FEMS Microbiology Letters 58 (1989) 111-114 111 Published by Elsevier

FEM 03486

Role of glutamine as a direct co-repressor of glutamine synthetase in Rhodobacter capsulatus ElF1

F r a n c i s c o R o m e r o , A n t o n i o Q u i n t e r o and J o s 6 M a n u e l Roldf in

Departamento de Bioquimica, Biologla Molecular y Fisiologia, Facultad de Ciencias, Universidad de Cbrdoba, Cbrdoba, Spain

Received 7 October 1988 Accepted 14 November 1988

Key words: Glutamine synthetase; Photosynthetic bacterium; Amino acid levels; Rhodobacter capsulatus

1. S U M M A R Y

High per formance liquid chromatography (HPLC) has been used to determine the internal levels of amino acids in Rhodobacter capsulatus ElF1 cells, subjected to different treatments and nutritional conditions. Glutamine synthetase ac- tivity and enzyme concentration correlated nega- tively with the level of glutamine, suggesting that glutamine per se acts as a co-repressor in the enzyme synthesis. Moreover, addition of the specific inhibitor L-methionine-D,L-sulfoximine, that produced an increase in enzyme concentra- tion, specifically promoted a depletion of in- tracellular glutamine.

tive) forms, depending on nutritional conditions [1]. GS synthesis is repressed by ammonia ad- dition [2]. In a previous paper we have proposed that glutamine (or a metabolic product of gluta- mine), but not ammonia, acts as a co-repressor in GS synthesis [3]. In this work, we summarize the results obtained when quantifying the internal levels of amino acids in Rhodobacter capsulatus ElF1 cells, subjected to different treatments. Our data suggest that glutamine per se is the direct co-repressor of GS synthesis, ruling out the direct role of other amino acids in the process.

3. MATERIALS A N D M E T H O D S

2. I N T R O D U C T I O N

Glutamine synthetase (GS) (EC 6.3.1.2.) of photosynthetic bacteria is regulated by different control mechanisms. The enzyme can be present in adenylylated (inactive) or deadenylylated (ac-

3.1. Growth of organisms Rhodobacter capsulatus ElF1 was cultured

anaerobically at 30 °C and saturated light condi- tions in the RCV medium described by Weaver et al. [4]. All the nitrogen sources used were added at a concentration of 1 g / l , except dissolved nitro- gen.

Correspondence to: J.M. Roldfin, Departamento de Bioquimica, Biologla Molecular y Fisiologia, Facultad de Ciencias, Uni- versidad de Crrdoba, E-14005 Crrdoba, Spain.

3.2. High performance liquid chromatography The internal pool of amino acids was extracted

from cells at mid log phase of growth, according to the method described by Herbert et al. [5], with

0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies

112

sl ight modif ica t ions . Nor leuc ine was a d d e d as an in terna l s t andard . A m i n o acids were dansy la t ed and separa ted by H P L C accord ing to the p ro toco l of Tapuh i et al. [6]. Quan t i t a t i on of amino acids was pe r fo rmed by fol lowing the abso rbance at 254 nm of their dansy la t ed derivat ives. The concent ra - t ion of a m i n o acids was de t e rmined f rom the ra t io of areas of each pa r t i cu la r amino acid der ivat ive and the in ternal s t a n d a r d area peak.

3.3. Enzyme and protein determinations Crude extracts were ob t a ined as previous ly de-

scr ibed [3]. Mn2+-dependen t t ransferase ac t iv i ty was de t e rmined by the me thod of Shapi ro and S t a d t m a n [7]. A unit of activity, U, is the a m o u n t of enzyme that t r ans fo rms 1 /~mol subs t ra te per minute . Pro te in was quan t i t a t ed by the me thod of Lowry et al. [8], using bovine serum a lbumin as a s t andard .

4. R E S U L T S A N D D I S C U S S I O N

A m m o n i a ass imi la t ion in purp le non-su l fur bac te r ia is p r e d o m i n a n t l y ca ta lyzed b y two en- zymes act ing sequent ia l ly : g lu tamine synthe tase and g lu tama te synthase [1]. G S p lays an i m p o r t a n t role in the regula t ion of n i t rogen ass imi la t ion by these o rgan isms [1,9]'.

On the basis of immunoe lec t rophore t i c mea- surements of G S levels, we have demons t r a t ed tha t a m m o n i a i tself is no t a co- repressor of G S synthesis in R. eapsulatus [3]. However , a l though we p r o p o s e d that such a co- repress ion effect could be exer ted by g lu tamine , the involvement in the process of o ther a m i n o acids, me tabo l i c p roduc t s of g lu tamine , was no t ru led out. The aim of the presen t work was to e luc ida te the poss ib le role of each amino acid in the regula t ion of the enzyme synthesis.

P re l iminary exper iments showed that add i t i on of 2-oxo-glu tara te , subs t ra te of g lu tamate syn- thase, to cell cul tures growing on ammonia , p ro - m o t e d a s ignif icant increase in bo th G S act ivi ty and p ro te in (da ta not shown). This inc rement can be due to a decrease in g lu tamine in ternal levels p r o m o t e d b y the enhancemen t of the g lu t ama te synthase ca ta lyzed reac t ion and suggests the specif ic i ty of g lu tamine as a co-repressor .

