pseudomonad B-16. In line with these studies, this investigation into the types of amino acid transport systems present in marine pseudomonad B-16 was initiated. Two systems that mediate the uptake of neutral amino acids in this marine bacterium were distinguished and are described in this report. (These studies were submitted by J.E.F. in partial fulfilment of the requirements for the ' Present address: Division of Microbiology, National Insti- Ph.D. degree at McGill University, Quebec, tute for Medical Research, Mill Hill, London NW7 1AA, Canada, 1974. Part of this work was presented at the 23rd Annual Meeting of the Canadian England. 1177

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FEIN AND MACLEOD

Society of Microbiologists, Edmonton, Alberta, 19 to 22 June 1973.) MATERIALS AND METHODS Organism, media and growth conditions. The organism referred to as marine pseudomonad B-16 (ATCC 19855, NCMB 19) has been classified by the Torry Research Group, Aberdeen, Scotland, as a Pseudomonas sp. type IV and, more recently, as Alteromonas haloplanktis by Reichelt and Baumann (31). Eight morphological variants of this organism have been distinguished (11). The wild-type strain used in this investigation has been designated variant 3. Fresh cultures were maintained as described elsewhere (11). Cells for transport studies were grown in a complex liquid medium (11). Succinate minimal medium (J. A. Gow and R. A. MacLeod, manuscript in preparation) was used during the isolation of mutants.

Isolation of mutants defective in AIB transport activity. The rationale behind the use of D-serine for the selection of amino acid transport-negative mutants has been described (35). Wild-type cells grown in succinate minimal medium were treated with N-methyl-N-nitro-N'-nitrosoguanidine as described elsewhere (Gow and MacLeod, in preparation). After mutagenesis, the cells were plated on solid succinate minimal medium containing 0.05 M DL-serine. After 4 days of incubation at 25 C, D-serine-resistant strains were selected, purified twice, and screened for the capacity to transport '4C-labeled a-aminoisobutyric acid (AIB) by using the standard technique described below. The two AIB transport-deficient mutants isolated by this procedure were designated DAG 157 and DAG 163. Forty-three additional AIB transport-negative mutants were subsequently isolated by a modified version of this technique. Exponential-phase cells of the wild-type strain grown in a complex liquid medium were harvested by centrifugation, washed free from the medium by use of a salts solution consisting of 0.3 M NaCl, 0.01 M KCl, and 0.05 M MgSO4 (complete salts solution), and plated on solid succinate minimal medium supplemented with 0.07 M DL-serine. The cells were then immediately exposed to ultraviolet light for 15 to 20 s (Havonia ultraviolet lamp type 16200, Havonia Chemical and Manufacturing Co., Newark, N.J.). After 5 days of incubation at 25 C, D-serine-resistant strains were transferred to a complex solid medium containing "4C-labeled AIB (12.5 ,uM, 0.2 pCi/jimol) and screened for their capacity to transport "4C-labeled AIB, by the autoradiographic technique of Zwaig and Lin (46). Isolation of leucine transport-negative mutants. Cells of variant 3 (wild type) grown to stationary phase in succinate minimal medium were harvested by centrifugation and suspended in fresh medium to their original concentration. A 20-ml portion of culture was irradiated with ultraviolet light in an open petri dish until 99.9% of the population was nonviable (29-s exposure; Havonia ultraviolet lamp type 16200). A portion of the irradiated culture was then incubated for 6 h at 25 C on a rotary shaker. The

J. BACTERIOL. selection of transport-negative mutants was based on the tritium suicide method (23, 24). L-[3H]leucine (5 Ci/mmol; 0.5 mCi/ml of culture) was added to 2 ml of culture, and incubation was continued for 90 min. The cells were washed twice in complete salts solution, suspended in the complex liquid medium and, after 38 h of storage at 4 C, plated onto the complex solid medium and incubated at 25 C for 2 days. Survivors of the suicide selection procedure were replica plated (21) onto a similar medium containing L-[14C]leucine (12.5 qM; 0.2 HCi/glmol) and screened via the autoradiographic technique of Zwaig and Lin (46) for their capacity to transport the labeled amino acid. Possible transporti-negative mutants were isolated, purified twice, and screened for L-["4C]leucine transport capacity by the standard membrane (Millipore Corp., Bedford, Mass.) filtration technique. Transport studies using the Millipore membrane filtration technique. Midlogarithmic-phase cells were grown and prepared for transport studies as previously described (11). Unless otherwise indicated, cells were added at a final concentration of 100 Ag (dry weight) per ml to a reaction mixture consisting of buffered complete salts solution [0.3 M NaCl, 0.01 M KCl, 0.05 M MgSO4, and 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2) containing 1 mM phosphate added as H3PO4] and an isotopically labeled amino acid (either '4C or 3H) as indicated in the text. Chloramphenicol (100 og/ml) was also added where indicated. Incubations were carried out at 25 C in 50-ml Erlenmeyer flasks with mechanical agitation. At desired intervals after the addition of cells to a reaction mixture, a 0.5-ml aliquot was withdrawn, filtered on a 0.45-jm HA membrane filter (Millipore Corp.), and washed with 5.0 ml of complete salts solution (20 C). The membrane filters plus adhering cells were placed in glass scintillation vials and dried. A 5-ml portion of scintillation fluid containing 5.0 g of 2,5-diphenyloxazole per liter of toluene was added to each vial. The radioactivity remaining on each filter was determined by using a Nuclear-Chicago Isocap/300 liquid scintillation spectrometer. Initial rates of uptake were determined from the linear portion of curves obtained by plotting uptake versus time at 30-s intervals for 2 to 3 min. Approximations of this rate could be obtained by measuring uptakes after the first minute of incubation and correcting for background. When approximating the initial rate, reactions were carried out in serological tubes containing 0.6 ml of suspension, under conditions as described above. A comparison of uptake by wild-type and mutant strains was made on a dry weight basis. To obtain equal dry weights of cells, a rough approximation of the dry weights was obtained from a curve relating optical density of the suspension to dry weight. Since this did not give a sufficiently accurate estimate of dry weight for subsequent calculations, a viable count was also made on the suspension and, unless otherwise indicated, corrections for the dry weight were made on the basis of the fact that 1 mg of cell dry weight was found to correspond to approximately 4 x

VOL. 124, 1975

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

10' colony-forming units when suspensions were plated on complex solid medium and incubated at 25 C for 36 h. The average value for the intracellular volume of these cells was 1.6 al/mg of cell dry weight (42).

