Immunological studies of glutamine synthetase in Frankia-Alnus incana symbioses

FEMS MicrobioltlgyLetters 91 (1992) 141-146 a'~ It~92 Federation of European Microbiological Societies I}378-10t~7/t~2/$05.(l~) Published by Elsevier

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FEMSLE 04792

Immunological studies of glutamine synthetase in Frankia-Alnus incana symbioses Per-Olof Lundquist and Kerstin Huss-Danell Department o]"Plant Phvsiolot,%Unit'ersityof UInet~i.Umct[, Sweden (Received 21 October I~)t)l) (Revision received 23 December It;91) (Accepted 27 December It~t~l)

Key words: Actinorhiza; Ammonium assimilation; Immunodetection; Nitrogen fixation; Root-nodule symbiosis; Western blot; Frankia; AInus htcana

1. SUMMARY We have investigated the presence and form of glutamine synthetase (GS) in Frankia vesicle cluster preparations of two actively nitrogen-fixing Frankia-Ahu,s h~cana root-nodule symbioses and in cultured Frankia sp. strain Cpll (HFP070101). The symbioses contained Frankia Cpii or the local source of Frankia. We used Western-blot analysis with antisera raised against three types of GS. in symbiotic Frankia GS protein was not detected at a significant level when either antisera against Rhodospirillum n~bnim GS or antisera against Rhizobium meliloti GSll were used. In cultured Frankia Cpll GS1 was detected both when grown with NH]- or N 2 as nitrogen source, and GSII was detected when grown on N 2. Antiserum raised against the nodule-specific GSnl o f Phaseohls t'ulgaris crossreacted with a 43-kDa polypeptide corresponding to plant GS in root-nodule extracts from Abuts, and with a 41Correspondence to: P.-O. Lundquist, Department of Plant Physiology, University of Ume~, S-901 87 Ume~, Sweden.

kDa polypeptide corresponding to GSII in cultured Frankia Cpll grown on N,. We conclude that both GS! and GSII are repressed in symbiotic Frankia and that NH~ produced through nitrogen fixation is assimilated by the plant in Frankia-Ahuls h~cana symbioses. It thus appears that vesicle formation, synthesis of nitrogenase and synthesis of GS are separately regulated in symbiotic Frankia and that the plant has to supply symbiotic Frankia with organic nitrogen in some form in addition to the carbon.

2. INTRODUCTION

Frankia is an actinomycete forming so-called actinorhizal, N2-fixing, root-nodule symbioses, but can also fix nitrogen for its own growth in culture. In cultures of Frankia sp. strain HFPArI3 grown on N 2 and labelled with 13NH~ the main NH~ assimilation was through glutamine synthetase (GS) [1]. Frankia sp. strain Cpll, as well as Rhizobhtm spp., Bradyrhizobium japonicum and Agroba~terium spp. which also interact with plants, and the actinomycetc Streptomyces spp.,

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have two different forms of GSs, GS1 and GSll [2-5]. GS! of Frankia and rhizobia are similar to the common prokaryotic GS. GSI of Frankia consists of 12 subunits of about 59 kDa [3] and is expressed during growth under both nitrogen-rich and nitrogen-starved conditions [6]. In contrast, GSII consists of 8 subunits of about 36 kDa for Rhizobhmz meliloti [7] to about 43 kDa for Frankia [3] and is derepressed in conditions of nitrogen starvation [3,6]. GSIi genes have high sequence similarities to the genes of eukaryotic GSs [8,9]. In a nitrogen-fixing symbiosis, the host benefits from the symbiont by utilizing the fixed nitrogen. Assimilation of ammonium in Frankia symbioses is however poorly known. When GS was immunolocalized in Ahms gluthlosa root nodules using antibodies made against the chloroplast localized GS of spinach, crossreactivity was found in the plant part of the infected cells [10]. Purification of GS from A. gluthlosa root nodules yielded two fractions where the major fraction was suggested to be of plant origin and the minor fraction was not identified [10]. Studies on GS and glutamate synthase activities in preparations of symbiotic Frankia failed to detect activity [11]. The aim of the present study was to immunologicaily investigate and characterize the presence of the different forms of GSs in a strain of cultured Frankia and in two Frankia-Ahlus utcana symbioses. For comparative purposes, soybean and E. colt were also included in the study.

