JOURNAL OF BACTERIOLOGY, July 1992, p. 47904797 Vol. 174, No. 14 0021-9193/92/144790-08$02.00/0 Copyright X 1992, American Society for Microbiology Cloning and Mutagenesis of the Rhizobium meliloti Isocitrate Dehydrogenase TIMOTHY R. McDERMOTT' AND MICHAEL L. KAHN1,2* Institute ofBiological Chemistry' and Department ofMicrobiology,2 Washington State University, Pullman, Washington 99164-6340 Received 18 February 1992/Accepted 21 April 1992 The gene encoding Rhizobium meliloti isocitrate dehydrogenase (ICD) was cloned by complementation of an Escherichia coli icd mutant with an R. meliloti genomic library constructed in pUC18. The complementing DNA was located on a 4.4-kb BamHI fragment. It encoded an ICD that had the same mobility as R. meliloti ICD in nondenaturing polyacrylamide gels. In Western immunoblot analysis, antibodies raised against this protein reacted with R. meliloti ICD but not with E. coli ICD. The complementing DNA fragment was mutated with transposon TnS and then exchanged for the wild-type allele by recombination by a novel method that employed the Bacillus subtilis levansucrase gene. No ICD activity was found in the two R. meliloti icd::Tn5 mutants isolated, and the mutants were also found to be glutamate auxotrophs. The mutants formed nodules, but they were completely ineffective. Faster-growing pseudorevertants were isolated from cultures of both R. melloti icd::Tn5 mutants. In addition to lacking all ICD activity, the pseudorevertants also lacked citrate synthase activity. Nodule formation by these mutants was severely affected, and inoculated plants had only callus structures or small spherical structures.

In the symbiosis between legumes and rhizobia, the host tarate. Although no data were reported, an R meliloti plant provides the bacteria with reduced carbon as an energy ax-ketoglutarate dehydrogenase mutant was said to be Fix- source. The bacteria use this energy to reduce atmospheric (10). nitrogen to ammonia, which they release to the plant. The We are interested in how the bacteroid TCA cycle is qualitative nature of this energy source has been the focus of regulated and have selected ICD for our initial studies much research. Sucrose is the major photosynthate trans- because it is a regulated enzyme at a branch point in the TCA ported from the shoot to the nodule (41), but biochemical cycle (15), because it is regulated by aerobiosis in other evidence suggests that neither sucrose nor hexoses obtained gram-negative bacteria (23, 24), and because it is a relatively from sucrose degradation are important sources of energy simple enzyme with a single type of subunit that, in other for the bacteroids (reviewed in references 7 and 34). In bacteria, is encoded by a single gene (2). In this study, we support of this conclusion, mutants of various species of report the isolation of the gene that encodes ICD in R rhizobia with defects in sugar metabolism have been found meliloti and the symbiotic properties of mutants with defects to be effective in symbiosis (7, 34). By contrast, dicarboxylic in this gene. acids appear to be important carbon sources in the estab- lishment of an effective symbiosis. Succinate and malate are found at high concentrations in nodules (14, 45, 55), are MATERIALS AND METHODS actively transported across the peribacteroid membrane (20, Bacterial strains and plasmids. The strains of R. meliloti 56), are taken up by bacteroids (11, 20, 42, 50), and are and Escherichia coli, phage, and plasmids used in this study quickly oxidized to CO2 after uptake (47). Dicarboxylic acid are shown in Table 1. R meliloti was grown on yeast transport (dct) mutants of Rhizobium meliloti (5) and R. extract-mannitol medium (YMB [53]) or on minimal manni- leguminosarum biovars viciae (3, 12) and trifolii (44) are all tol medium (53) supplemented with arabinose (5 g/liter), Fix-. glutamate (1.1 g/liter), and filter-sterilized antibiotics as Succinate and malate are intermediates in the tricarboxy- indicated. Strains of E. coli were grown on either LB or M9 lic acid (TCA) cycle, and thus the TCA cycle is implicated as mineral salts (49) medium. M9 