Metabolism of Basic Amino Acids in Pseudomonas putida

Abstract Pseudomonas putida P2 (ATCC 25571) grown on l-lysine or dl-pipecolic acid is induced for a high velocity lysine transport system. Double reciprocal plots of l-lysine transport in induced cells are linear. Km and Vmax values are 7.3 x 10-6 m and 130 nmoles per min per mg for lysine-grown cells and 9.3 x 10-6 m and 42 nmoles per min per mg for cells grown on pipecolate. Lysine transport is optimal at 30° and at pH 6.5 to 7.5, and is inhibited by azide and by cyanide. Induction of the transport system is accompanied by an increase in the levels of enzymes of lysine catabolism. d-Lysine and the d- and l-isomers of arginine and ornithine inhibit l-lysine transport, and l-ornithine and l-arginine are transported by lysine-grown cells. Structural features important for inhibition of l-lysine transport include α-amino and α-carboxyl groups and a positive charge removed from the α-carbon by three to four methylene groups. Similar Michaelis constants, temperature profiles, and responses to a wide range of amino acids and amines are found for l-lysine transport by cells grown either on lysine or pipecolate.

V,mS,,,, = 100 nmoles per min per mg) and is inhibited by D-or L-lysine, D-or L-ornithine, and by D-arginine, but not by L-arginine nor by other amino acids.
L-Lysine and L-ornithine accumulate at ratios of intracellular to extracellular amino acid concentration of 750 and 18,000, respectively, suggesting that transport is an active process.
Active transport of L-arginine by cells grown on L-arginine occurs via a low K,, L-arginine-specific system (K, = 5.2 X lo-'M; Ir,,, = 11 nmoles per min per md.
Transport, which is not inhibited by any substrate analog tested, is linear for at least 7 min, is abolished by CNor N3-, and generates ratios of internal to external arginine as high as 3350.
Pseudomon,as putida 1'2 (ATCC 25571) can utilize a variety of amino acids Including lysine, arginine, and ornithine as sole sources of carbon and nitrogen.
We have previously described the pathways for catabolism of lysine (1) and arginine (2) in this organism and the presence in cultures grown on L-lysine of a general basic amino acid transport system (3) which transports lysine, ornithine, and arginine with K, values of greater than 1OV M. We report here the presence, in cells grown on L&sine, of an additional transport system for basic amino acids. L-Ly-* This work was supported by Grant GB-8321 from the Sational Science Foundation and a grant from the David Ross Foundation of l'urduc University.
It is Journal Paper 4568 from the Purdue University Agricultiral Experiment Station. $ Present address, Central Analytical Laboratory Service, Inc., 8337 Telegraph Road, Pica Rivera, Calif. SOBGO.
sine-grown cells also possess a low K, system for L-lysine (K, = 4.1 X lop7 M) and L-ornithine (K, = 1.3 X 10m7 M). Cultures grown on L-arginine appear to lack the general basic amino acid transport system but are induced for a low K, system specific for L-arginine (K, = 5.2 X lo-* M). MATERIALS ASD METHODS L-[U-1%]Xrginine and L-[U-%]ornithine were from New England Nuclear Corp. and L-[U-14C]lysine was from Calbiochem. L-Arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was from Sigma. ar-N-Methyl-L-arginine and L-arginine amide were from Fox Chemical Co. All other amino acids and amines were from previously listed commercial sources (l-3). cy-N-Methyl-Larginine, L-homoarginine, and n-arginine were purified by descending paper chromatography in butanol-l-glacial acetic acid-H,O 120:30:50 (v/v) and L-arginine amide was purified by electrophoresis at pH 6.4 prior to use.
Electrophoresis---Conditions for isolation of amino acids by electrophoresis at pH 6.4 were as previously described (2,3).