(Cat. No. HFP()7t)i01)was grown on it succinate medium with ammonium [15] on a rotary shaker at 120 tom and 30°C. Cultures were transferred to new NH~-containing medium or to medium with only N 2 as nitrogen source, and were collected and washed with sterile distilled water on a 100-ram nylon filter after 3 and 4 days, respectively. Soybean (Glycine max) plants, inoculated with Bradyrhizobiton japonicum (Baliyfixt!aboratoriet, Swedish University of Agricultural Science, Uppsala, Sweden), were grown in perlite with additions of an N-free nutrient solution for 8 weeks in a greenhouse.

3. MATERIAL AND METHODS

3.2 Protein extracts Nodules (0.5 g) were frozen in liquid nitrogen and homogenized in a mortar to a fine powder. After addition of 3 ml of anaerobic buffer containing 4% PVPP, 50 mM Tris. HCI (pH 7.5), 5 mM DTT, 2 mM EDTA and 1 mM Na2S204, the suspensions were centrifuged at 20(10 g for 5 rain and the supernatant again at 100(10×g for 5 min. Proteins were precipitated in 8(1% acetone at -20°C for 3 h, pelleted at 10000×g for 5 rain, evaporated to dryness and dissolved in 200 ml of 2.5% SDS, 50 mM Tris. HCI (pH 8.0) and 1 mM EDTA. Protein from Frankia vesicle cluster preparations and Frankia cultures were extracted as described earlier [13]. Purified E. colt GS was purchased from Sigma. Protein concentrations were determined with the bicinchoninic acid assay (Pierce; the 37°C-protocol) with bovine serum albumin as reference.

3.1. Plant material, Frankia resicle clusters and Cldlllres Rooted cuttings of grey alder (Ablus hwana(L.) (Moench) inoculated with the local source of Frankia [12] or with Frankia Cpll (see below), were grown in a climate chamber for 9-12 weeks as described earlier [13]. Frankia vesicle clusters were prepared from root nodules as described earlier [13,14] and in addition further washed five times in 0.1% Triton X-100, 50 mM Tris. HCI (pH 8.0), 5 mM DTT and 1 mM EDTA by resuspending and pelleting at 10000×g for 5 rain at 4°C. Cultured Frankia sp. strain Cpll

3.3. Western-blot analysis SDS-PAGE (12% T, 2.7% C) in a MiniProtean II Dual Slab Cell (Bio-Rad), blotting and immunochemical detection were as described earlier [13]. That equal amounts of protein were loaded to each lane of the SDS-PA gels was evident from Coomassie and silver-stained gels (data not shown). Biotinylated Iow-molecuJar mass markers were detected with an avidin-alkaline phosphatase conjugate (Bio-Rad). The primary antisera were as d.escribed in the figure legend.

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4. RESULTS

4.1. Glutamhze synthetase I Antiserum made against GS of fhc phototrophic bacterium Rho. ntbmm was used to investigate the presence of GS! protein. The antiserum crossrcacted well with a 57-kDa polypeptide of extracts from Frankia Cp! I culture grown with NH~- or with N, as nitrogen source (Fig. IA, lanes 5 and 6). The crossreaeting band of purified E. coli GS (data not shown) ,,,,as of the same molecular mass as that of Frankh~, thus confirming the similarity. No crossreaction was detected to any polypeptide of the extract from Frankia Cpll grown in symbiosis (Fig. IA, lane 4). The local source of Frankia grown in symbiosis (Fig. IA, lane 3) showed a faint crossreaction for a polypeptidc of about the same molecular mass as the crossreacting polypeptide of Cpll cultures (Fig. 1A, lanes 5 and 6). This may indicate the presence of a GSI polypeptide in this source of symbiotic Frankia, although at a much lower concentration than in Cpll in culture. The local source of Frankia was studied only as a symbiont since isolation and reinoculation of isolates have not yielded the

A

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original phenotypcs hydmgenase ( - ) , spore ( + ) in symbiosis [12]. The crossreactive polypeptides of about 61 and 59 kDa in the extract of the local source of Frankia (Fig. IA, lane 3) may be due to unspecific binding to abundant polypeptides present only in this source of Frankia. An SDS-polyacrylamide gel stained for total protein (data not shown) showed that the two types of symbiotic Frankia indeed had a slightly different protein pattern in this region. No crossreaction was detected to the soybean nodule extract using the anti-Rho, rubn,n GS antiserum (data not shown).