medium contained glucose as a major catabolic sequence. Enzymes of the TCA cycle, the carbon source and ammonium chloride as the primary such as citrate synthase (CS) (31), isocitrate dehydrogenase nitrogen source plus histidine (15 ,ug/ml), tryptophan (40 (ICD) (22, 28, 31), fumarase (28), malate dehydrogenase (22, p,g/ml), and thiamine (2 ,ug/ml). Filter-sterilized antibiotics 28), and a-ketoglutarate dehydrogenase (48), have been and glutamate were added as needed. For long-term storage, found in bacteroids of various species of Rhizobium and strains ofR. meliloti and E. coli were grown to mid-log phase Bradyrhizobium. Radioactive metabolite conversion studies in appropriate selective medium, diluted with an equal by Stovall and Cole (54) implied a fully functional TCA volume of sterile 50% glycerol, and stored at -70°C. cycle, but evidence for the decarboxylating leg of the TCA Cloning of R. meliloti icd and isolation of mutants. Con- cycle was not obtained until Salminen and Streeter (47) struction of the R meliloti gene bank and subsequent DNA reported that significant amounts of label from [2,3-14C]suc- manipulations followed the protocols of Sambrook et al. cinate accumulated in glutamate, indicating that in soybean (49). Aliquots of chromosomal DNA were partially digested bacteroids the TCA cycle is complete, at least to oa-ketoglu- with restriction enzyme Sau3A, and the pooled digests were fractionated by sucrose gradient centrifugation. The 4- to 8-kb fragments were then ligated to pUC18 (60) that had * Corresponding author. been digested with BamHI and calf intestinal alkaline phos- 4790 VOL. 174, 1992 R MELILOTI ISOCITRATE DEHYDROGENASE GENE 4791

TABLE 1. Strains of E. coli and R melioti, phage, and plasmids used Strain, phage, Relevant genotype or characteristics Relevant " Reference or source or plasmid RI meliloti 104A14 Wild type 53 A39S 104A14::TnSicd ICD- Glut- Kanr This study B49S 104A14::TnSicd ICD- Glut- Kanr This study A39L 104A14::TnSicd gItA (?) ICD- Glut- Kanr CS- This study B49L 104A14::TnSicdgkA (?) ICD- Glut- Kan' CS This study E. coli S17-1 Pro- Mob' 52 DL39 ilvE12 tyrB507 aspC13 Sup' 4 DEK2038 F- A- his-4 thi-1 rspL31 lacBKI rfiB recA ICD- Glut- Peter Thorsness aceKi icd-11 RR1 supE44 hsdS20 ara-14 proA2 lacYI galK2 High-efficiency transformation 49 rpsL20 xyl-5 mtl-l Phage X467 TnS Kanr 8 Plasmids pUC18 Penr 60 pTK509 pBR322 with E. coli icd Penr Peter Thorsness pTM1 pUC18 with R meliloti icd Pen' This study pRK311 tetA Tetr 9 pUM24 nptI sacB sacR Kanr Suc, 43 pMK413 tetA sacB sacR Tetr Suc, This study pTM2 pMK413 with R. meliloti icd Tetr Suc, This study pTM3 pRK311 with R melioti icd Tetr This study pTM2::TnSA39 icd::TnS mutant of pTM2 Suc5 Tetr Kanr This study pTM2::Tn5B49 icd::TnS mutant of pTM2 Suc5 Tetr Kanr This study I Glut, glutamate; Pro, proline; Suc, sucrose. phatase (Boehringer). Transformation ofE. coli RR1 yielded mM DL-isocitrate, 0.4 mM NADP+, 3.3 mM MgCl2, 10 mM approxmately 10,000 independent transformants that con- potassium phosphate buffer (pH 7.4), and enzyme extract in tained inserts. Plasmid DNA was purified from these colo- a final reaction volume of 1.0 ml. nies and used as the gene bank in the initial selection. For (ii) CS. CS was assayed by measuring the formation of transposon TnS mutagenesis, we used X467::TnS as outlined coenzyme A-sulfhydryl by the increase in A412 as described by de Bruijn and Lupski (8). by Sere (51). Assay mixes contained 200 mM Tris (pH 8.1), A mutated allele is often recombined into R meliloti by first using a broad-host-range plasmid to bring the allele into the strain and then using a second plasmid of the same incompatibility group to destabilize the first plasmid (46). By EOR continuing to select for the mutated allele, the desired p_iEcoRI recombinant can be recovered, since it will stably maintain / ~~~~onV ~% Hindlll the selected marker. Although the technique works in R sac meliloti 104A14, plasmid incompatibility is not very strong, Ps and this procedure can be tedious, especially if the mutant grows more slowly than the wild type (26). An alternative method was developed