Chemicals. 1- 4C-labeled AIB, L- [3- 3H lalanine, D[1- 14C lalanine, L- [4,5-_H ]leucine, [carboxyl[1-_ 4C alanine, L- [4,5-'H Ileucine, and most of the other "4C-labeled amino acids (uniformly labeled) were purchased from New England Nuclear Corp., Boston, Mass. D- [3- 4C ]serine was purchased from Amersham/Searle, Arlington Heights, Ill. Unless otherwise specified, "4C-labeled amino acids were diluted with nonradioactive amino acids to final specific activities of 0.22 MCi/gmol. All unlabeled amino acids, dipeptides, and amino acid structural analogues were obtained from Sigma Chemical Co., St. Louis, Mo., with the exception of L-isoleucine (allo-free) and D-alanine, which were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio, and methylamine hydrochloride, which was purchased from Fisher Scientific Co., Fairlawn, N.J. Chloramphenicol was purchased from Parke, Davis and Co., Ltd., Brockville, Ontario.

RESULTS

Transport interactions between neutral amino acids. The prior observation of Drapeau et al. (10) that a number of neutral amino acids were able to inhibit the uptake of "'C-labeled AIB in marine pseudomonad B-16, presumably by competitive inhibition, suggested the possible existence of an amino acid transport system having low substrate specificity. As a preliminary step towards exploring further the nature of amino acid transport in this bacterium and towards gaining an understanding of the numbers and kinds of transport systems present, the earlier competition study was extended to include a survey of the competitive interactions among seven neutral amino acids during membrane transport. Almost every amino acid

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tested as a potential competitor had the capacity to inhibit the uptake of each of the other amino acids (Table 1). The strong mutual inhibitory interactions between L-leucine and L-valine and between L-serine and L-threonine indicated the possible existence of two amino acid transport systems with respective affinities for the branched-chain and hydroxyamino acids. There was, however, a certain degree of overlap between the substrate specificities of these indicated systems. The strong inhibition of AIB and L-alanine uptake by all seven amino acids tested suggested the possible existence of a third transport system with the capacity to recognize a wide range of neutral amino acids. AIB is a structural analogue of alanine and is not metabolized by the marine pseudomonad (10). To further clarify the picture of neutral amino acid transport in marine pseudomonad B-16, a search was initiated for mutants defective in AIB and leucine transport. Characterization of AIB transport-negative mutants. A number of mutants defective in their capacity to transport AIB were isolated by the D-serine resistance technique of Schwartz et al. (35). This technique was selected owing to the ability of D-serine to compete with AIB for uptake (see Table 3) and to inhibit the growth of the marine pseudomonad in succinate minimal medium. As in a number of other bacteria (8), D-serine was found to interfere with the biosynthesis of pantothenic acid in the marine pseudomonad (J. Fein, Ph.D. thesis, McGill Univ., Montreal, Quebec, Canada, 1974). In Fig. 1, the capacity of the wild-type strain and AIB transport-negative mutants DAG 157 and DAG 163 to transport "4C-labeled AIB is compared. At 150 AM AIB, the transport capacity in these mutants was reduced by over 80%. Similar results were obtained with the other

TABLE 1. Inhibitory interactions among seven neutral amino acids for uptake in the marine pseudomonad B-16 Inhibition of uptakea (%) Competitor

L-Leucine L-Valine L-Alanine AIB

Glycine L-Serine L-Threonine

L-Leucine'

L-Valine

L-Alanine

AIB

Glycine

L-Serine

L-Threonine

98 91 40

96 95 75 9 22 35 57

43 43 97 80 75 85 74

70 64 98 99

2 0 95 91 97 89 90

8 14 44 48

39 28 38 37 48 93 96

23 11 27 40

98 99 96

50 97 80

aCells were incubated 10 min with labeled substrate (5 x 10 - M) in buffered complete salts solution containing chloramphenicol (100 ug/ml) and one of the unlabeled inhibitors (5 x 10-3 M). Percent inhibition was based on uptake observed in the absence of added inhibitors. ° "4C-labeled substrate.

1180 Z

-

FEIN AND MACLEOD B

A

32(

E E w

16( 0I

J. BACTERIOL.

to screen for leucine transport-negative strains among the survivors by the autoradiographic method of Zwaig and Lin (46). Three leucine transport-negative mutants were obtained in this manner (strains LIV 1, LIV 2, and LIV 3). To further characterize these mutants, they

NC S

D-ALA I

20

40

160

6t 60 0 60 20 40 M NUT ES

FIG. 1. Comparative transport of "4C-labeled AIB by: (A) cells of the wild-type strain (0) and mutant DAG 157 (U); (B) cells of the wild-type strain (0) and mutant DAG 163 (0). The assay was conducted as described in the text, with "4C-labeled AIB at 150 1M (0.22 gCi/umol).

80~

mutants that were isolated. Fresh membrane 0 10 20 vesicles of mutants DAG 157 and DAG 163, E prepared by the method of Sprott and MacLeod (38), were unable to transport AIB, even in the presence of suitable electron donors (unpublished data). E To examine further the nature of the system I.. 0~ that transports AIB, the uptakes of various -o "4C-labeled amino acids by the wild-type strain and by mutant DAG 157 were compared (Fig. 2 4-' 0 and Table 2). In these studies, the "4C-labeled amino acids tested were added to the suspending medium at a final concentration of 1.5 x 10-' M. As illustrated (Fig. 2), the transport of glycine, D-alanine and D-serine, in addition to that of AIB, was significantly reduced in the mutant. The rates of transport of a number of other amino acids into the mutant did not, on the other hand, appear to be significantly different from the rates observed in the wild-type strain (Table 2). It would, therefore, appear that the transport defect expressed in AIB transport-negative mutants of marine pseudomonad B-16 is of a similar nature to that expressed in a dagA strain of Escherichia coli (7, 34). For this reason, the system responsible for the uptake of D-alanine and related amino acids in marine pseudomonad B-16 has been referred to as the DAG system, and the mutants have 10 0 20 been named with the prefix DAG. This terMinutes minology should in no way be taken to imply FIG. 2. Comparative transport of glycine, D-alathat this system is identical to the one described nine, and D-serine by wild-type cells and cells of in E. coli. Characterization of leucine transport-neg- mutant DAG 157. Experimental conditions as in the legend to Fig. 1, with "4C-labeled amino acids added ative mutants. The approach taken to isolate to the reaction mixture at 150 0.22 leucine transport-negative mutants was first to ,ICi/Mlmol; D-serine and D-alanine,uM 1.1(glycine, /lCi/lmol). select against leucine transport-positive cells in Symbols: (U) wild-type cells; (0) cells of mutant the mutagenized culture with L- [3H ]leucine by DAG 157. Uptakes were measured in the presence the tritium suicide technique (23, 24) and then of chloramphenicol (100 gg/ml).

