FEMS Microbiology Letters 56 (1988) 127-130 127 Published by Elsevier

FEM 03352

Uptake of glutamine and glutamate by the dinitrogen-fixing cyanobacterium Anabaena sp. PCC7120

Enr ique Flores and M. Isabel M u r o - P a s t o r

Instituto de Bioqulmica Vegetal y Fotoslntesis, Universidad de Sevilla y CSIC, Facultad de Biologla, Sevilla, Spain

Received 11 July 1988 Accepted 19 July 1988

Key words: Amino acid transport

1. SUMMARY

Whole cells of the dinitrogen-fixing cyanobac- terium Anabaena sp. PCC7120 exhibited K m val- ues for L-glutamine and L-glutamate of 33 #M and 0.5 mM, respectively. Vm~ of uptake was ca. 30 nmol mg -1 (chlorophyll) min -1 for both amino acids. The similar pattern of sensitivity to other amino acids exhibited by both transport activities suggests that a common transport system is in- volved in glutamine and glutamate uptake by this cyanobacterium.

2. IN TR ODUC TI ON

In cyanobacteria, regulation by ammonium of nitrate assimilation [1-3] or of dinitrogen fixation [4] requires ammonium metabolism via glutamine synthetase. Therefore, glutamine, or a derivative of it, is probably involved in nitrogen control in these organisms [5,6]. This has focussed attention on glutamine uptake by dinitrogen-fixing cyano- bacteria [7-9]. Anabaena cylindrica was reported

Correspondence to: Enrique Flores, Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y CSIC, Facul- tad de Biologia, Apdo. 1113, 41080-Sevilla, Spain.

to show a K m for glutamine of 14.3 /~M [8]. Anabaena variabilis, on the other hand, has been shown to possess two transport systems for glutamine with K m values of 13.8 /~M and 1.3 mM [7]; in this strain, glutamine and glutamate appear to share a common component for trans- port [7].

We are currently interested in nitrogen metab- olism in Anabaena sp. PCC7120, the dinitrogen- fixing cyanobacterium in which genetic analysis is now possible [10]. In strain PCC7120, glutamine has been shown to reverse the in vivo inhibition of glutamine synthetase by methionine sulfoximine [11], but a characterization of the process of glutamine uptake has not been reported. We have studied, for strain PCC7120, the affinity of whole cells for glutamine and glutamate and the specific- ity of the system involved in the uptake of these amino acids.

3. MATERIALS AND METHODS

Anabaena sp. strain PCC7120 (ATCC27893) was grown in medium B G l l [12] or BG11 o ( B G l l without NaNO3) supplemented with 10 mM NaHCO 3 (medium BG11C or BGl loC , respec- tively). Liquid cultures were incubated at 30°C under white light, with shaking. Ceils were

0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies

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harvested by low-speed centr i fugat ion at room

temperature, washed, and finally resuspended in 25 m M N - t r i s ( h y d r o x y m e t h y l ) m e t h y l g l y c i n e - N a O H buffer, pI-I 8.1 (Tricine buffer). The trans- port assays were started by mixing cells (about 5 /~g of chlorophyll [13] per ml, final concent ra t ion) with a solut ion of amino acid(s) (f inal concentra- t ions as indicated in each experiment). The assays

were carried out at 3 0 ° C in the light (100 W .

m - a ) . Incuba t ion t ime was 10 min, unless other- wise indicated. L-(U-14C)glutamine or L-(U-

14C)glutamate were purchased from Amersham

(specific activity, 342 /xCi//~mol) and incorpo-

rated into the assays at a final concen t ra t ion of 0.1-0.5 /~Ci/ml. Samples (0.4-1.0 ml) from the assay mixtures were filtered ( 0 . 4 5 - ~ m pore size Millipore HA filters) and the cells on the filters washed with ca. 10 ml of Tricine buffer. The

filters were immersed in scinti l lat ion cocktail and their radioactivi ty measured. Radioact ivi ty re- ta ined by boiled cells was used as a blank.

4. RESULTS

Cells of Anabaena sp. PCC7120 supplemented with 10 /~M L-(14C)glutamate or 100 /~M L-

(14C)glutamate took up either amino acid at a rate

C . _

E

O

V

O E C

0.4 /

A

0.2 / A

/ ~O ~ ° x ~ o~o ~.- o

| I |

0 20 40 60 S -1 (r .M)-~

Fig. 1. Lineweaver-Burk plot of the effect of the concentration of gluta~ne on the rate of g]uta~ne up t~e (~) , or of the concentration of glutamate on the rate of glutamate uptake (~), by nitrate-~own cells of Anabaena sp. PCC7120. v, rate of uptake; s, concentration of substrate (glutamate or g]uta- ~ne).