in which a vector that allows direct selection is used to obtain cells that have lost the plasmid carrying the mutated allele. pMK413 (Fig. 1) contains the pMK413 SetA Bacillus subtilis levansucrase , which inhibit the LSmal growth of gram-negative bacteria when they are incubated in 23kb media that contain sucrose (17, 21). To construct pMK413, the 1.7-kb cos fragment of pRK311 (9) was replaced by the 3.8-kb BamHI fragment from pUM24 (43), which contains \_oriT/ the B. subtilis sacR and sacB genes, which encode levansu- crase, and the nptl gene, which confers kanamycin resis- ESol la a2 tance (KanT). The nptI gene was subsequently deleted by SaiHl using PstI. E. coli DEK2038(pMK413) and R meliloti PstI 104A14(pMK413) do not grow on media that contain tetra- Hindlil cycline (to select for plasmid retention) and 5% sucrose. FIG. 1. Restriction map of pMK413. The cos fragment was Enzyme assays. (i) ICD. ICD activity in cell extracts was deleted from pRK311 (9) and replaced with the sac cassette from measured spectrophotometrically. The rate of NADP+ re- pUM24 (49) as described in Materials and Methods. Arrow indicates duction was measured at 340 nm. Assay mixes contained 0.5 orientation of lacZ relative to the polylinker site. 4792 McDERMOTT AND KAHN J. BACTERIOL.

0.2 mM 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 0.1 mM containing 0.02 M Tris base and 0.1 M NaCl (pH 8.0), and acetyl-S-CoA, 0.5 mM potassium oxaloacetate, and enzyme stored at -20°C. extract in a final volume of 1.0 ml. DTNB and acetyl-S-CoA Western immunoblots. Wild-type and ICD- mutant strains were prepared fresh daily in 0.5 M potassium phosphate of R meliloti were grown in minimal mannitol-glutamate- buffer. arabinose medium, with appropriate antibiotic selection. Activity stains in nondenaturing polyacrylamide gels. The Cells were collected by centrifugation, washed once in protocol of Kittell et al. (29) was modified to include stacking breaking buffer, and then lysed by sonication. Centrifuged and resolving phases in the separation system. Nondenatur- extracts were subjected to SDS-PAGE (12% acrylamide ing (native) gels contained 10% acrylamide (9.7% acryl- [49]), transferred to nitrocellulose by electrophoresis, amide, 0.3% bisacrylamide) in the resolving phase and 5% probed with anti-ICD antibodies, and visualized by a chemi- acrylamide (4.85% acrylamide, 0.15% bisacrylamide) in the luminescent detection system (Amersham). Purified E. coli stacking phase. Gels were made up in 0.25 M Tris (pH 8.3) ICD protein was a generous gift from Peter Thorsness. and polymerized by using ammonium persulfate and Prestained SDS-PAGE standards (Bio-Rad) were used to TEMED (N,N,N',N'-tetramethylethylenediamine). Gels estimate molecular weight. The protein concentration of were prerun in 0.375 M Tris (pH 8.3) for 2 h and then, just extracts used in this experiment, as well as those listed prior to sample loading, the prerun buffer was replaced by a above, was determined with the Bio-Rad protein assay kit. running buffer containing 25 mM Tris base and 190 mM Plant growth and inoculation. Alfalfa (Medicago sativa) glycine, pH 8.5. Cell extracts were mixed 3:1 (vol/vol) with cultivar Ladak was used for all symbiotic performance buffer of diluted in 5 ml of studies. Seeds were surface sterilized by soaking in 5% sample (2 mg xylene cyanol 40% sodium hypochlorite for 5 min, rinsed several times in sterile glycerol), loaded into the gel, and electrophoresed for ap- distilled water, and then germinated on YMB agar. Seedlings proximately 1.5 h at 60 mA constant current. Gels were showing no signs of contamination were transferred to sterile removed from the glass plates and submerged in a staining growth box units (four seedlings per box) and inoculated solution containing 180 mM Tris (pH 7.9), 0.1 mM NADP+, immediately. Each growth box unit consisted of three Ma- 15 mM MgCl2, 8 mM sodium isocitrate, 0.4 mM 3-(4,5- genta (Sigma GA-7 vessel) plant tissue culture boxes ar- dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, ranged as a Leonard jar (58), with the top box inverted to and 0.5 mM phenazine