aI)

VOL. 124, 1975

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

TABLE 2. Uptake of various a-amino acids by marine pseudomonad B-16 wild type and by mutant DAG 157 Initial rate of uptakea (nmol/min per mg of cell dry wt) Wild type DAG 157

Amino acid

AIB

Glycine

4.3±0.2 2.3 i 0.3

1.0±0.2 0.7 ± 0.3

L-Alanine L-Serine L-Threonine

8.3 ± 1.4 9.2 0.1 6.3 i 0.3

5.1

L-Arginine L-Histidine L-Leucine L-Valine

2.3 i 0.7 2.2 ± 0.9 3.9 ± 0.1 4.3 i 2.5

2.6 ± 0.9 ± 3.9 ± 2.9 ±

L-Aspartate L-Glutamate L-Isoleucine L-Lysine

2.3 ± 0.5 3.0 ± 0.4 5.4 ± 0.4 2.0 ±0.6

1.5 ± 0.6 2.2 ± 0.2 4.5 ± 1.3 1.5 ±0.3

7.9 ± 2.7 3.1 4.9 ± 1.4 1.4 0.4 0.7

1.5

0.8 ± 0.4 L-Tyrosine 1.5 ± 0.6 aCells (300 ,g/ml) were incubated with each of the "4C-labeled amino acids (1.5 x 10-4 M) in buffered complete salts solution containing chloramphenicol (100 ;g/ml). Initial rates of uptake (based on three experiments) were determined from the slopes of 3-min time course plots (uptake versus time) as described in the text. To allow rapid filtration and washing of cells, Whatman GF/A prefilters were employed, together with the 0.45-Am membrane filters.

LEU

AIB

1181

were screened by the Millipore membrane filtration method for their capacities to transport

several neutral amino acids when the latter were present in the incubation medium at a concentration of 150 gM (Fig. 3). Each mutant was found to have impaired capacity to transport L-isoleucine, L-valine, and L-alanine, in addition to L-leucine, suggesting the possible existence of a common transport system for these amino acids in marine pseudomonad B-16. The transport of AIB in the three mutants, on the other hand, did not appear to be defective under these conditions. The term LIV will be used to name the L-alanine branchedchain amino acid transport system distinguished here but, again, the terminology should not be taken to imply identity to systems of similar specificity reported in other bacteria. Kinetics of AIB transport in wild-type and mutant strains. When the kinetics of AIB transport in the wild-type strain and in mutants DAG 157 and LIV 1 were studied, the results illustrated in Fig. 4 were obtained. With the

wild-type strain, a biphasic Lineweaver-Burk plot (22) with a break at approximately 1.6 mM was obtained for transport over a wide range (2.5 x 10-i to 8.0 x 10-3 M) of AIB concentra-

tions (Fig. 4A). By the method of Neal (27), approximate values for the Michaelis constants for transport via the high-affinity and low-affinity processes were determined to be 2.7 x 10-5 and 2.5 x 10-i M AIB, respectively. That the

