Soluble and membrane-bound nitrate reductase from Bradyrhizobium japonicum bacteroids

Plant

Phyviol.

Biochen~.

, 1998,

36 (4). 279-283

Soluble and membrane-bound japonicum bacteroids Maria

J. Delgado’,

Manuel

FernBudez-Lbpez2,

nitrate reductase from Bradyrhizobium Eulogio

J. Bedmar’*

’ Departamento de Microbiologia de1 Suelo y Sistemas Simbioticos, Estacion Experimental de1 Zaidin, CSIC. P.O. Box 419. 18080 Granada, Spain. ’ Present address: V.I.B. Department of Genetics. Liniversiteit Gent, K.L. Ledeganckstraat 35. 9000 Gent, Belgium. * Author to whom correspondence should be addressed (fax 34 58 129600: e-mail [email protected]) (Received May 30, 1997; accepted July 26. 1997)

Abstract - Nitrate reductase (NR) activity was detected in the cytosol and membranes from bacteroids of Bradyrhizobiumjqonicum strain L-236 isolated from nodules of soybean (G!vcirze max. Merr., cv. Williams) plants that were only Ns-dependent. The molecular mass (M) of the NR from each source was estimated by gel filtration to be about 76 kDa. Treatment of plants with 20 mM KNO, induced NR activity in the bacteroid cytosol, but did not affect rates of activity in the membranes. Furthermore, the M of the cytosolic enzyme was 160 kDa, while that of the NR from the membranes was 76 kDa. When bacteroids that had been isolated from nodules of plants not receiving nitrate were incubated microaerobically (2 % O2 v/v) in both the absence and presence of nitrate, the M of the membrane-bound NR enzyme was calculated to be 230 kDa: in bacteroids incubated in the absence of nitrate. the M of the soluble enzyme was 76 kDa, whereas that of bacteroids incubated with nitrate was about 160 kDa. Incubation of bacteroids under aerobic conditions with nitrate induced NR activity in the soluble fraction, and the M of the enzyme was 160 kDa; in the absence of nitrate the M of the soluble NR was 76 kDa. No NR activity was found in membranes from aerobically incubated bacteroids. 0 Elsevier, Paris Bacteroids

I symbiosis I microaerobiosis

I nitrate

I nitrate

reductase

I Bradyrhizobium

M, molecular mass I NR, nitrate reductase I PMSF, phenylmethyl-sulphonyl Tris, 2-amino-2-(hydroxymethyl)-1-3-propanediol.

Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite. the initial step of nitrate utilization. In the Rhizobiaceae, the metabolism of nitrate may occur by assimilation via ammonia into cellular nitrogen: bradyrhizobia speciesmay also, in the absenceof oxygen, denitrify nitrate [24]. Some legumes,such as Phaseolus. Pisum and Lotus sp., expressNR activity only in the soluble fraction of their nodules, whereas Glycine, Vignu and Medicago sp. may exhibit NR activity in both the bacteroids and the nodule cytosol [ 1, 151.Evidence that bacteroids of many Rhizobium and Brudyrhizobium speciescontain both soluble and particulate NR has also been obtained [ 1, IS]. The presence of constitutive (nonnitrate induced) NR in bacteroids was first demonstrated by Evans 171:Kennedy et al. [ 1I] subsequently showed that NR activity was present in both soluble Physiol.

Biochem..

0981-9428/98/04/O

I Glycine max.

I PVPP, polyvinylpolypirrolidone

I

and membrane-bound fractions of B. japonicum bacteroids.

1. INTRODUCTION

Plant

fluoride

japonicum

Elsevier.

Paris

The soluble NR from bacteroids of B. ,japyonicum hasbeen partially purified, and its molecular mass(M) was about 70 kDa, as estimated by gel filtration [ 111. With similar procedures, the M of the soluble NR from microaerobically nitrate-grown cells of B. ,juponicum was calculated to be 69 kDa [4], and that of R. melifoti cells grown aerobically with nitrate was 58 kDa [22]. More recently, an M of 236 kDa has been reported for the membrane-bound NR in cells of B. juponicum cultured microaerobically with nitrate [6]. In contrast to free-living cells, no data exist in the literature on the M of the membrane-bound NR in the symbiotic forms of the Rhizobiaceae. In this paper we report on the acti-vity and estimation of the constitutive and nitrate-induced, soluble and memhranebound NR enzymes from B. jqwnirum bacteroids.

280

M. J. Delgado

2. RESULTS

et al.

