FEMS Microbiology Letters 119 (1994) 209-214 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05993

Ornithine cyclodeaminase activity in Rhizobium meliloti

Maria Jos6 Soto, Pieter van Dillewijn, Jos6 Olivares and Nicolas Toro *

Departamento de Microbiolog£a, Estacidn Experimental del Zaidln, Consejo Superior de Investigaciones Cienffficas, C / Prof. Albareda 1, 18008 Granada, Spain

(Received 16 February 1994; revision received 21 March 1994; accepted 25 March 1994)

Abstract." Deamination of L-ornithine to L-proline by ornithine cyclodeaminase is an unusual enzyme reaction that has been shown to occur in only a few bacteria. Rhizobium meliloti strains GR4, 2011 and 41 are able to use ornithine as the sole carbon and nitrogen source. The main pathway of ornithine utilization in strain GR4 depends on ornithine cyclodeaminase activity. In addition, this enzymatic activity has been found to be dependent on NAD ÷ and L-arginine similar to Agrobacterium ornithine cyclodeami- nases. The ornithine cyclodeaminase activity is also expressed in R. meliloti strains 2011 and 41 growing with L-ornithine.

Key words: Rhizobium meliloti; L-Ornithine; L-Proline; Ornithine cyclodeaminase

Introduction

Ornithine cyclodeaminase (EC 4.3.1.12; OCD) converts L-ornithine directly into L-proline with the release of ammonia. This unusual enzymatic activity has been demonstrated in some anaerobic bacteria [1,2], in two Pseudomonas strains [3,4], in Agrobacterium tumefaciens [5-8] and recently in Rhodobacter [9]. In A. tumefaciens, OCD cat- alyzes the last step in a pathway leading from nopaline or octopine through L-arginine and L- ornithine to L-proline. The reaction mechanism, although not well understood, requires NAD ÷ as cofactor and is regulated by L-arginine. Nopaline

* Corresponding author. Tel: (58) 121011; Fax: (58) 129600.

and octopine tumor inducing (Ti) plasmid-en- coded OCDs have many properties in common, but there are also significant differences in the deduced amino acid sequences and in the regula- tion of enzyme activity [7]. It has recently been reported that mammalian/z-crystallin shows simi- larity at the amino acid sequence level with the ornithine cyclodeaminases encoded by the Ti plasmid of A. tumefaciens. However, no enzy- matic activity was detected with kangaroo lens /z-crystallin under conditions optimal for Agrobacterium OCDs [10]. Similarly, in Rhizo- bium meliloti strain GR4, the product of the recently identified nodulation efficiency nfeC lo- cus located on large plasmid pRmeGR4b, showed homology with the Agrobacterium OCDs but did not reveal any activity (N. Toro, unpublished re- sults).

SSDI 0378-1097(94)00139-1

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In this work we report that R. meliloti strain GR4 is able to convert L-ornithine to L-proline by an ornithine cyclodeaminase which shows similar enzymatic properties to that of A. tumefaciens. In vivo studies of ornithine utilization by strain GR4 suggest that the ornithine catabolic pathway pro- ceeds via proline formation. Similar results were obtained with R. meliloti wild-type strains 2011 and 41 indicating that ornithine cyclodeaminase activity could be involved in a general catabolic pathway of ornithine utilization in R. meliloti.

Materials and Methods

Bacterial strains and growth conditions The strains used in this study (Table 1) were

cultured at 30°C in TY medium [11] or minimal medium (MM) [12] with mannitol and L-gluta- mate or just L-ornithine as carbon and nitrogen source.

OCD incubation Enzyme extracts from R. meliloti were pre-

pared as described [5]. Routine assays contained, in a final volume of 20 ~l: 55 /zg of protein, 3 mM EDTA, 0.5 mM NAD +, 0.1 mM L-arginine, 0.1 mM unlabelled L-ornithine, 20 t iM L[1- 14C]ornithine (2 G B q / m m o l , Amersham) and 20 mM potassium phosphate, pH 8.5. After incuba- tion at 30°C for 30 rain, the reactions were

stopped by mixing with 5/~1 of 50% trichloroacetic acid. After removal of precipitates by centrifuga- tion, supernatants were extracted with 50 p.l of chloroform to remove lipids. The evaluation of OCD activity was performed by thin-layer chro- matography, high-voltage paper electrophoresis [6,8] and descending paper chromatography. For the latter the mobile phase used was n-buta- nol : acetic acid : water (12 : 3 : 5). The release of ammonium ion concomitant to proline formation was measured using the glutamate dehydrogenase method as described [6].

Ornithine transaminase Ornithine transaminase (EC 2.6.1.13) assay was

performed and evaluated as the OCD activity, except that 0.4 gmol of 2-oxoglutarate and 0.01 /xmol of pyridoxal-5'-phosphate were added to the reaction mixture.

In vivo utilization of L[1-14C]ornithine IL meliloti cells grown in MM with L-ornithine

as the sole carbon and nitrogen source were suspended in 120/zl of MM containing 2.5/~M of L[1-14C]ornithine at an optical density of 0.5. After 5, 10 and 20 min incubation, extracts from 20 /~1 samples were prepared as described [13]. Electrophoresis was carried out on Whatman 3M paper at 1700 V for 7 min, in water : formic acid : acetic buffer (910 : 30 : 60).

Table 1

Bacterial strains used in this work

Strain Characteristics Source

R. meliloti GR4

G R M 8

41

2011

Wild-type; Nod + Fix+; harbours two large plasmids (pR meGR 4a and pRmeGR4b) in addition to the symbiotic megaplasmids

pRmeGR4a- and pRmeGR4b-cured derivative of GR4 obtained by heat t reatment; Nod + Fix +

Wild-type; Nod + Fix +

Wild-type; Nod + Fix +

This laboratory

This laboratory

A. Kondorosi

J. Denari6

10"

9 . . . . . .