E

IL

w 100

12 N.cn

~~~w

w

z

3

I w 0

.

MINUTES FIG. 3. Relative rates of uptake of AIB, L-alanine, L-leucine, L-isoleucine, and L-valine in the wild-type strain (W) and L-leucine transport-negative mutants LIV 1 (1), LIV 2 (2), and LIV 3 (3). Washed cells (final concentration, 100 ug/mo were added at zero time to a reaction mixture consisting of buffered complete salts solution, chloramphenicol (100 Ag/ml), and one of the "4C-labeled amino acids (1.5 x 10-4 M; 0.22 uCi/Atmol).

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low-affinity process was not simple diffusion is shown by the data presented in Fig. 4B in which the kinetics of AIB transport in the wild-type strain was examined in greater detail at high substrate concentrations (8.0 x 10-i to 1.67 x 10-2 M). In contrast to the results obtained with the wild-type strain, the double-reciprocal plot for AIB transport in mutant DAG 157 was linear over the concentration range 1.0 x 10-I to 1.33 x 10-2 M (Fig. 4C). The apparent Michaelis constant for this transport was 1.4 x 10-3 M, in close correspondence to the value obtained for the low-affinity process in the wild-type strain. Each of the other D-serine-resistant, AIB transport-negative mutants was also found to be missing the high-affinity process. It can, therefore, be concluded that high-affinity AIB transport in the wild-type strain is mediated by the DAG system. Similar kinetic studies have also been carried out with strain LIV 1 (3.3 x 10-5 to 1.33 x 10-2 M AIB). As illustrated in Fig. 4D, a linear reciprocal plot with an apparent k, of 2.8 x 10- 5 M was obtained, corresponding to the k, for AIB transport by the DAG system in the wild-type strain. It would, therefore, appear that the LIV system was responsible for the low-affinity AIB transport detected in the wild-type strain. Substrate specificity of the DAG transport system. A series of competition experiments was carried out to determine in more detail the substrate specificity of the DAG transport system. Cells of the wild-type strain were incubated for 1 min with 2.5 x 10-5 M "4C-labeled AIB, both in the presence and absence of unlabeled a-amino acids, dipeptides, and structural analogues at 5 x 10-4 M. At the low AIB concentration selected, no uptake was found to occur via the LIV system (Fig. 4). Strong inhibition of high-affinity AIB uptake was produced by many of the a-amino acids tested (Table 3). A large group of neutral amino acids inhibited uptake 89% or more. A second group containing both neutral and basic amino acids had intermediate activity. The acidic amino acids L-aspartate and L-glutamate, as well as L-isoleucine and L-lysine and the dipeptides glycylglycine and DL-alanylglycine, however, were not effective inhibitors of this system. Since L-glutamine and L-asparagine were found to be strong inhibitors of AIB uptake, it is possible that the negative charge on the carboxyl side groups of L-glutamic and L-aspartic acids reduced the capacity of these amino acids to inhibit high-affinity AIB transport. The kinetics of the above inhibitions were all

Il

I

A

0.24 0.16

K~~ha=- 2.7 x 10 5M ~~~~Kla= 2.5 x 10 m

g ~~~ L 0.08 4

0

0.12

E ov o' E E C

0.081

-1> 0.041 o

0.15

0.30

0.45

2.5

5.0

7.5

0.60

C 1.2

0.81 0.41 0

_

I_

10.0

1

[AIB]

mMM

c

E .0E

E - 1>

10 1

[AIB]

mMM

FIG. 4. Double-reciprocal plots of the initial rate of 14C-labeled AIB transport by the wild-type strain and by mutants DAG 157 and LIV 1, as a function of the extracellular AIB concentration. Initial velocities of transport were determined as in Table 2. Cell dry weights were based on optical density. (A and B) Transport activity in the wild-type strain; (C) transport activity in strain DAG 157; (D) transport activity in strain LIV 1; Kh0 and KLa, Michaelis constants for high-affinity and low-affinity AIB transport, respectively.

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

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TABLE 3. Comparative inhibitory action of various a-amino acids, dipeptides, and structural analogues on the transport of AIB by the DAG transport system in the wild-type strain of marine pseudomonad B-16 Inhibition

Competitor

of AIB transporta

._2

(%)

Glycine L-Alanine D-Alanine L-Serine D-Serine L-Homoserine L-Glutamine L-Asparagine L-Threonine L-Methionine L-Phenylalanine L-Arginine L-Histidine L-Leucine D-Threonine L-Valine L-Aspartate L-Glutamate L-Isoleucine L-Lysine N-glycylglycine N-DL-alanylglycine a-Amino group-modified compounds: N-formylglycine N-methylglycine N-acetylglycine N,N-dimethylglycine L-Proline

Acetate Succinate a-Ketobutyrate a-Ketoglutarate

#-Alanine DL-,8-Aminoisobutyrate

99 97 95 95 94 94

c

90 89 89 63 54 52 51 42 38 37 14 9 8 5 1 7

0

2 4 3 1 No. of R Carbon Atoms

FIG. 5. Effect of stereospecificity and chain length on the capacity of neutral amino acids to inhibit AIB transport via the DAG transport system in wild-type cells of marine pseudomonad B-16. Experimental conditions as described in Table 3. Inhibitors: glycine (R = 0); D-alanine and L-alanine (R = 1); D-a-aminoN-butyrate and L-a-amino-N-butyrate (R = 2); D-norvaline and L-norvaline (R = 3); and D-norleucine plus L-norleucine (R = 4). Symbols: 0, D isomers; 0, L isomers. R, side-chain carbon atoms.

8 8 3 1 1

(_5)b

(- 10)

(-8) (-1) 2

1

a-Carboxyl group modified compounds: Methylamine Glycinamide 2-Aminoethanol a Cells were incubated for 1 min in buffered complete salts solution containing "IC-labeled AIB (2.5 x 10-5 M; 0.98 ACi/JLmol), chloramphenicol (100 Ag/ml), and one of the unlabeled competitors (5 10-4 M). Percent inhibition was based on uptake observed in the absence of added inhibitor. ,'The negative values in parentheses indicate stimulation of transport. x

found to be competitive by Lineweaver-Burk plots. The stereospecificity of the DAG transport system was examined by determining the effec-

tiveness of a series of straight-chain D- and L-a-amino acids as competitive inhibitors of high-affinity AIB transport (Fig. 5). Whereas the L-a-amino acids were strong to moderate inhibitors at all chain lengths tested, the D-aamino acids were poor inhibitors at chain lengths of three and four carbon atoms, corresponding to D-norvaline and D-norleucine. The strongest inhibitors were found to be glycine, Dand L-alanine, and L-a-amino-N-butyrate. D-aAmino-N-butyrate, although still a good competitor, was not as effective as its L isomer. These results suggest that the DAG transport system shows apparent stereospecificity only towards a-amino acids that contain bulky side chains. In these cases, it is the L configuration that is recognized. Since AIB, which has no a-hydrogen, and glycine, which lacks a side chain on the a-carbon, have high affinities for the DAG transport system, it would appear that neither an ahydrogen nor a side chain on the a-carbon is required for recognition by the binding site of this system. Various analogues with modified a-amino and a-carboxyl groups were tested as potential competitive inhibitors of the DAG system, and all were found to be inactive (Table 3). It would therefore appear that the minimal structural requirements that allowed so many a-amino acids to be recognized by the DAG system (Table 3) were the presence of a free a-amino group and a free a-carboxyl group on