Organism and Growth Conditions--P. putida P2 (ATCC 25571) was grown as previously described (l-3) with either 25 mM L-lysine, 25 mM L-arginine, or 25 mM uL-malate plus 2.5 mM ammonium sulfate as the sole source of carbon and nitrogen. Measurement of Transport--Transport of amino acids was measured in a manner similar to that described previously (3). Data for L-lysine and L-ornithine transport were obtained with cell suspensions at final concentrations of 10 and 2.0 pg, dry weight, per ml, respectively.
Data at low (0.05 to 0.4 P~VI) and at high (12 PM) L-arginine concentration were obtained with cell suspensions at final concentrations of 10 and 100 pg, dry weight, per ml, respectively.
Cells harvested in midexponential phase growth (100 to 120 Klett units) by centrifugation for 10 min at 6000 x g were resuspended in 150 ml of ionic medium (4), centrifuged again, and finally resuspended in ionic medium (4) at a concentration of 130, 12.5, or 2.5 pg, dry weight, per ml. The cell suspension, chilled to and maintained at 4", retained full transport activity for at least 8 hours. Chilled cell suspensions were first incubated by shaking at 30" for 9 min prior to use. This preliminary incubation is necessary to obtain reproducible rates of transport (3). After incubation, the cell suspension was transferred to a tube containing radioactive amino acid at the concentrations specified in table and figure legends. When transport of L-lysine, L-ornithine, or low (0.05 to 0.40 PM) concentrations of L-arginine was studied, 0.8 ml of cell suspension was added to 0.2 ml of L-arginine, L-lysine, or L-ornithine solu- were added to 0.5 ml of L-arginine solution (approximately 4 X 10" cpm). Unless otherwise stated, all incubations were at 30" at pH 7.0. After either 30 set (2 to 50 pM L-arginine concentration) or 15 set (all other concentrations of L-arginine, L-lysine, or L-ornithine), aliquots (0.2 ml for high concentrations of L-argilline; 0.5 ml for others) were transferred to 0.45 pm HAWP Millipore filters and vacuum-filtered.
'l'hc cells were immediately rinsed with 10 ml of ionic medium at room temltcrature and the filters were transferred to scintillation vials. Diosane fluor was added to the vials within 3 min after cells were placed on the filter (3). Radioactivity was determined with a 13eckman CI'12 100 liquid scintillation spectrometer.
Counting efficiency, determined by addition of internal stalldards, was unimpaired by the lxesence of the filter.
'l'ransl)ort rates are expressed as nanomoles of arnillo acid transported 1x.x min l)er mg of cells, dry weight. L-arginine amide (Table I) .I Guanidilloacetate, P-guanidillopropionate, y-guanidinobutyrate, a-amino-n-caproate, agmatine, L-canavanine, I)-or L-glutamate, L-glutamine, glycille, L-histidine, I)-or L-isoleucine, I)-or L-l~henylala~~ir~e, L-lxoline, I)-or L-threonine, and L-valine were without inhibitory effect. l'ransport of 12.4 pM L-arginine by cells grown on L-lysine thus al)pears to occur via the general basic amino acid transport system responsible for transport of similar concentrations of L-lysine. The Ii;, for transport of L-arginine is 4.8 X'lOV M  We nest studied the ability of lysine-prowl1 ~~11s to accumulate lysine. Lysine-growu cells, 10 pg per ml, were lxrmittcd to accumulate L-[lPC]lysine, present initially at 0.20 PM coricci~tration, for 15 xc and intracellular lysinc was isolated as described lxeviously (3). The intracellular lysinc ~ollc,cntr:ltioli exceeded the initial extracellular concentration by 750.fold. Transliort of L-lysine by this low K, system thus al)l)ears to be active.
Transport of L-lysine by the low K, system is inhibited by r)-lysine, I)-or L-ornithillc, and to a lesser estellt by n-arpinine, but is not inhibited by L-arginine or other amino acids tested (Table II).