7 o ; io l'S io is

Time (hours)

Fig. 1. Ability of R. meliloti GR4 (zx) and derivative GR M 8 (o ) to grow with ornithine as the sole carbon and nitrogen source ( . . . . . . ) as compared with the same strains grown in

MM with mannitol and glutamate ( ).

Results and Discussion

R. meliloti GR4 is able to use ornithine as the sole carbon and nitrogen source. GR4 cells grown in MM with ornithine showed a doubling time of 340 min, whereas cells grown in MM containing mannitol and glutamate exhibited a doubling time of 240 min (Fig. 1). GR4 pRmeGR4a- and pRmeGR4b-cured derivative strain GRM8 was also able to grow with ornithine as the sole car- bon and nitrogen source. However, this mutant strain showed a slower growth rate as compared with the parental in MM regardless of the carbon and nitrogen sources (Fig. 1). Therefore, the abil- ity of GR4 to utilize ornithine does not depend on any of the two cryptic plasmids harboured by this strain. Other R. meliloti strains assayed such as 41 and 2011 were also able to grow with ornithine as the sole carbon and nitrogen source but they exhibited a slower growth rate to that of strain GR4 (data not shown).

Crude extracts from wild-type GR4 and the cured derivative GRM8 were assayed in vitro for ornithine cvclodeaminase activity. Assays per- formed with crude extracts from cells grown in MM with mannitol and glutamate were negative and no utilization of L[1-14C]ornithine occurred. However, when we used crude extracts from el-

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ther GR4 or GRM8 cells grown with ornithine as the sole carbon and nitrogen source the forma- tion of a product which comigrates with proline was observed. Formation of proline was concomi- tant with the release of equimolar amounts of ammonia. Crude extracts could be frozen and thawed without significant loss of activity. How- ever, the conversion rates of ornithine into pro- line differed from one crude extract preparation to another. In addition, conversion of ornithine into proline in extracts from either wild-type GR4 or mutant GRM8 was dependent on NAD ÷ and L-arginine (Fig. 2) as occurs with the ornithine cyclodeaminase activity of A. tumefaciens. The apparent K m value (2 mM) for ornithine in the presence of 0.5 mM NAD + and 0.1 mM L-arginine was similar for GR4 and GRM8 strains, suggest- ing that the enzymatic activity detected was the same.

Ornithine can be metabolized by several en- zyme reactions, and most often conversion of ornithine into proline proceeds by transamination t o glutamic acid via 1-pyroline-5-carboxylate [8]. However, these intermediate products were not detected in our assays. Furthermore, when we added pyridoxal phosphate and 2-oxoglutarate to the reaction mixture, required for the transami-

V

8 "

7-

~ 6 - Q.

~,5-

t l .

~ 3 -

~E2-

0 5 10 15 20 25 30 Tlme (mi nutes)

Fig. 2. OCD in vitro activity on extracts of R. meliloti GR4 (zx) and GRM8 (o) grown in MM medium with ornithine as the sole carbon and nitrogen source. The reaction mixture contained NAD ÷ 0.5 mM and L-arginine 0.1 raM. Data are

normalized to nmol/mg protein.

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1 2 3

P r o - - ~

Or.---- |

Fig. 3. Ornithine utilization in vivo by R. meliloti GR4. Identi- fication of intermediates of ornithine catabolism. GR4 (lane 1) and GRM8 (lane 2 and 3). Similar results were obtained after 5, 10 and 20 min incubation with laC-labelled ornithine.

Orn, ornithine; Pro; proline.

nation reaction, no labelled products derived from L[1-14C]ornithine utilization were observed. Therefore our results indicate the presence of an ornithine cyclodeaminase in GR4 and GRM8 cell extracts.

To test whether utilization of ornithine in vivo by strain GR4 proceeds via proline, cells of GR4 and the derivative strain GRM8 were pre-grown on ornithine, exposed to L[1-14C]ornithine for 5, 10 and 20 min, and extracted with acetic acid. The cell extracts from either wild-type GR4 or derivative mutant GRM8 contained only labelled ornithine and proline (Fig. 3). These results strengthen the idea that the cyclodeaminase is involved in the main pathway of ornithine degra- dation in these bacterial strains.

Similar to strain GR4, the conversion of or- nithine into proline was also detected in extracts of R. meliloti strains 2011 and 41 (data not shown). The fact that this catabolic activity is not exclu-

sive for GR4 and that it can be detected in other R. meliloti strains indicates that OCD may repre- sent the main pathway of ornithine metabolism in this rhizobial species.

The significance of ornithine metabolism, and particularly of this OCD activity in the Rhizo- bium-legume symbiosis is unknown. In Gram- negative bacteria, proline plays an important role in maintaining the osmotic equilibrium and may alter the permeability of the cytoplasmic mem- brane, allowing solutes to permeate the cells more easily [14]. In plants, ornithine is a required inter- mediate for the biosynthesis of arginine, and it is also utilized in the biosynthesis of other plant secondary products such as alkaloids and poly- amines [15]. It has been suggested that proline metabolism in legume root nodules could supply an important fraction of the energy requirement for N 2 fixation [16]. Further investigations of R. meliloti OCD should provide interesting insights into the importance of the ornithine catabolic pathway in the Rhizobium-legume symbiosis.

Acknowledgements

We thank J. Sanjuan for comments on, and corrections to, the manuscript. This work was supported by Comision Asesora de Investigaci6n Cientlfica y T6cnica, Grant BIO93-0677 and by the EEC Bridge Contract BIOT-0159-C.M.J.S was supported by a MEC fellowship.

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