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these compounds. Since L-serine and L-threonine were as effective inhibitors of AIB uptake as L-alanine and L-a-amino-N-butyrate were, one can conclude that the presence of a hydroxyl group on the ,B carbon does not affect the affinity of an amino acid for the DAG transport system. On the other hand, the fact that Lvaline and L-isoleucine are less effective inhibitors than their straight-chain analogues suggests that the presence of a methyl group attached as a branch to the ,B carbon of an amino acid considerably reduces the affinity of this system for that amino acid. The finding that many of the competitive inhibitors of AIB transport via the DAG system were transported into mutant DAG 157 at rates comparable to those observed in the wild-type strain (Table 2) warranted further investigation. In particular, it was important to establish whether the various competitors were transported via the DAG system or were merely recognized by the binding site. To examine this question, a detailed investigation into the transport of L-alanine was carried out. This amino acid was one of the strongest competitive inhibitors of AIB transport via the DAG system (Table 3), yet the rates of L-alanine uptake by DAG 157 and by the wild-type strain were identical (Table 2). Evidence that L-alanine was, in fact, a substrate of the DAG transport system was first obtained in a competition study in which the effects of glycine, AIB, and various unlabeled D- and L-amino acids on the initial rate of L- [3H ]alanine uptake in the wild-type strain and in the mutant DAG 157 were compared (Table 4). Although the rates of uptake of L-alanine by the two strains were about equal when the concen-

tration of L-alanine in the medium was 150 yM, with 25 AM L-alanine in the medium the mutant took up only 66% as much amino acid as the wild type. This pattern of uptake in the mutant has been observed repeatedly. Table 4 also shows that when the concentration of L-alanine in the medium was 25,uM the various amino acids added as inhibitors reduced the initial rate of L-alanine uptake by the wild-type strain from 31 to 76%. At the same L-alanine concentration, L-alanine uptake by mutant DAG 157 was inhibited only slightly or not at all by AIB, glycine, and the D-amino acids tested but to nearly the same extent as in the wildtype strain by L-serine and L-a-amino-Nbutyrate. In Table 3 and Fig. 5, it is shown that glycine and short-chain D-amino acids are strong competitive inhibitors of AIB transport via the DAG system. The inability of these same amino acids to inhibit L-alanine transport in the mutant strain when L-alanine is present in the medium at 25 MM suggests that L-alanine is transported into marine pseudomonad B- 16 by at least two transport systems and that one of these is the DAG system. When the L-alanine concentration in the medium was increased from 25 to 150 AM, both D-serine and D-aamino-N-butyrate became less effective inhibitors of L-alanine uptake by the wild-type strain (Table 4). This suggests that, as the extracellular L-alanine concentration is increased, the contribution made by the DAG transport system to the total uptake of L-alanine by the wild-type strain becomes less significant. Multiplicity of L-alanine transport systems. Preliminary evidence has suggested that L-alanine transport in marine pseudomonad

TABLE 4. Comparative inhibition of L-alanine uptake in marine pseudomonad B-16 wild type and mutant DAG 157 by various a-amino acids S

Strai n

L-(H-

Initial rate of

Inhibitor

concn (AM)

uptake'

concn

(nmol/

(mM)

AIB

Glycine

D-Alanine

D-Serine

D-ANW

L-Serine

L-ANBc

alanine|

min per mg)

Inhibition of initial rate of L-alanine uptake

)with inhibitor:

Wild type DAG 157

25 25

5.3 ± 0.4 3.5 0.3

0.5 0.5

55 23

60 14

52 0

54 9

31 0

71 48

76 56

Wild type DAG 157

150 150

11.0 ± 0.5 12.0 ± 1.1

1.5 1.5

Npd NP

NP NP

NP NP

30 8

13 17

53 57

81 75

L- [9H Jalanine; 8 jACi/Amol. Based on 1-min incubation of cells with L- [3H lalanine in buffered complete salts containing chloramphenicol (100 Ag/m1). cInhibition by D- and L-a-amino-N-butyrate (ANB). dNP, Not performed. a

"

VOL. 124, 1975

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

B-16 is mediated by two independent transport systems, the DAG transport system and an uncharacterized system with apparent stereospecificity for L-amino acids (Table 4). From the evidence presented in Fig. 3 and 4, it would appear that this latter system is the LIV transport system. This interpretation is supported by the observation that L- [3H]alanine uptake in a mutant with a defective DAG transport system (strain DAG 157) was competitively inhibited by AIB, with an apparent K1 of 2.0 x 10-' M

1185

(Fig. 6). This value for K, corresponds well with the previously observed Michaelis constant (Kt = 1.4 x 10-3 M) for AIB transport via the LIV transport system (Fig. 4). A clear demonstration of heterogeneity and of the involvement of the DAG and LIV transport systems in L-alanine uptake was obtained in a competition study in which the capacity of D-alanine to inhibit the uptake of L-alanine in mutants lacking either system was measured (Fig. 7). Although D-alanine was found to have little effect on L-alanine uptake by strain DAG 157, except at very high inhibitor concentrations, low levels of D-alanine (1 mM) caused a marked inhibition of L-alanine uptake in strain LIV 1. Since D-alanine is transported almost

E E a I

9

5 -W41

2CL

I

L-ALA] -mM

0

.rc

FIG. 6. Competitive inhibition of L-alanine uptake in mutant DAG 157 by AIB. Double-reciprocal plot of the initial velocity of L-alanine uptake versus the extracellular L-alanine concentration, measured in the presence and absence of unlabeled AIB. Initial rates were based on 1-min incubation of cells (100 ,sg/ml) with L- ['Hjalanine (7.92 MCiIumol) in a reaction mixture containing buffered complete salts, chloramphenicol (100 Mg/ml), and unlabeled AIB where indicated. Symbols: 0, no inhibitor; 0, 500,uM AIB; *, 2.5 mM AIB.

4 .j

D-Alanine Conc.

(mnM)

FIG. 7. Effect of D-alanine on the initial rates of L-alanine uptake in strains DAG 157 (a) and LIV 1 (A). Cells were incubated with 100 MM L-['HJalanine for 1 min in a reaction mixture containing buffered complete salts, chloramphenicol (100 g/mlo, and unlabeled D-alanine as indicated.

A 1.0 I

co

0 ° 0.6

Wild Type 0.4

-l> 0.2

0

I

I

I

20

40

60

I 80

I

100

0

20

40

60

80

100

I