AND DISCUSSION

As indicated by earlier work [ 1 I], NR activity was present in both soluble (cytosol) and particulate (membrane) fractions from bacteroids of B. ju~~~nicum L-236 isolated from root nodules of soybeans that were only N2-dependent. The specific activity of the cytosol was 3 times higher than that of the membranes (table I). The elution pattern from Sephacryl S-200 columns showed that only a single NR was present in each soluble and particulate fraction, and indicated that the M of the enzyme from either source was about 76 kDa (ruble I). This molecular mass is close to that of 66 for kDa the constitutive (non-nitrate induced) soluble NR found in B. juponicum bacteroids [ 1 I]. When soybean plants were supplied with 20 mM KNO,, the specitic activity of the soluble NR from bacteroids was induced 6.9-fold, as compared with that found in the cytosol of bacteroids isolated from plant nodules not treated with nitrate. while that of the membranes remained unaffected (tuble I). Although previous work has established that exposure of soybeans to high (IO-20 mM) KNO, induces NR in bacteroids [2], no data were presented to show whether the soluble or particulate fraction could be responsible for the increase in bacteroidal NR activity. Results in table I indicate that such an increase was due to the enhancement of cytosolic NR. Application of the membrane fraction of bacteroids isolated from plant nodules treated with nitrate to the Sephacryl column, yielded a peak similar to that of the cytosol and of membranes of bacteroids isolated from plants not treated with nitrate, with a corresponding M of about 76 kDa (ruble I). However, the elution profile of the

Table I. Soluble isolated aerobically protein.

and membrane-bound from root nodules of G/yrine and microaerobically [2 % Values represent the mean SD

cytosolic fraction indicated that the M of the enzyme was about 160 kDa (table 0. Incubation of bacteroids isolated from nodules of N,-dependent soybeans under 2 % (v/v) O2 in a medium without nitrate did not affect rates of specific activity of the soluble NR enzyme, as compared with those found before incubation, but there was a 23-fold increase in the activity of the membrane-bound NR. This result agrees with those indicating that the presence of nitrate, or other nitrogenous oxides, is not always necessary for induction of particulate NR. and that significant levels of enzyme may be present as a consequence of microaerobiosis [IO]. When nitrate was included in the microaerobic incubation medium there was a 6-fold and a 54-fold increase in rates of soluble and particulate activity, respectively, as compared with those found before incubation (ruble I). After microaerobic incubation, in the absence of nitrate in the incubation medium, the elution pattern for the enzyme activity in the soluble fraction of bacteroids revealed that the M of the enzyme was 76 kDa (table I). However, the elution profile of the cytosol from bacteroids incubated microaerobically with nitrate indicated that the M of the NR enzyme was about 160 kDa (table I). Microaerobic incubation of bacteroids, regardless of the presence or the absence of nitrate in the incubation medium, also produced a change in the M of the membrane-bound NR, which was estimated to be 230 kDa (table I). No NR activity was detected in membranes from aerobically-incubated bacteroids, regardless of the presence or the absence of nitrate in the incubation mixture (table I). After aerobic incubation in the pre-

nitrate reductase (NR) activity and their molecular mass (MI in bacteroids of 8. jq>on&m. Bacteroids were MU plants treated or not with 20 mM KNO,. Bacteroids from N2-dependent plants were further incubated O2 (v/v)] for 36 h in a medium with or without 5 mM KNO,. Activities are expressed as nmol NOE.h-‘.rng-’ of three replicates. Molecular mass (M) is expressed in kDa. nd. not detected.

Treatment

Fraction Cytosol NR

Bacteroids Incubated Incubated Incubated Incubated Incubated lncuhated Bacteroids

from Nz-dependent plants microaerobically without nitrate microaerobically with nitrate microaerobically f nitrate plus 100 mg.ml-’ chloroamphenicol aerobically without nitrate aerobically with nitrate aerobically f nitrate plus 100 mg.ml ’ chloramphenicol from plants treated with 20 mM KNO,