methosulfate (1). Depending on the provide an aseptic barrier. specific activity of ICD in the samples, activity bands were Plants were cultured in a walk-in growth room at a seen within 1 to 20 min. temperature of 28°C with a 16-h photoperiod. Light was Protein purification and antibody production. For purifica- supplied by Philips 40W Agro-Lites at an intensity of 100 tion of R meliloti ICD, we modified the purification method ,umol of photons m-1 s-. The nutrient solution was modi- of Nambiar and Shethna (36). DEK2038(pTM1) was inocu- fied from that described by Wych and Rains (59) and lated into 18 liters of LB-penicillin (200 mg/liter) broth, contained 1.0 mM K2SO4, 0.5 mM KH2PO4, 0.25 mM grown to late log phase, and then harvested and washed by K2HPO4, 0.5 mM MgSO4- 7H20, 2.0 mM CaSO4. 2H20, 25 filtration and centrifugation. Cells resuspended in breaking ,uM KCl, 13 FM H3BO3, 1.0 ,uM MnSO4. H20, 1.0 ,uM buffer (25 mM Tris, 3 mM dithiothreitol [pH 7.5]) were lysed ZnS04 7H20, 0.25 I'M CUSO4- 5H20, 2.5 ,uM by sonication and cleared by centrifugation at 100,000 x g CoCl2 6H20, 20 ,uM FeCl2 6H20, and 0.25 ,uM for 1 h. Protamine sulfate was added to the supernatant Na2MoO4. 2H20. The solution pH was adjusted to 6.5 with (1:10, protamine sulfate to total protein), and after 1 h the KOH and HCl. precipitate was removed by centrifugation. The supernatant Nodulation and nitrogen fixation. Plants were harvested 5 was brought to 45% (wt/vol) ammonium sulfate and centri- to 6 weeks after inoculation. Nitrogen fixation was estimated fuged. The supematant was then brought to 70% (wt/vol) by acetylene reduction, using methods similar to those ammonium sulfate and recentrifuged, and the pellet was outlined by Somerville and Kahn (53). Whole plants were resuspended in 0.05 M Tris-HCl (pH 7.5) and desalted transferred from the growth boxes to 40-ml glass tubes. Each through a column (2.5 by 40 cm) of Bio-Gel P-6DG (Bio-Rad, tube was plugged with a rubber stopper outfitted with a Richmond, Calif.). Further purification was accomplished by rubber septum, and then 10% of the air was replaced with chromatography on DEAE-Sepharose (eluted with a 0.0 to acetylene. After 1 h, gas samples were removed and stored 0.5 M KCl gradient) and Affi-Gel Blue (Bio-Rad) (eluted with in Vacutainer tubes (Becton Dickinson) until analyzed by a single step of 2 mM NADP). The protein was then gas chromatography. Nodules were removed, counted, and fractionated by nondenaturing polyacrylamide gel electro- weighed, and plant shoots were detached, dried at 65°C, and phoresis (PAGE) (see below), and the gels were stained for weighed. ICD activity. The band of ICD activity was cut out and Plasmid and transposon stability during symbiosis was electrophoresed in a sodium dodecyl sulfate (SDS)-poly- assessed by testing nodule occupants for antibiotic resis- acrylamide gel. The gel was then stained with Coomassie tance and glutamate auxotrophy. Fresh nodules were sur- blue, which revealed two protein bands with molecular face sterilized in 1% sodium hypochlorite and washed five masses of approximately 25 and 53 kDa. The 53-kDa band times with sterile distilled water. They were then crushed in was judged to be ICD from the similarity of the molecular YMB broth, serially diluted, and plated onto minimal man- mass to that of ICD from E. coli (16) and because the 53-kDa nitol medium containing glutamate and arabinose. Colonies protein was clearly the predominant protein in all purifica- were then screened for resistance to appropriate antibiotics tion steps (determined by SDS-PAGE), which would be and glutamate auxotrophy. expected if the protein was encoded by a highly expressed gene. The 53-kDa band was cut out and used to immunize a RESULTS female New Zealand White rabbit. Blood was harvested, allowed to clot, and then cleared by centrifugation. Anti-ICD Cloning and mutagenesis. E. coli DEK2038, an icd mutant polyclonal antibodies were then partially purified by precip- and glutamate auxotroph (Table 1), was transformed with itation in 40% ammonium sulfate, dialyzed against a buffer the R. meliloti genomic library and incubated on M9 agar VOL. 174, 1992 R MELILOTI ISOCITRATE DEHYDROGENASE GENE 4793