[L-ALA] -mM FIG. 8. Kinetics of L-alanine uptake in marine pseudomonad B-16. (A) Double-reciprocal plot of the initial rates of L- ['H]alanine uptake in the wild-type strain (0), as a function of the extracellular L-alanine concentration. Initial velocities were determined as in Table 4. L- ['HJalanine, 7.92 MCi/Mgmol. (B) As in (A) with strains DAG 157 (A) and LIV 1 (U).

1186

J. BACTERIOL.

FEIN AND MAcLEOD

exclusively by the DAG transport system, L-aianine uptake in strain LIV 1 must also occur by this system. Similarly, the finding that L-alanine transport is resistant to inhibition by low levels of D-alanine in strain DAG 157 and partially resistant in the wild-type strain (Table 4), but not in the mutant LIV 1, suggests that L-alanine transport in strain DAG 157 is mediated by the LIV transport system. The heterogeneity of L-alanine transport activity was not revealed in kinetic studies with the wild-type strain. As illustrated in Fig. 8A, the reciprocal plot of the initial rates of L-alanine uptake versus the extracellular L-alanine concentration was linear over the concentration range tested (10 to 200 ,tM). The failure to observe biphasic kinetics cannot, however, be considered as evidence against heterogeneity since linear plots are derived in cases in which heterogeneity involves multiple transport systems with similar kinetic parameters for a substrate (4). That this was the case for L-alanine uptake via the DAG and LIV transport systems is shown in Fig. 8B. In this experiment, mutants DAG 157 and LIV 1 were used to study L-alanine transport by each system separately. From the reciprocal plots, K, values of 4.6 x 10-5 and 1.9 x 10- M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for L-alanine. Substrate specificity of the LIV transport system. The substrate specificity of the LIV transport system was investigated by measuring the capacity of a number of amino acids and structural analogues to inhibit the transport of L-[3H]alanine or "4C-labeled AIB in strain DAG 157. From the results presented in Table 5, it would appear that this system has highest affinity for neutral a-amino acids in the L configuration. Sprott and MacLeod (38) have recently demonstrated that L-alanine is rapidly metabolized by marine pseudomonad B-16. It is unlikely, however, that the competition studies with L- [3 H]alanine (Table 5) reflect competition for metabolism rather than for transport since a similar pattern of inhibition was observed when measuring the uptake of "4C-labeled AIB. The competition studies were carried out in the presence of chloramphenicol to prevent protein biosynthesis. To the extent studied, the minimal structural requirements for reactivity of an amino acid with the LIV transport system were found to be more complex than those reported above for activity with the DAG transport system. Modification of either the position or the structure of

TABLE 5. Comparative inhibitory action of various amino acids and structural analogues on the transport of L-alanine and AIB in mutant DAG 157

Inhibitor

Inhibi- Inhibition of L-- tion of L-alaAIB uptake a uptake'

(371)

(%

a-Amino acids: L-Alanine

L-a-Amino-N-butyrate L-Serine L-Norvaline L-Norleucine L-Leucine L-Valine L-Homoserine L-Threonine L-Isoleucine L-Methionine

a-Aminoisobutyrate L-Glutamine

Glycine D-Norleucine L-Glutamate

D-a-Amino-N-butyrate D-Norvaline D-Alanine

L-Phenylalanine

L-Arginine D-Threonine

D-Serine L-Histidine

L-Asparagine L-Lysine

a-Carboxyl group-modified compounds: Methvlamine

Glycinamide a-Amino group-modified compounds 2-Aminoethanol N-methylglycine L-Proline

N-formylglvcine Acetate a-Ketobutvrate a-Ketoglutarate 3-Alanine

88 85

65 5.3

94 82 74

50 49 48 48 46

45 45 38 35 34

70

30 23 21 20 16 16 15 15 1.3 12 10 2

17

25

32

4 4

5 4 9 (- 19)c 17 ( (- 4) 7

17

a Cells were incubated for 1 min in buffered complete salts solution containing L-13H]alanine (100 uM: 15.2 pCi/pmol), chloramphenicol (100 gg/ml), and one of the unlabeled inhibitors (2.5 mM). Percent inhibition was based on the uptake observed in the absence of any added inhibitor. 'Experimental conditions as above, with '4C-labeled AIB (1.4 mM; 0.55MCi/Ugmol) as labeled substrate and unlabeled inhibitors added at 14 mM. "The negative values in parentheses indicate stimulation of transport.

the a-amino or a-carboxyl group of an amino acid abolished the capacity of the modified compound to inhibit amino acid transport by the LIV transport system (Table 5). The weak inhibition of L-alanine transport in strain DAG 157 by glycine, as opposed to the strong inhibi-

VOL. 124, 1975

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

tion by L-alanine and L-a-amino-N-butyrate, suggests that the presence of an aliphatic group in the a, position of an amino acid (corresponding to the position of the methyl group in L-alanine) may play an important role in the recognition process. Similarly, a hydrogen atom in the a, position of an amino acid (correspondto the position of the a-hydrogen in L-alanine) may be preferred to another group since AIB and the D-amino acids were weak inhibitors. The strict stereospecificity of the LIV transport system has been demonstrated (Fig. 7 and Table 5). The branched-chain amino acids L-valine and L-leucine showed the same strong capacity to inhibit uptake as their straightchain analogues L-norvaline and L-norleucine, suggesting that the LIV transport system recognizes either type of side chain equally well. On the other hand, the finding that L-serine and L-threonine were weaker inhibitors of uptake than were L-alanine and L-a-amino-N-butyrate suggests that the presence of a hydroxyl group on the side chain of an amino acid interferes to some extent with recognition. Requirement for Na+ for transport via the DAG and LIV systems. The absolute requirement for Na+ for the active transport of AIB and various metabolites in marine pseudomonad B-16 has been well documented (10, 45). In an attempt to find features that could further distinguish the DAG and LIV transport systems, the optimal level of Na+ for the transport of AIB via each system was determined (Fig. 9). Leakage of intracellular solutes at low Na+ concentrations (below 0.3 M) was prevented by maintaining a constant molarity with added Li+ (45). To obtain transport via the DAG system alone, cells of the wild-type strain were incubated with "4C-labeled AIB added to the suspending medium at 1.5 x 10-4 M. At this concentration, transport via the LIV system would have been negligible (Fig. 4). Transport by the LIV system alone was measured with strain DAG 157, using 7.5 x 10-3 M '"C-labeled AIB. The quantitative requirements of the two transport systems for Na + were found to be similar, with optimal transport activity being observed in the region of 0.3 M NaCl. In the complete absence of Na+, neither system exhibited any capacity to transport AIB (data not shown). DISCUSSION Inhibitory interactions among a number of neutral amino acids competing for uptake into marine pseudomonad B-16 were found to be quite complex, suggesting that a number of

A

1187

I

100

50

2

a

I'a

~0 so

Ox

I

I O0

MtCcowfsian

0.6

(A )

