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 Gene 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 phenotype" 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 genes, 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.