Tn5 Insertions A B C A39 B49 B x x B V V I I

1 kb FIG. 2. Partial restriction map and insertion points of TnS (A39 FIG. 3. Native PAGE of extracts from DEK2038 (pTK509) (A), and B49) in the fragment of R meliloti DNA that rescues the DEK2038(pTM1) (B), and R. meliloti 104A14 (C). Each lane was glutamate auxotrophy of E. coli DEK2038 and encodes ICD activ- loaded with approximately 60 pg of protein. See Materials and ity. The fragment bounded by the XhoI sites is still capable of Methods for procedural details. rescuing DEK2038. B, BamHI; X, XhoI.

cell extracts and found wild-type CS activity (750 nmoll medium without glutamate. Ten transformants that grew on min/mg of protein) in the small-colony types of both icd::TnS this medium were subcultured on LB agar containing peni- mutants, but no CS activity in the large-colony-type isolates. cillin (200 p,g/ml). All Glut' Penr isolates contained a plas- Introducing pTM2 into the small-colony-type isolates mid derived from pUC18 that had a single BamHI digestion (ICD- only) relieved the glutamate auxotrophy and restored pattern. Complementation was confirmed by transforming ICD activity. pTM2 also restored ICD activity in the large- DEK2038 with one of these plasmid preparations (designat- colony-type TnS mutants, but did not affect glutamate aux- ed pTMl), and the presence of ICD was verified by enzyme otrophy (results not shown). This is consistent with their assays. Extracts from these clones had high ICD specific CS- phenotype. activities, with rates of 400 ,umol/min/mg of protein being Activity stains and Western blot analysis. Activity stains in typical. The glutamate auxotrophy of DEK2038 caused by native gels showed that the electrophoretic mobility of the its icd was extremely stable, and we did not ICD activity encoded by the cloned DNA was identical to encounter a single instance of DEK2038 reverting to an that of the ICD in R. meliloti 104A14 extracts but different ICD+ phenotype. from that ofE. coli ICD (Fig. 3). Polyclonal antibodies raised In subcloning analysis, pTM1 and pMK413 were digested against the purified protein were used as probes in Western with BamHI, mixed and ligated with T4 DNA ligase, and blot analyses to determine the immunological properties of then used to transform DEK2038. pTM2, isolated as a the cloned protein relative to ICD in crude cell extracts of tetracycline-resistant (Tetr) plasmid that complemented the wild-type R meliloti 104A14 as well as authentic E. coli glutamate auxotrophy of DEK2038, contained a 4.4-kb ICD. The anti-ICD serum reacted with a peptide of approx- BamHI fragment from pTMl. Site-directed TnS mutagenesis imately 53 kDa in R. meliloti 104A14 and B49S(pTM3) was accomplished by infecting E. coli DL39(pTM2) with extracts but not in B49S extracts and failed to react with X467::TnS. Plasmids collected from Tetr Kanr colonies of the purified E. coli ICD (Fig. 4). To verify that the purified E. infected cells were transformed into DEK2038. Seven of coli ICD had been eluted from the gel and that the lack of approximately 300 Tetr Kanr DEK2038 transformants were detection was not due to nontransfer, the electroeluted gel glutamate auxotrophs, suggesting that the TnS had inserted and a companion replica gel were silver stained. Transfer of into the R meliloti icd gene. BamHI restriction digests of proteins was nearly complete (results not shown). plasmids isolated from these glutamate auxotrophs revealed Symbiosis phenotype. The R meliloti ICD- mutants A39S two distinguishable TnS insertions, A39 and B49, located and B49S were totally ineffective (Table 2). The dry weight approximately 770 bp apart (Fig. 2). Further analysis has of 5-week-old plants inoculated with the wild-type R meli- shown that the glutamate auxotrophy of DEK2038 can be loti 104A14 was approximately four times that of plants rescued by a 2.7-kb XhoI fragment contained in the cloned inoculated with A39S or B49S. Uninoculated control plants R meliloti DNA (Fig. 2). did not form nodules in any of the tests. In the initial plant To exchange these TnS for the wild-type icd tests, the large-colony-type icd mutants formed callus struc- alleles in 104A14, pTM2::TnSA39 and pTM2::TnSB49 were transformed into S17-1 and then conjugated into R. meliloti 104A14. Tetr Kanr colonies were then streaked onto YMB-5% sucrose-kanamycin (20 mg/liter) plates, and the A sucrose-resistant colonies were screened for Tets and gluta- B C D mate auxotrophy. Several Kanr Sucr Tet' glutamate auxo- 106 kD trophs were identified for each TnS insertion. 