Table 1

Glutamine synthetase activity and intracellular amino acid levels in Rhodobacter capsulatus ElF1 grown on different nitrogen sources

Nitrogen sources

KNO 3 NHaC1 N 2 Glu Gin Ala

GS a 2.30 0.67 0.61 1.21 1.04 1.31 Asn - 0.31 0.02 Gin 0.05 0.32 0.47 0.32 0.20 0.14 Ser 0.46 0.13 0.10 0.13 0.33 0.02 Asp 0.13 0.16 0.06 0.13 0.13 - Glu 15 8.08 15.1 6.57 6.35 0.50 Gly 1.33 1.18 0.89 0.90 0.90 0.26 Thr 0.30 0.45 1.18 0.28 0.24 0.13 Ala 1.72 1.95 0.82 1.07 3.37 1.15 Pro 0.35 0.44 0.74 0.39 0.49 0.10 Met 0.11 0.14 0.02 0.04 0.04 0.02 Val 0.63 0.50 0.27 0.39 0.03 0.13 Trp - 0.12 - - 0.01 Arg 0.20 0.29 0.60 0.31 0.27 0.22 Leu 1.15 12.5 11.7 5.7 2.55 3.53

Values are the mean of four different experiments, a GS activ- ity in U/mg protein. Amino acid concentrations in nmoles/mg protein. ( - ) , Not detected. Isoleucine and phenylalanine showed a common peak and are not represented. Data for lysine, histidine and tyrosine are not represented due to their high variability.

Tab le 1 shows the Mn2+-dependen t G S act iv i ty and amino acid concen t ra t ions in cells grown on dif ferent n i t rogen sources. In all cases, g lu t ama te was the mos t a b u n d a n t amino acid in R. capsula- tus cells, as prev ious ly descr ibed in o ther species of bac te r ia [5,10,11]. I t could be due to the centra l role of g lu t ama te in the synthesis of o ther a m i n o acids [12]. Glycine , threonine, a lan ine and leucine also showed high levels when c o m p a r e d with o ther amino acids.

The values of G S t ransferase ac t iv i ty in cells grown on a given n i t rogen source are ind ica t ive of enzyme concen t ra t ion [3]. F r o m d a t a p resen ted in Table 1 it could be p r o p o s e d that, wi th the excep- t ion of cul tures grown in g lu tamate , the higher G S act ivi ty (and prote in) , the lower g lu t amine in- t racel lu lar levels. Cor re la t ion coeff ic ients be tween G S and concen t ra t ion of amino acids ind ica te the highest negat ive cor re la t ion for g lu tamine ( - 0.85), fol lowed by leucine ( - 0 . 8 0 ) . The o ther a m i n o acids showed coeff ic ient values lower than 0.60 (ei ther posi t ive or negative). These resul ts comple -

01 34

P r e

0 1 2 3 4 0 1 2 3 4

GIR Glu

1234 0123~

Gly Tbr

0 1 2 3 ~ 0 1 Z 3 a

Val Le,,

113

Fig. 1. Effect of L-methionine-D,L-sulfoximine (MSX) addition on the intracellular concentration of some free amino acids in R. capsulatus cells. Ammonia grown cultures were supple- mented with 100 ~M MSX. Values in each case are relative to the highest amino acid concentration following the treatment. (0, control before addition; 1, 30 min after addition; 2, one hour after addition; 3, two hours after addition; 4, four hours after addition).

ment the previously reported decrease in GS when glutamine was added to cell cultures [3].

Addition of L-methionine-D,L-sulfoximine (MSX) to R. capsulatus cells promotes a drastic inactivation of GS which is simultaneous with an increase in GS protein [3]. Fig. 1 shows repre- sentative examples of changes in internal amino acid concentrations after treatment of cells with the inhibitor. From all the amino acids that corre- lated negatively with GS (glutamine, threonine, proline, leucine), only glutamine concentration values became negligible along the experiment. Consequently, depression of GS appears to be parallel with the depletion of internal glutamine. The other amino acids studied followed a pattern of concentration changes resembling that ex- hibited by glutamate: after an initial increase, amino acid levels decreased to values similar to the control before addition. The depletion of in- ternal glutamine in Rhodopseudomonas acidophila cells treated with MSX has been previously de-

scribed [5]. However, no relationship of this pro- cess with GS de-repression was proposed.

In conclusion, the data presented suggest that co-repression of GS synthesis in R. capsulatus ElF1 is specifically exerted by glutamine. The direct involvement of other amino acids in the process can be ruled out.

ACKNOWLEDGEMENTS

We thank Dr. J. Diez Dapena for his critical reading of the manuscript and very helpful discus- sions.

Supported by Comisi6n Asesora de In- vestigaci6n Cientifica y T6cnica, Spain (Grant No. PB-086-0167-CO3-02) and Instituto Nacional de la Salud, Spain, which also provided a fellowship for F.R.

REFERENCES

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