Table 1

Effect of some amino acids and amino acid analogs on gluta- mine and glutamate uptake by nitrate-grown cells of Anabaena sp. PCC7120

Addition % Inhibition of uptake of:

L-Glutamine L-Glutamate

L-Cysteine 67 76 L-Serine 59 76 L-Glutamate-7-monohydroxamate 57 68 L-Asparagine 53 74 L-Alanine 53 31 L-Histidine 50 49 3,- Benzyl-L-glutamat e 48 61 L-Methionine 41 39 Glycine 33 21 L-Threonine 26 22 L-Valine 22 21 L-Leucine 21 16 L-Citrulline ] 8 24 L-Proline 16 7 L-Tryptophan 12 15 L-Phenylalanine 12 14 L-Isoleucine 12 9 L-Arginine 11 0 L-Tyrosine 10 25 L-Ornithine 7 0 L- Methionine-D,L-sul foximine 6 26 L-Aspartate 4 53 L-Lysine 2 0 a-Methyl-DL-glutamate 0 30 ~-Hydroxylysine 0 6 D-Glutamate 0 2 L-Glutamine - 68 L-Glutamate 6 -

To study glutamine uptake, 10/~M L-(14C)glutamine was used as a substrate and the inhibitors were added at 0.5 mM; the uninhibited uptake was 63.4 nmol/mg of chlorophyll in 10 rain. To study glutamate uptake, 100 ~tM L-(~aC)glutamate was used and the inhibitors were added at 5 mM; the unin- hibited uptake was 52.2 mnol/mg of chlorophyll in 10 rain.

of about 10 n m o l . m g - l ( c h l o r o p h y l l ) m i n 1. A t ime-course s tudy of the process showed that up- take was l ineal for at least 16 min (data not shown). Nitrate- or N2-grown cells showed similar uptake activities.

Aff ini ty of whole cells for L-glutamine and L-glutamate was studied for amino acid concentra- t ions ranging from 12/~M to 1.28 raM. K m values were 33 /~M and 0.5 m M for g lu tamine and glutamate, respectively (Fig. 1). Vma X was similar for both uptake processes, about 30 n m o l - m g 1

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Table 2

Cumulative nature of inhibition by cysteine and aspartate of the uptake of glutamate by nitrate-grown cells of Anabaena sp.

PCC7120

Concentration of % L-Glutamate uptake Expected activity

added amino acid Added amino acid: in the case of cumulative inhibition

(raM) L-Cysteine L-Aspartate L-Cysteine and L-aspartate

0.5 13.9 43.4 5.7 6.0 1.0 8.2 38.2 3.2 3.1 2.0 4.2 31.1 1.7 1.3 4.0 2.2 24.3 1.0 0.5

The uptake of 100 /~M L-(14C)glutamate was measured in the absence or in the presence of the indicated concentrations of each L-cysteine or /and L-aspartate. 100%, the activity observed in the absence of any inhibitor, was 220 nmol/mg of chlorophyll in 10 min. The colunm on the right shows the expected uptake activities in the case of cumulative inhibition, calculated as follows: (activity in the presence of cysteine/activity without inhibitory amino acid) × (activity in the presence of aspartate/activity without inhibitory amino acid) × 100 (see [14]).

(chlorophyll)min -1 (Fig. 1). No evidence for a second affinity component was observed for either glutamine or glutamate transport.

The effect of some analogues and natural amino acids on glutamine and glutamate uptake was investigated (Table 1). A strong correspondence between the inhibition of glutamine uptake and that of glutamate uptake was found for most of the compounds tested. However, L-aspartate, a- methyl-D,L-glutamate and L-methionine-o,~-sulf- oximine inhibited glutamate uptake much more than glutamine uptake. In addition, whereas L- glutamate inhibited very tittle L-glutamine uptake, ~-glutamine inhibited 68% the uptake of ~-gluta- mate.

The nature of the inhibition of glutamate up- take exerted by the simultaneous presence of L-cy- steine (an amino acid inhibiting both glutamine and glutamate uptake) and L-aspartate (which in- hibited much more glutamate than glutamine up- take) was investigated (Table 2). The results indi- cate that inhibition was cumulative in nature, since the fraction of the uninhibited uptake activity found in the presence of the two inhibitors corre- sponded to the product of multiplying the frac- tions observed separately with each inhibitor [14].

5. DISCUSSION

Rates of transport of glutamine and glutamate similar to those reported for other heterocystous

cyanobacteria [7,8,15] have been observed for Anabaena sp. PCC7120. Also, the affinity of this cyanobacterium towards glutamine or glutamate is similar to that exhibited by other Anabaena strains [7,8]. However, wheras A. variabilis has been re- ported to possess two transport systems, one for glutamine and one for glutamate, only one affinity component for each amino acid has been observed in our studies with strain PCC7120.