80 For each insertion, two colony types were distinguished and purified: a slow-growing, small-colony type (A39S and 49.5 B49S), and a faster-growing, large-colony type (A39L and B49L). Both colony types were glutamate auxotrophs and 32.5 - lacked ICD activity. Probing total genomic preparations of 27.5 each colony type with pTM2 in Southern blot analyses 18.5 - showed identical fragment shift patterns and a loss of the FIG. 4. Western blots of purified E. coli ICD (A), R meliloti wild-type allele (results not shown). ICD- mutant B49S (B), R meliloti B49S(pTM3) (C), and R meliloti A similar difference in colony morphology has been ob- wild-type 104A14 (D). Apparent molecular masses of prestained served with E. coli icd mutants (32). The smaller E. coli SDS-PAGE standards are shown in kilodaltons. Each well of the colony type was found to have CS activity, whereas the corresponding gel was loaded with a total of 100 Lg of protein except larger colony type lacked CS activity. We assayed for CS in for purified E. coli ICD, which was loaded with 20 F±g of protein. 4794 McDERMOTI AND KAHN J. BACTERIOL.

TABLE 2. Symbiosis of wild-type and ICD- mutants TABLE 3. Nodulation by R meliloti ICD- and CS- ICD- of R. meliloti 104A14 mutants on alfalfa growing in nutrient solution containing FeC13- 6H20 or ferric citrate as the iron source Plant dry Nodule sp Plasmid retention (% Strain wt act (jLmol/h/ antibiotic-resistant Strain Phenotype Iron sourcea No. of (mg/plant)' nodule)'g of colonies) nodules/plant" 104A14 Wild type FeC13 * 6H20 3.4 ± 0.4 104A14 (wild type) 23.2 ± 3.3 13.7 ± 1.7 Ferric citrate 3.8 + 0.7 104A14(pTM3) 25.2 ± 4.8 12.2 ± 1.2 67b A39S ICD- FeC13- 6H20 4.6 + 1.7 104A14(pRK311) 22.9 ± 4.9 11.1 ± 3.1 71b Ferric citrate 2.7 ± 0.8 A39S 5.3 ± 0.7 1.2 + 1.0 100c A39L CS- ICD- FeC13 6H20 0.3 ± 0.2 A39S(pTM3) 18.1 ± 1.8 13.5 ± 3.1 82" Ferric citrate 0.9 + 0.7 A39S(pRK311) 6.6 + 0.3 0.6 ± 0.1 56b B49S ICD- FeCl3 6H20 4.5 ± 0.6 A39L 4.9 + 0.2 NDd 100C Ferric citrate 4.6 ± 0.8 B49S 5.6 ± 0.7 0.5 ± 0.1 100c B49L CS- ICD- FeCl3 * 6H20 0.1 ± 0.1 B49S(pTM3) 22.5 ± 5.3 12.6 ± 3.0 63b Ferric citrate 0 B49S(pRK311) 6.6 ± 0.4 0.5 ± 0.1 50b B49L 6.1 ± 0.7 ND 100c a Iron was added to 1.2 ,g of Fe per ml. Not inoculated 5.2 ± 0.8 "Plants were harvested 21 days after inoculation. a Data reported as mean + standard error of four replications for each treatment. b Tetracycline resistance. moderately stable; an average of 66 and 77%, respectively, c Kanamycin resistance. of the tested nodule isolates were Tetr. d ND, not determined. We investigated the possibility that citrate may be used as an iron siderophore and that the type of iron source con- tained in the plant nutrient solution may influence nodulation by the CS- ICD- isolates. Growth boxes were soaked with tures, as opposed to the small-colony-type isolates, A39S dilute HCl prior to autoclaving to eliminate traces of iron and B49S, which formed white nodules that were morpho- residues. All nutrients were individually filter sterilized and logically similar to the nodules induced by the wild-type added separately to each growth box after autoclaving. The strain 104A14. The number of viable cells isolated from pH of the nutrient solution was adjusted to 6.5 after auto- nodules formed by A39S and B49S (2.6 [±1.1] x 106/mg of claving but before the addition of iron. Regardless of iron nodule; mean + standard error of three experiments, A39S source, the fast-growing CS- ICD- mutants formed few and B49S combined) was similar to that from wild-type nodules, whereas the slow-growing ICD- mutants nodulated nodules (1.11 [+0.24] x 106/mg of