FIG. 9. Optimal Na+ concentrations for transport via the DAG and LIV systems. (A) DAG transport system. Cells of the wild-type strain were incubated with "C-labeled AIB (150 ,M; 0.22 MCi/mol in buffered complete salts solution containing Na+ at the concentrations indicated (5.0 x 10-2 to 1.0 Al). The amount of Na+ transferred with washed cells to the assay medium was negligible (less than 40 Mmol). At concentrations of Na+ below 0.3 M, the molarity was kept constant at 0.3 M with added LiCl. The initial rates of AIB transport were measured as in Table 2. The maximum rate of uptake observed was 1.7 x 104 counts/min per mg of cell dry weight. (B) LIV transport system. Cells of DAG 157 were incubated with "4C-labeled AIB (7.5 mM; 0.11 gCi! umol) and Na+ (1.0 x 10-2 to 4.0 x 10-1 M) under conditions identical to those described above. The maximum rate of transport measured was 2.7 x 102 counts/min per mg of cell dry weight.

transport systems with overlapping affinities involved in the transport of these substances. In the study reported here, two neutral amino acid transport systems with considerable

were

overlap in substrate specificities were distinguished and characterized. The DAG transport system plays a major role in the uptake of glycine, D-alanine, D-serine, and AIB and, to a lesser extent, L-alanine and possibly other related amino acids. This system was found to be defective in a number of D-serine-resistant mutants. The LIV transport system, on the other hand, was found to be most active with L-leucine, L-isoleucine, L-valine, and L-alanine, with some affinity for other neutral the L configuration. Mutants tem were also isolated. The

amino acids with missing this sysLIV system was

1188

FEIN AND MAcLEOD

found to be highly stereospecific and in this regard was best distinguished from the DAG transport system. Both systems appeared to be constitutive (unpublished data) and both required Na+ for activity (0.3 M Na+ being optimal for each). Based on the average value for the intracellular volume (42), it can be shown that the two transport systems are able to concentrate amino acids intracellularly against a concentration gradient. This capacity is lost when cells are treated with KCN (10; unpublished data). Transport systems basically homologous to the DAG and LIV transport systems have been reported in a variety of nonmarine bacteria (14, 29). These systems are distinguished from the systems in the marine pseudomonad B-16, however, by their lack of a demonstratable requirement for Na+ for activity. The functions of Na+ in metabolite transport in this bacterium have been investigated (15, 37, 38, 42, 43). Schwartz et al. (35) observed that, in mutants of E. coli W selected for their resistance to growth inhibition by D-serine the transport of D-serine and glycine was reduced by over 93% and the transport of L-alanine by over 65% when compared with the transport of these amino acids by the parent strain. The studies of Kessel and Lubin (19) with E. coli W demonstrated that D-alanine and D-cycloserine were also transported by the D-serine-glycine-L-alanine transport system and that L-alanine might also be transported by an additional transport system. Studies with strains of E. coli K-12 resistant to D-cycloserine (44) and to D-serine (7) have given rise to similar findings. More recently, Oxender and co-workers (30, 34) have examined the transport of alanine, glycine, and serine in E. coli K-12 in more detail and have indicated that two or more transport systems may be involved in the uptake of these amino acids. One system serves mainly to transport D-alanine, glycine, and D-serine and, to some extent, L-alanine. This system is missing in strains carrying the dagA mutation. A second system (LIV-I system) transports L-alanine, L-threonine, the branched-chain L-amino acids, and possibly L-serine. This system appears somewhat similar to the LIV system of marine pseudomonad B-16. The kinetics of glycine transport in E. coli K-12 were found to be biphasic, suggesting that additional routes of uptake might be available to this amino acid at higher substrate concentrations (34). The existence of multiple transport systems for glycine in marine pseudomonad B-16 is also probable since transport of this amino acid (at 150 MM) was not completely

J. BACTERIOL.

abolished in transport-negative mutant DAG 157. In E. coli B/r, Templeton and Savageau (40) have reported that L-alanine was transported by a system similar to, but with broader specificity than, the LIV-I system of E. coli K-12 and have indicated that L-threonine and L-serine may also be transported by an additional system specific for these amino acids. Evidence suggested that L-homoserine was transported by the former system only. L-Homoserine was found to be a strong competitive inhibitor of both the DAG and LIV transport systems in marine pseudomonad B-16. Systems similar to the alanine-glycine-Dserine-D-cycloserine transport system of E. coli have also been reported in Bacillus megaterium (26), B. subtilis (5), Streptococcus challis (31), and Mycobacterium tuberculosis (9). The system described in B. megaterium also appears to transport AIB. In S. faecalis (faecium), a system that can operate by either exchange diffusion or by coupling metabolic energy has been implicated in the transport of glycine, L-alanine, L-serine, and L-threonine (1, 3). Analogues with modified a-amino, a-carboxyl, or a-hydrogen groups were weak inhibitors of this system, as were amino acids with long carbon side chains and those with a D configuration (3). In contrast, a transport system having relatively unspecific structural discrimination among neutral D-amino acids and L-amino acids has been described in S. faecalis R (17). Transport systems basically similar to the LIV transport system of marine pseudomonad B-16 have also been reported in a variety of nonmarine bacteria. Cohen and Rickenberg (6) first reported the existence of a stereospecific transport system for leucine, isoleucine, and valine in E. coli K-12. This was followed by reports of similar systems in P. aeruginosa (18), B. subtilis (20), Staphylococcus aureus (36), and in a nontumorigenic strain of Agrobacterium tumefaciens (2). More recent studies with E. coli K-12, however, indicate that the pattern of branched-chain transport in bacteria is far more complex than was originally thought. Rahmanian et al. (30) were able to distinguish three transport systems for L-leucine in this bacterium, the LIV-I, LIV-II, and LIV-III systems. The LIV-I system, whose substrate specificity was described above, was found to be the major component of L-leucine uptake (K, = 0.2 gM). This system was sensitive to osmotic shock and was repressed in cells grown in the presence of leucine. The LIV-binding protein appears to be associated with this system. Though very similar to the LIV-I system in substrate specificity, the LIV system of marine

VOL. 124, 1975

AMINO ACID TRANSPORT IN A MARINE PSEUDOMONAD