This result contrasts with the specificit\-of the high ZC, system which is inhibited by the I) or L isomers of all three amino acids (3)    (1.08 X lo6 cpm). The total quantity of radioactivity taken up, expressed as nanomoles of L-arginine per mg of cells, was calculated from aliquots removed at the indicated times. low7 M) system for L-ornithine transport with a Vmax of 100 nmoles per min per mg (Fig. 5).
Transport of L-ornithine by this low K, system also appears t,o be an active process. Lysine-grown cells were permitted to accumulate L-[U-Wlornithine, present initially at 0.18 pM concentration, for 15 set in an experiment otherwise identical with that described above for accumulation of L-lysine. Following isolation of intracellular ornithine by electrophoresis at pH 6.4, a ratio of intracellular to initial extracellular ornithine of 18,000 was obtained.
The pattern of inhibition of L-ornit'hine transport resembled that for inhibition of transport of n-lysine by the low K, system. n-Lysine, n-ornithine, and to a lesser extent n-arginine and L-ly- sine, but neither L-arginine nor any other amino acid tested inhibited L-ornithine transport (Table  II) to be at least 77% the L isomer by conversion to L-ornithine by treatment with L-arginase (Table III).
Transport of L-arginine by arginine-grown cells thus appears to be an active.process.
The data of Table  III also suggest that the size of the internal arginine pool is essentially independent of the external L-arginine concentration.
Kinetic Parameters for Transport of z-Arginine-The initial rate of L-arginine transport by cells grown on L-arginine was studied at various concentrations of n-arginine (Fig. 1). K, was 5.2 x 1OP M and V,,, was 11 nmoles per min per mg.
Induction of L-Arginine-specific Transport System---The ability of cells grown on malate plus ammonia and exposed to L-arginine to transport L-arginine begins to rise after 4 hours and then increases rapidly (Fig. 7). The transport rate at 14 hours was 9.6 nmoles per min per mg or 9.6 times that of uninduced, malate-grown cells.
Specificity of L-Arginine Transport-The ability of lysine, ornithine, and structural analogs of L-arginine to inhibit transport of 0.04 to 0.07 pM L-arginine was tested. Transport of L-arginine is unaffected by even a 70.fold molar excess of D-arginine, D-or L-lysine, or D-or L-ornithine.
The next higher and lower methylene homologs of L-arginine, cr-N-methyl-L-arginine, and L-arginine amide also are without inhibitory effect (Table  I) Guanidinoacetate, P-guanidinopropionate, y-guanidinobutyrate, cY-amino-n-caproate, agmatine, L-canavanine, n-or L-glutamate, L-glutamine, glycine, L-histidine, D-or L-isoleucine, D-or L-phenylalanine, L-proline, D-or L-threonine, and L-valine were without inhibitory effect. When the kinetics of uptake of 2 to 50 PM L-arginine by arginine-grown cells was studied, no saturation was observed, and compounds which effectively inhibit transport of 12.4 pM L-arginine by lysine-grown cells (Table I) were without significant inhibitory effect. Cells grown on L-arginine thus appear not to possess a general basic amino acid transport system similar to that found in lysine-grown cells, but rather to transport L-arginine by an L-arginine-specific system.

DISCUSSION
In P. putida, the basic amino acids lysine, ornithine, and arginine are transported by at least three systems (Table IV).
One is a general system with relatively high K, values and broad specificity.
The other two systems have lower K, values and narrower substrate specificities.
We previously reported that P. putida grown on L&sine is induced for transport of lysine, ornithine, and arginine and that all three amino acids appear to be transported by the same general system. Transport of L-lysine by this general s)-stem is inhibited by the D and L isomers of all three basic amino acids (3) and by the next higher and lower homologs of L-arginine. That the transport of L-arginine by lysine-grown cells can also occur via this general system is suggested by the similarities in K, values and by the ability of D-or L-lysine, D-or L-ornithine, D-arginine, and the next higher and lower homologs of L-arginine to inhibit both L-lysine and L-arginine transport (Table IV). The other transport system of P. putida grown on L-lysine appears also to involve actire transport since it is dependent on energy production and catalyzes accumulation of amino acids against a concentration gradient. This system, termed the diamino acid system, transport L-lysine and its next lower homolog, L-ornithine.