activity

3.2 k 0.3 3.4 5 0.5 20.4 r 3. I nd 2.2 f. 0.2 46.2 f 3.x nd 22.2 + 3.2

Membranes M

NR

76 76 I60

l.I* 0.3 25.2 f 4.1 60.0 + IO3 nd nd nd nd 1.2+0.1

76 I60 I60

activity

M 76 230 230

76

Nitrate

sence of nitrate the soluble fraction had 21 times higher specific NR activity than that from bacteroids incubated without nitrate (table 0. The elution pattern for the enzyme activity from bacteroids incubated with nitrate indicated that the M of the enzyme was 160 kDa, whereas that of the enzyme from bacteroids incubated without nitrate was 76 kDa (ruble r). Inclusion of chloramphenicol in the aerobic and microaerobic incubation medium, with or without nitrate, prevented the development of NR activity in cytosol and in membrane fractions, and negligible values of NR activity were detected after incubation (table Z). Chloramphenicol itself did not inhibit NR activity as addition of the antibiotic to assay reactions containing samples expressing NR activity gave similar values to those found without antibiotic (data not shown). Substitution of chloramphenicol for rifampitin in the incubation medium gave similar results (data not shown). Inhibition of induction of NR activity by chloramphenicol and rifampicin indicated a de novo synthesis of the 160 kDa and the 230 kDa NR enzyme species. Rigaud [ 191 and Giannakis et al. [9] have also demonstrated an induction of NR activity in microaerobic preparations of Phaseolus vulgaris and Glytine max bacteroids, respectively, when incubated for at least 10 h. There are two main types of NR in bacteria associated with nitrate assimilation and dissimilation, respectively [3, 10, 231. Previous researches have established that 0, is the main factor controlling synthesis of particulate NR enzymes in Rhizobium and Bradyrhizobium grown aerobically, anaerobically, or symbiotically [4, 171. From our results, both microaerobiosis and the presence of nitrate were required for full expression of the cytosol activity and that of the membrane-bound NR. Vairinhos et al. [24] and Delgado et al. [6] also noted that high rates of NR activity in free-living cells of B. japonicum depended on the presence of nitrate. However. the fact that microaerobiosis alone induced the membrane-bound NR activity and did not affect rates of activity of the soluble enzyme in microaerobically-incubated bacteroids (table I), suggests that the membrane-bound enzyme could be involved in nitrate respiration. Since nitrate was required for induction of soluble NR in bacteroids incubated either aerobically or microaerobically, and in bacteroids from plants treated with nitrate (fable I), it is possible that the soluble enzyme could be implicated in nitrate assimilation. Our results also indicate that the constitutive (non-nitrate induced), 76 kDa NR enzyme found in cytosol and in

reductase

in B. japonicum

281

bacteroids

membranes from N,-dependent plants may not be operational under physiological conditions. This suggestion is based on the fact that, except for the membrane-bound enzyme in bacteroids from nitrate-treated plants, the M of the enzyme in the soluble fraction was 160 kDa, and that of the particulate fraction was 230 kDa under all other conditions examined (table I). Moreover, a 76 kDa enzyme was never detected together with the 160 kDa and 230 kDa enzymes after Sephacryl elution. The reason why NR activity was induced in bacteroids after isolation and microaerobic incubation in both the absence and the presence of nitrate, and the lack of a similar response in situ, with nitrate supplied to the root medium (table I) cannot be deduced from the present results. Although bacteroids are capable of nitrogen fixation using nitrate as an electron acceptor for energy generation [21], since 20 mM KNO, treatment inhibits nitrogenase activity in nodulated soybean plants [2], the functioning of the ATP-producing, respiratory NR would be no longer required. In constrast, the assimilatory, soluble NR would be induced to provide the plant with ammonia coming from nitrate reduction. It might also be possible that some other conditions in pfuntu prevent induction of the membrane-bound NR. In fact, it has been shown that symbiotic baterial genes can be regulated by the host plant [8].

3. CONCLUSION Both soluble and membrane-bound NR activity can be found in bacteroids isolated from N,-dependent soybean plants. Being a substrate-inducible enzyme, nitrate utilization by bacteroids would result in an increase of the activity of each or both NR enzymes. We conclude that nitrate is metabolized by the soluble NR because no induction of the membrane-bound NR activity was observed after nitrate treatment of nodulated soybeans. Bacteroids from nodules of N,dependent plants showed an increase in both soluble and membrane-bound NR after isolation and further incubation under microaerobic conditions in the presence of nitrate. Thus expression of bacteroidal membrane-bound NR may be under the control of the host plant. vol.

36 (4)

1998

282

M. J. Delgado et al.

4. METHODS 4.1. Bacterial

strain and culture conditions. Bv~al\,vhi:~~ strain L-236 (51 was used in this study. Bacteria were grown aerobically in liquid LMB medium [ 131 foi 5 d at 28 “C. The cells were collected by centrifugation at 8 000 x g at 4 “C for IO min. washed twice in SO mM TrisHCR buffer {pH 7.5). and finally resuspended in 5 ml (I 0 cells.ml- ) of the same buffer before using for inoculations. hiutnjaponicum