nodule; mean + standard at levels similar to the wild type (Table 3). Nodules formed error of two experiments). by the CS- ICD- mutants were poorly defined calluslike The symbiotic characteristics of the fast-growing large- structures or very small spherical bumps on the roots that colony-type isolates have not been fully resolved. Plants appeared to cease growth at an early stage of development. inoculated with these mutants were stunted (Table 2) and resembled uninoculated controls in that they appeared to be nitrogen stressed. The calli and small spherical structures DISCUSSION formed by these mutants reduced little acetylene. Since they Using a complementation strategy, we have identified a were difficult to quantitatively remove from the roots, nod- 4.4-kb BamHI fragment of K meliloti DNA that rescues the ule weight, and therefore specific nodule activity, was not glutamate auxotrophy of the E. coli icd mutant DEK2038 determined. and encodes a protein that gives high ICD activity in Plants inoculated with strains containing pMK413 or DEK2038. Antibodies raised against the cloned protein did pTM2 had fewer nodules, and few of the bacteroids isolated not react with E. coli ICD or with extracts obtained from R from these nodules contained plasmids (7% plasmid reten- meliloti ICD- mutants. However, Western blot analysis tion rate). We speculated that this might be due to the sac showed that these antibodies did react with a single band in cassette in these plasmids, which may cause problems for cell extracts of wild-type R meliloti 104A14 and ICD- the invading rhizobia in the presence of photosynthetically mutants rescued with pTM3. Finally, activity stains in native derived sucrose in the roots. Because of these problems, we gels showed that the cloned protein was identical in mobility found it necessary to subclone the R meliloti icd fragment to authentic R meliloti ICD but was different from E. coli into pRK311 to give pTM3 (Table 1). pTM3 complemented ICD. The above evidence strongly argues that the gene we the symbiotic defect in A39S and B49S and restored plant have cloned is R meliloti icd. dry weight and nitrogen fixation (acetylene reduction) to Insertion of TnS into the complementing DNA fragment wild-type levels (Table 2). The symbiosis phenotype was eliminated ICD activity. The approximately 770-bp spacing unaffected by pRK311, nor did it appear to be influenced by between the inserts argues that at least one of the trans- extra copies of icd in the bacteroids [104A14(pTM3), Table posons is located in the structural portion of the gene, which, 2]. based on molecular mass estimates of the protein (53 kDa), Nodule isolates were checked for plasmid and transposon should be approximately 1.4 kb long. In other work, we have retention (Table 2). We did not detect any spontaneous used native PAGE gels to investigate ICD mobility patterns resistance of 104A14 to the levels of kanamycin and tetra- in both free-living and bacteroid cells of R. meliloti 104A14. cycline used. For both A39S and B49S, all of the nodule We typically find a single band in free-living cell extracts and isolates screened were Kanr and Glut-, indicating that the two bands in bacteroid extracts (35). We have been unable to transposon is very stable. Although the calli formed by A39L find any staining activity in extracts from bacteroids isolated and B49L contained substantially fewer bacteria, all isolates from nodules formed by A39S or B49S (35), which suggests were also Kanr and Glut-. Both pRK311 and pTM3 were that the second band observed in wild-type bacteroid ex- VOL. 174, 1992 R. MELILOTI ISOCITRATE DEHYDROGENASE GENE 4795 tracts is not due to a second icd that is expressed under mutant. Citrate can be used in iron uptake, but a majority of symbiotic conditions. Consistent with this interpretation, the Bradyrhizobium japonicum (19) and R. leguminosarum Southern blots with pTM2 as the probe show only a single biovar trifolii (52) strains tested do not use citrate as a band of hybridization (35). We speculate that the second siderophore. R. meliloti mutants defective in the production ICD