The fact that both glutamine and glutamate uptake exhibited similar patterns of sensitivity to some amino acids and amino acid analogs sug- gests that a common permease might be involved in the transport of the two amino acids. L-Gluta- mate, as well as L-aspartate, methionine sulfoxi- mine and methyl-glutamate (the three of which inhibited glutamate uptake), did not inhibit glutamine uptake. This could be the consequence of a very low affinity of the permease towards the acidic amino acids and methionine sulfoximine. An alternative possibility, that strain PCC7120 has a second glutamate transport system such as the high-affinity glutamate/aspartate transport system of Nostoc sp. [15], is not supported by our results, which show a single affinity component for glutamate uptake. In addition, our results show a cumulative inhibition of glutamate uptake by L-cysteine and L-aspartate, suggesting that the L- cysteine- and the ~-aspartate-sensitive uptake of glutamate are carried out by the same permease. In the case of two independent transport systems

130

(each one sensitive to only one of the amino acids), an additive type of inhibition would be expected.

If the g lu tamine /g lu tamate permease were able to t ransport those amino acids inhibiting gluta- mine and glutamate uptake, it would be a rela- tively low-specificity permease. For instance, L- glutamate-~,-monohydroxamate and L-asparagine are probably t ransported into the cell, since the former inhibits growth of strain PCC7120 (unpub- lished observation), and the latter serves as a nitrogen source (A. Herrero and E. Flores, unpub- lished). Therefore, it is possible that, in strain PCC7120, glutamine and glutamate uptake take place via a rather general amino acid permease. Such a permease would however not t ransport basic amino acids, since arginine, lysine, 8-hy- droxylysine and ornithine exhibited very low in- hibition of glutamine or glutamate uptake. In this context, it is of interest that t ransport systems specific for basic amino acids have recently been observed in strain PCC7120 (A. Herrero and E. Flores, submitted).

Our results suggest that selection for resistance to L-glutamate-y-monohydroxamate , rather than for methionine sulfoximine [7], can be useful in order to isolate mutants lacking the g lu t amine / glutamate t ransport system.

A C K N O W L E D G E M E N T S

Research supported by the Comisi6n Inter- ministerial de Ciencia y Tecnologia (Grant No.

87020). The skillful secretarial assistance of Ms. Pepa P6rez de Le6n is much appreciated. We thank Dr. An ton ia Herrero for a critical reading of the manuscript .

R E F E R E N C E S

[1] Flores, E., Guerrero, M.G. and Losada, M. (1980) Arch. Microbiol. 128, 137 144.

[2] Herrero, A., Flores, E. and Guerrero, M.G. (1981) J. Bacteriol. 145, 175-180.

[3] Herrero, A., Flores, E. and Guerrero, M.G. (1985) FEMS Microbiol. Lett. 26, 21-25.

[4] Stewart, W.D.P. and Rowell, P. (1975) Biochem. Biophys. Res. Commun. 65, 846-856.

[5] Romero, J.M., Flores, E. and Guerrero. M.G. (1985) Arch. Microbiol. 142, 1-5.

[6] Thomas, J., Meeks, J.C., Wolk, C.P., Shaffer, P.W., Austin, S.M. and Chien, W.-S. (1977) J. Bacteriol. 129, 1545-1555.

[7] Chapman, J.S. and Meeks, J.C. (1983) J. Bacteriol, 156, 122-129.

[8] Rowell, P., Enticott, S. and Stewart, W.D.P. (1977) New Phytol. 79, 41-54.

[9] Thiel, T. and Leone, M. (1986) J. Bacteriol. 168, 769-774. [10] Wolk, C.P., Cai, Y., Cardemil, L., Flores, E., Hohn, B.,

Murry, M., Schmetterer, G., Schrautemeier, B. and Wil- son, R. (1988) J. Bacteriol. 170, 1239-1244.

[11] Haselkorn, R., Mazur, B., Orr, J., Rice, D., Wood, N. and Rippka, R. (1980) in Nitrogen Fixation (Newton, W.E. and Orme-Johnson, W.H., eds,), Vol. 2, pp. 259-278, University Park Press, Baltimore.

[12] Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M. and Stanier, R.Y. (1979) J. Gen. Microbiol. 111, 1-61.

[13] Mackinney, G. (1941) J. Biol. Chem. 140, 315-322. [14] Segel, I.H. (1975) Enzyme Kinetics, pp. 497-498, John

Wiley and Sons, New York. [15] Strasser, P. and Falkner, G. (1986) Planta 168, 381-385.