The D and L isomers of lgsine, ornithine, and Dbut not L-arginine, or the next higher or lower homologs of L-arginine, inhibit transport of both L-lysine and L-ornithine. Both L-lysine and L-ornithine appear to be transported either by the same system or by systems with one or more common components.
This interpretation is suggested by the similarities in K, values and by inhibition by the same substrate analogs  Transport of Lysine, Ornithine, and Arginine Vol. 247, No. 8 (Table IV). The third transport system, present in argininegrown cells, has the lowest k' t)L and appears to be absolutely specific for active transport of n-arginine.
The K, values for transport by all three systems fall within the range reported for transport of amino acids by other bacteria. \Vhile the I',,,,, values range from equivalent to far in excess of those reported elsewhere, P. putida, unlike most bacteria, utilizes transported amino acids not only for protein biosynthesis but also for production of ATP and of all amphibolic intermediates.
The most striking difference between the three transport systems is their substrate specificity, which ranges from relatively broad to absolutely specific (Table IV).
The absolute specificity of the n-arginine-specific system is suggested by the failure of any guanidino compound tested, including n-arginine and the next higher and lower homologs of L-arginine, to significantly inhibit n-arginine transport.
Other amino acids also are without inhibitory effect. The diamino acid transport system exhibits intermediate specificity.
n-Lysine and its next lower homolog L-ornithine both are transported, and the D isomers of lysine and ornithine and n-arginine inhibit transport of either n-amino acid. Inhibition data suggest that n-lysine, n-ornithine, and n-arginine also may be transported by the diamino acid transport system. The effect of substrate analog inhibitors on transport of n-arginine by the general system, taken together with data reported previously for inhibition of transport of n-lysine (3), permits assessment of the specificity of the general amino acid transport system. Transported substrates appear to possess the following minimal structure. R iAl&+ (c!xI?&_, The unsubstituted carboxyl is required (agmatine and n-arginine amide do not inhibit) as is al-o an unsubstituted a-amino group (guanidinoacetate, P-guanidinopropionate, y-guanidinobutyrate, N-oc-acetyl-n&sine (3) and cr-A-methyl-n-arginine do not inhibit) although the configuration about the ac carbon atom may be either D or L (n-lysine, n-ornithine, and n-arginine inhibit).
A positively charged group is required distal to the carboxyl group (cr-amino-n-caproate does not inhibit) and this must be either a free amino group (N-e-acetyl-n-lysine (3) does not inhibit) or a guanidino group.
The number of methylene carbon atoms separating the a-amino group from the terminal nitrogen may range from 3 to 5 (n-ornithine, n-lysine, and n-arginine are transported; n-a-amino-y-guanidinobutyrate and n-homoarginine inhibit), but the terminal methylene carbon may not be replaced by an oxygen atom (while n-homoarginine inhibits, n-canavanine does not).
An analogy may be drawn between transport of basic amino acids by P. putida and of n-lysine, n-ornithine, and n-arginine by Escherichia co& K-12, which also is mediated by three distinct transport systems (6). Both organisms possess a low K, system specific for n-arginine transport (K, = 2.6 X lo-* M (E. coli) and 5.2 X 10vs RI (P. putida)), and substrate analogs are not inhibitors of n-arginine transport,. E. coli also possesses a general basic amino acid transport system which, in contrast to that of P. putida (3), is not inhibited by the D isomers of the transported amino acids. Finally, while P. putida possesses a diamino acid transport system, E. coli possesses an n-lysine-specific system for which no analogy was detected in P. putida.