band found in bacteroid extracts might be due to and uptake of rhizobactin can still form nodules (18), which posttranslational modification. suggests that R. meliloti may use other pathways to acquire The R meliloti ICD- mutants isolated in this study iron. High concentrations of citrate have been found in completely lacked ICD activity, were glutamate auxotrophs, alfalfa nodule cytosol (14), and this would argue that a and formed ineffective nodules. While the Fix- phenotype of bacteroid CS- phenotype would be of little consequence in these mutants was not totally unexpected, we would not iron acquisition. However, the alfalfa peribacteroid mem- have been surprised by a Fix' or partially Fix' phenotype. brane may be impermeable to citrate, and iron transport Johnson et al. (25) found that R. meliloti bacteroids show across the peribacteroid membrane may require extracellu- little isocitrate lyase activity, but we speculated that by lar reduction and complex dissociation, as is required for disrupting the TCA cycle at ICD, such a lesion might iron uptake by soybean roots (6). If this were the case, then "force" carbon through the glyoxylate shunt. When E. coli bacteroid synthesis of citrate may be very important in is grown in acetate, carbon flow is directed through the acquiring iron from the peribacteroid space. glyoxalate shunt as a consequence of phosphorylation of An alternative explanation for the Nod- phenotype of the ICD by ICD kinase-phosphatase (15). Under these condi- CS- icd mutants might be that the intracellular pool of tions, ICD activity is reduced, increasing the intracellular acetate accumulates to concentrations at which the acetyl isocitrate concentration to a level at which isocitrate lyase, modifications in the extracellular polysaccharide are quanti- which has a higher Km for isocitrate, can compete against tatively affected. This could be important given that extra- ICD for the substrate (37). While we do not know if R. cellular polysaccharide composition is critical for successful meliloti 104A14 uses the same mechanism, this strain will invasion (33). We are currently trying to characterize the grow on acetate as the sole source of carbon (39), suggesting nodulation phenotype of these putative g1tA mutants, clone that it is capable of operating this anaplerotic sequence. R. meliloti git4, and study the regulation of this gene. Another inference that can be drawn from the symbiosis phenotype of these mutants concerns the role of glutamate in ACKNOWLEDGMENTS the symbiosis. It has been proposed that amino acids play a key role in the exchange of nutrients between the host and We thank Larry Wilhelm for construction of the R. meliloti bacteroids (27). In this hypothesis, glutamate catabolism and genomic library, and Derek Wood and Karen Liljebjelke for expert the shuttle were to be technical assistance. We also thank Peter Thorsness for generously malate-aspartate suggested important providing us with purified E. coli ICD protein and Peter Thorsness in transferring reducing equivalents from host to bacteroid. and Tony Dean for sending us E. coli icd mutants, including The R. meliloti icd mutants isolated in this study are strict DEK2038. glutamate auxotrophs. Since they are Nod', the host must This work was supported by grants from the USDA Competitive be directly or indirectly providing enough glutamate for cell Research Grant Office and by the Agricultural Research Center at growth and maintenance. However, the Fix- phenotype Washington State University. suggests that either a-ketoglutarate or glutamate is not supplied in large quantities or that glutamate catabolism is of REFERENCES relatively little importance. This is consistent with studies 1. Aebersold, P. B., G. A. Winans, D. J. Teel, G. B. Milner, and showing that the rates of glutamate transport across the F. M. Utter. 1987. Manual for starch gel electrophoresis: a soybean (57) and common bean (20) peribacteroid membrane method for the detection of genetic variation, p. 1-19. National are much slower than those of dicarboxylic acids. 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