pseudomonad B-16 was not repressed by the growth of cells in a rich medium (complex medium). Furthermore, the LIV transport system appears to be functional in isolated membrane vesicles of marine pseudomonad B-16 (38), and no evidence has been obtained for the association of a LIV-binding protein with this system. The LIV-II system of E. coli K-12 (K, = 2 AM L-leucine) was specific for leucine, isoleucine, and valine. This system was not sensitive to osmotic shock treatment and was not repressed by growth in the presence of leucine. The L system (K, = approximately 0.2 qM L-leucine) was specific for leucine, sensitive to osmotic shock treatment, and repressed by leucine. The L-binding protein appears to be associated with this system. An extensive study into the multiplicity of branched-chain amino acid transport systems in E. coli K-12 was also carried out recently by Guardiola et al. (12, 13). From kinetic and genetic evidence, these workers described a "very-high-affinity" system and two "highaffinity" systems for L-leucine, L-isoleucine, and L-valine and three "low-affinity" transport systems, each specific for either L-leucine, L-isoleucine, or L-valine. The "very-high-affinity" system was inhibited by L-threonine, L-methionine, L-alanine, and by several analogues, including D-serine and D-leucine, and was repressed by growth in the presence of methionine. The "high-affinity" systems appeared to be sterospecific and one ("high-affinity-2") was inhibited by L-threonine. The possibility of multiple transport systems for L-leucine, L-isoleucine, and L-valine in marine pseudomonad B-16 would also appear likely since mutants with defective LIV transport systems exhibited some capacity for transporting these amino acids when the latter were present in the suspending medium (at a 1.5 x 10-4 M concentration). Competition studies have indicated that the branched-chain amino acids have some affinity for the DAG system, but whether they are in fact transported by this system has yet to be determined. In addition, the possible existence of other, more-specific transport systems for one or more of the branched-chain amino acids, or for that matter other neutral amino acids studied here, can not be ruled out at this time. With the DAG and LIV transport-negative mutants that we have isolated, we hope that some of these questions can be resolved. The isolation of a double mutant with defective DAG and LIV transport systems would also be helpful. The extremely low affinity of the LIV transport system for AIB (K, = 1.4 x 10' M)

1189

explains why several earlier workers from this laboratory (42, 45) did not detect the heterogeneity of AIB transport in this microorganism. In their studies, the highest AIB concentration used in the suspending medium never exceeded 150 ,M, well below the level at which measurable transport by the LIV transport system can be obtained. Thus, in all previous studies of AIB transport into marine pseudomonad B-16, except one, transport by the DAG system was being measured (42, 43, 45). The one exception was a transport study using a thick cell suspension technique in which the AIB concentration used in the experiments was 3 mM (41). While this manuscript was in preparation, Reizer and Grossowicz (33) demonstrated possible heterogeneity in the uptake of AIB in a thermophilic bacillus. The specificity of AIB uptake in the thermophile was similar to that reported here for AIB transport via the DAG transport system of marine pseudomonad B-16. AIB transport in the thermophile, however, showed no requirment for Na+ for activity (33). ACKNOWLEDGMENTS This work was supported by a grant from the National Research Council of Canada. J.E.F. wishes to thank the National Research Council of Canada for a postgraduate scholarship, and McGill University for a McConnell Predoctoral Fellowship. LITERATURE CITED 1. Asghar, S. S., E. Levin, and F. M. Harold. 1973. Accumulation of neutral amino acids by Streptococcus faecalis: energy coupling by a proton-motive force. J. Biol. Chem. 248:5225-5233. 2. Behki, R. M., and R. M. Hochster. 1966. Metabolism of amino acids in Agrobacterium tumefaciens. 1. Uptake of L-valine by resting cells. Can. J. Biochem. 44: 1477-1491. 3. Brock, T. D., and G. Moo-Penn. 1962. An amino acid transport system in Streptococcus faecium. Arch. Biochem. Biophys. 98:183-190. 4. Christensen, H. N. 1966. Methods for distinguishing amino acid transport systems of a given cell or tissue.

Fed. Proc. 25:850-853. 5. Clark, V. L., and F. E. Young. 1974. Active transport of D-alanine and related amino acids by whole cells of Bacillus subtilis. J. Bacteriol. 120:1085-1092. 6. Cohen, G. N., and H. V. Rickenberg. 1956. Concentration specifique reversible des amino acides chez Escherichia coli. Ann. Inst. Pasteur Paris 91:693-720. 7. Cosloy, S. D. 1973. D-Serine transport system in Escherichia coli K-12. J. Bacteriol. 114:679-684. 8. Cosloy, S. D., and E. McFall. 1973. Metabolism of D-serine in Escherichia coli K-12: mechanism of growth inhibition. J. Bacteriol. 114:685-694. 9. David, H. L. 1971. Resistance to D-cycloserine in the tubercle bacilli: mutation rate and transport of alanine in parental cells and drug-resistant mutants. Appl. Microbiol. 21:888-892. 10. Drapeau, G. R., T. I. Matula, and R. A. MacLeod. 1966. 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.

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and indirect selection of bacterial mutants. .J. Bacteriol. 63:399-406. 22. Lineweaver, H., and D. Burk. 19:14. The determination of enzyme dissociationi constanits. .J. Am. Chem. Soc. 56:658-666. 23. Lo, T. C. Y., M. K. Rayman, and B. D. Sanwal. 1972. Transport of succinate in Escherichia coli. 1. Biochemical and genetic studies of transport in whole cells. *J. Biol. Chem. 247:6323-6:3:31. 24. Lubin, M. 1959. Selection of auxotrophic bacterial mutants by tritium-labeled thvmidine. Science 129:838839. 25. MacLeod, R. A. 1965. The question of the existenice of specific marine bacteria. Bacteriol. Rev. 29:9-23. 26. Marquis. R. E., and P. Gerhardt. 1964. Respiration-coupled and passive uptake of a-aminoisobutyric acid, a metabolically inert transport analogue. by Bacillus megaterium. J. Biol. Chem. 239::3.361-:3:3 1. 27. Neal, J. L. 1972. Analysis of Michaelis kinietics for two independent saturable membrane transport functions. J. Theor. Biol. 35:113-118. 28. Oxender, D. L. 1972. Amino acid transport in microorganisms, p. 1:3:3-185. In L. E. Hokin (ed.). Metabolic pathways, vol. 6. Metabolic transport. Academic Press Inc., New York. 29. Pall. M. L. 1969. Amino acid transp)ort in Neurospora crassa. 1. Properties of two amino acid transport systems. Biochim. Biophys. Acta 173:11:3-127. .30. Rahmanian, M., D. R. Claus, and 1). L. Oxender. 197:3. Multiplicity of leucine transport systems in Escherichia coli K-12. J. Bacteriol. 116:1258-1266.

31.

Reichelt. .J. L., anid P. Baumann. 197:3. Change of the name Alteromonas marinopraesans (Zobell and

Upham) Baumann

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