4.2. Ghaamine synthetase 11 Specific crossreaction of the antiserum made against GSII of Rhi. meliloti was detected only to a 41-kDa polypeptide, most likely being GSII of Fmnkia, in extracts of N_,-grown Cpll culture (Fig. IB, hme 5). In the first report of GSII in Frankia the subunit molecular mass was shown to be 43 kDa [3]. in the present study a higher total acrylamide concentration and other mo!ecular mass standards were used. The deduced amino acid sequence [9] gives a calculated subunit molecular mass of 42 kDa. Similar crossreaction as to the N~-grown Cpll culture was neither

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21.5 " ~ 14.4 : Fig. 1. A. Western blot using antiserum raised against GS of Rhodospirillum mbnon. Lane I, biotinylated molecular maw markers 14.4-97.4 kDa; lane 2, extract of A. bwana root nodule containing the local source of Frankia: lane 3. Frankia local ~mrce vesicle ,.'luster; lane 4, Frankia Cpll vesicle cluster; lane 5. Nz-grown Fnmkia Cpll euhur¢: lane 6, NH,~-grown Frankia Cpll culture. The arrow indicates the position of 57 kDa. B. Western blot using antiserum raised against GSil of Rhizobium mefiloti [7]. Lanes. see A. The arrow indicates the position of 41 kDa. Total protein added to each lane was 4.0 rag. All samples were also analysed using only 1.3 mg total protein giving similar results but with lower intensities of all bands.

144

A

kDal 97.4 66.2

2

3

4

5

6

8 1

2

3

4

5

6

N

45.0 , ; ll!ll mA'D a m l

31.0 --21.5 ...14.4 ....

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Fig. 2. Western blot using antiserum raised against GSnl of Plut~eolus" cu/garis [27]. A. Lanes, see Fig. IA. The upper arrow and the lower arrow indicate the positions of 43 kDa and 41 kDa, respectively. Total protein added to each hme were 4.(1 rag, All samples were also analysed using only 1.3 mg total protein giving similar results but with lower intensities of all bands. !1. Lane I, biotinylated molecular mass markers 14.4-97.4 kDa; lane 2, Frankia Cpll vesicle cluster; lane 3, Frankia Cpll vesicle cluster mixed (4:1) with N2-grown Frankia Cpll culture: hme 4. N a-grown Frankia Cpll culture; lane 5, N2-grown Frankia Cpll culture mixed (1 : 1) with A. incana root-nodule extract; lane 6. A. incana root-nodule extract. Arrows as in A. Total protein added to lanes 2 and 3 were 4.(} mg and ta lanes 4 to 6 were 1.3 mg.

detected to extracts of NH~--grown cultures (Fig. IB, lane 6), nor to extracts of symbiotic Frankia of both strains tested (Fig. IB, lanes 3 and 4), A. btcana nodules (Fig. IB, lane 2) and soybean nodules (data not shown). The bands between 55 kDa and 66 kDa (Fig. IB) were most likely artifacts since they appear equally intense in all samples including the molecular mass markers. In Western blots where antiserum raised against the nodule-specific form of plant GS (GS,t) was used, crossreaction was again detected to a 41-kDa polypeptide of N2-grown Cpll culture (Fig. 2A, lane 5). This can be explained by the high similarities between the amino acid sequences of GSII and plant GSs [8,9]. Faint crossreaction with antiserum against GSnt to vesicle cluster preparations was detected for both Frankia strains (Fig. 2A, lanes 3 and 4). To examine this further, the extract of N2-grown Cpll was mixed with either the Cpll vesicle cluster extract or the A. incana root-nodule extract (Fig. 2B). This comparison showed that the crossreacting polypeptide of vesicle clusters (Fig. 2A, lanes 3 and 4, Fig. 2B, lane 2) was most likely contaminating plant GS and that no polypeptide corre-