4.2. Plant material and growth conditions. Seeds of soybean (G/ycine ~ZLI.YL. Merr., cv. Williams) were xurface-sterilized with 95 %j (v/v) ethanol for 3 min. rinsed thoroughly with sterile water and germinated before planting in autoclaved I -I Leonard jars assemblies filled with vermiculite. Plants (2 plants perjar) were inoculated at sowing (I ml, cell suspension per plant) and provided with sterile N-free nutrient solution [201. Five daya before the measurements, a set of plants was fed with the same nutrient solution supplemented with 20 mM KNO,. Plants were grown in a controlled envronmental chamber under conditions previously described 1st. 4.3. Bacteroid isolation and incubation conditions. Nodules (SO g fresh weight) were harvested from S-week-old plants. freed of vermiculite, surface-sterilized by rinsing several times with sterile water before and after immersion for 30 s in SO Q’ ethanol (v/v). Bacteroids were prepared by grinding nodules with SO mM Tris-HCI buffer (pH 7.5) containing 200 mM sodium ascorbate and 2.5 mM MgCI, at pH 7.5. The homogenates were mixed with PVPP ( Ii? OF the nodulea fresh weight), filtered through l’our layers of cheesecloth and centrifuged at 250 x R at 4 “C for 5 min to remove nodule debris. The resulting xupernatant was recentrifuged at 8 000 x s (4 “C l’or IO min). washed twice in SO mM TrisHCI buffer (pH 7.5). and finally resuspended in 40 ml of the same buffer supplemented wjith I mM PMSF to inhibit proteolysis. To determine the effects of oxygen and nitrate on soluble and membrane-bound NR activity, bacteroida were incubated aerobically and microaerobically [0,/A] (2:Y8. v/v)] at 28 “C for 36 h in a medium containing (per ml): 32.5 mmol Tris-HCI (pH 7.5). 5 mmol KNO,. 5 mmol glucose. 5 mmol sodium succinate, and 0.2 ml of bacteroids (0.3-0.5 mg protein) 16, 91. After incubation. the bacteroidz were collected by centrifugation as above, washed with SO mM Tris-HCI (pH 7.5) until nitrite could not bc detected in the supernatant. then resuspended in the hame buffer supplemented with I mM PMSE 4.4. Preparation of soluble and membrane fractions. Bacteroids were passed twice through a French press at about I20 MPa. Unbroken bacteroidx were removed by centrifugation at 8 000 x $ for IO min at 3 “C. Membrane and cytosol fractions were prepared hy further centrifupation of the supernatant al 250 000 x g for I h at -I “C. When required. the final supernatant was concentrated to about 5 ml by pressure filtration over an Amicon XM-SO mem-

brane. The membrane pellet was washed once with SO mM Tris-HCI (pH 7.5). and dispersed by syypension to a final protein concentration of about 10 mg.ml in identical buffer containing 4 5% (w/v) Triton X-100 and 0.1 mM PMSF. Following incubation on ice for I5 min, the solubilized membranes were obtained after removing of Triton X-100 insoluble material by centrifugation at 270 000 x g for 30 min. 4.5. Determination of molecular mass of nitrate reductases. Aliquots (5 mL) of the cytosol or Triton X-100 solubilized membranes from bacteroids were supplemented with 25 % (w/v) glycerol and, separately, applied to a Sephacryl S-200 column (2.6 x 100 cm. Pharmacia-LKB) equilibrated w’ith 50 mM Tris-HCI buffer (pH 7.5) added with I %a (w/v) Triton X- IO0 (for Triton X-I OO-solubilized membranes) oi not (for the cytosol fraction). Proteins were eluted with SO0 ml of the same buffer at a flow rate of 30 rnL,h-‘, and 3 ml fractions were collected. Nitrate reductase-containing fractions were pooled and concentrated to about 3 ml by pressure filtration as above. and stored at -25 “C. All chromatographic steps were carried out at 4 “C. The column was calibrated with Dextran Blue and the following marker proteins: catalase (232 kDa). y-globulin (160 kDa). bovineserum albumin (68 h-Da). o\albumin (43 kDa) and chymotrypsinogen (25 kDa). A plot of log M against the partition coefficient [ 121 was used to determine the M of the nitrate reducta\c enrymes. 4.6. Enzyme assay and protein determination. NR activity was assayed at 30°C by measurin, cr the reduction of nitrate to nitrite with dithionite-methyl viologen as the electron donor 161. The reaction was started by addition of the dithionite and terminated after S-IS min by vigorous shaking until samples had lost their blue colour. Nitrite was estimated after diazotation by adding the sulphanilamide-naphthylethylene diamine dihydrochloride reagent [ 161. Protein was determined as described by Markw,ell et al. j 141 using bovine serum albumin as a standard protein. Acknowledgements. Financial support was obtained from the Direccicin General de Jnvestigaci6n Cientifica y Tecnica (DCICYT), Grant No. PB94-01 17.

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vol. 36 (4) 1998