sponding to GSII could be detected in symbiotic Frankia using the anti-GS,t antiserum. 4.3. Plant glutambze synthetase Intense crossreaetion of the anti-GS~t antiserum to a 43-kDa polypeptide was detected for extracts of soybean (data not shown) and A. incana nodules (Fig. 2A, lane 2). The molecular mass of these polypeptides corresponds well with the subunits of legume GSs [16] and GS of A. glutinosa nodules [10], The faint crossreaction of anti-GS.nl to purified E. coli GS (data not shown) and to a polypeptide of similar size in extracts of NH~-grown Cpll and the local source of symbiotic Frankia (Fig. 2A, lanes 3 and 6) may be explained by the amino acid sequence similarity, however low, found between prokaryotic and eukaryotic GSs [17].

5. DISCUSSION The present study verifies that in cultured Frankia both GSI and GSII are synthesized under nitrogen-fixing conditions, and that only GSI

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is synthesized during growth on NH~. In contrast, our results show that during nitrogen-fixing symbiotic conditions, synthesis of both the GSI and the GSII proteins is r,:pressed in Frankia. Frankia grown in culture on N 2 or on glutamate develops vesicles, has nitrogenase and GSll activity [6]. GSll accounted for most of the GS activity during these conditions [6] and was present in vesicles, the nitrogenase containing cells, as well as hyphae [18]. In contrast, in the symbiotic stage Frankia has vesicles and nitrogenase activity but no GSll (Figs. I and 2). it thus appears that vesicle formation, synthesis of nitrogenase and synthesis of GS are separately regulated in Frankia. The symbiotic stage does not seem to be perceived as a nitrogen starved condition, as far as GSll is concerned, possibly modulated by metabolites made available by the host plant. Alternatively, the GS synthesis is specifically inhibited by the host. For B. japonicum and Rhi. meliloti it has been suggested that expression of nil genes and of glnll, coding for GSIi, is regulated by separate regulatory networks in free-living growth and in symbiosis [19,20]. GSI and GSI! mRNA were detected in soybean-B. japonicum nodules [19] so the corresponding genes are at least transcribed in that symbiosis. In organisms fixing nitrogen in free-living conditions nitrogenase synthesis is inhibited by the presence of NH~ in the culture medium and assimilation of the NH~- to glutamine is necessary for inhibition [21,22]. By avoiding assimilation of NH~- in a symbiotic nitrogen fixer through the lack of GS, the symbiont is prevented from sensing the high amount of nitrogen produced by nitrogen fixation and can thereby express a high nitrogen fixation activity, that becomes beneficial for the symbiosis. A third form of GS (GSIII) in RhL ineliloti has recently been characterized [23]. At present its importance in symbiosis has not been fully investigated. Unless there is a, yet unknown, addienzymes that tional GS or other NHa-assimilating + are expressed in Frankia during symbiotic conditions, the results of the present study suggest that the plant has to supply the symbiotic Frankia with organic nitrogen in some form in addition to the carbon.

Absence or reduction of the amount of GS protein in the symbiont seems to be a common phenomenon not only in rhizobial symbioses [7] but also in cyanobacterial symbioses [24,25], ~me cyanobacterial symbioses with cycads excepted [26]. The results in the present study extend this phenomenon to actinorhizal symbioses.

ACKNOWLEDGEMENTS We thank Dr J.V. Cullimore, University of Warwick, Coventry, U. K., Dr. S. Nordlund, University of Stockholm, Sweden, and Dr. M.L. Kahn, Washington State University, Pullman, WA, for providing antisera and Helene Ohlsson for technical assistance. This study was financially supported by the Swedish Natural Science Research Council and the Swedish Council for Fercstry and Agricultural Research.

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