Pleomorphic Forms of Bradyrhizobium Japonicum H
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1989, p. 666-671 Vol. 55, No. 3 0099-2240/89/030666-06$02.00/0 Copyright C 1989, American Society for Microbiology Physiological Characterization of Dicarboxylate-Induced Pleomorphic Forms of Bradyrhizobium japonicum H. KEITH REDINGt AND JOE EUGENE LEPOt* Department of Biology, The University of Mississippi, University, Mississippi 38677 Received 12 August 1988/Accepted 12 December 1988 When Bradyrhizobiumjaponicum 1-110 was transferred into medium containing 40 mM succinate or 40 mM fumarate, over 90% of the bacteria acquired a swollen, pleomorphic form similar to that of bacteroids. The induction of pleomorphism was dependent on the carbon substrate and concentration but was independent of the hydrogen ion and sodium ion concentration. Cell extracts of rod-shaped and pleomorphic cells contained enzymes required for sugar catabolism and gluconeogenesis. Variations in these enzyme profiles were correlated with the carbon source used and not with the conversion to the bacteroid-like morphology. Rod-shaped cells cultured on glucose or 10 mM succinate transported glucose and succinate; however, the pleomorphic cells behaved similarly to symbiotic bacteroids in that they lacked the ability to transport glucose and transported succinate at lower rates than did rod-shaped cells. Rhizobia form symbioses with a variety of leguminous pH and mono- and divalent cation concentrations. In addi- plants. The plants produce photosynthate, some of which tion, we report the activity profiles of carbohydrate-catabo- supplies the reductant and ATP required for nitrogen fixa- lizing enzymes and substrate uptake activities of rod-shaped tion. This substrate may limit nitrogen fixation rates (1, 14). and pleomorphic cells. Thus, we have compared the physi- Although sucrose and sugar alcohols are the most abun- ology of rod-shaped and pleomorphic cells with that of dant carbon-containing compounds in the nodule cytosol symbiotic bacteroids to determine whether the substrate- (14), dicarboxylic acids are the most likely class of com- induced morphological transformation in free-living cells is pounds to support symbiotic nitrogen fixation for the follow- accompanied by an altered physiology analogous to that of ing reasons: (i) bacteroids do not transport (16) or oxidize the bacteroids. We found that the pleomorphic cells had a (41) sugars, (ii) bacteroids do oxidize (9, 26, 41) and trans- carbon substrate catabolic enzyme profile similar to that of port (6, 11, 29) tricarboxylic acid cycle intermediates, (iii) the rod-shaped cells; however, such pleomorphic cells had mutants defective in sugar metabolism form effective sym- glucose and succinate transport capabilities characteristic of bioses (10, 31), and (iv) mutants defective in dicarboxylate bacteroids. metabolism form ineffective symbioses (6, 7, 30). The recent introduction of dicarboxylate transport (dct) genes from MATERIALS AND METHODS Rhizobium meliloti into Bradyrhizobium japonicum pro- duced a strain with enhanced succinate uptake and free- Organism and cultivation. B. japonicum strains USDA living nitrogen-fixing activities (2). 1-110 and USDA 136 were obtained from the U.S. Depart- Early in the symbiotic association, the rhizobia develop ment of Agriculture culture collection at Beltsville, Md. structurally and physiologically into bacteroids. The bacteria Unless otherwise indicated, all studies were performed with change from a rod shape to a swollen, pleomorphic form (4) strain 1-110. in which cell division ceases (13). Free-living Rhizobium Bacteria were maintained on agar slants of hydrogen trifolii acquire a similar morphology when cultured in media uptake medium (HUM; 22) containing 20 mM sodium glu- containing succinate (42), as do rhizobia cultured in the conate as the sole source of carbon. Experimental cultures presence of alkaloids (43) or yeast extract (17, 18, 35, 40). were grown in a modified HUM broth supplemented with The physiological differentiation, aside from the develop- biotin (1 mg/liter) and NH4Cl (1 g/liter). Other carbon ment of the nitrogen-fixing mechanism, consists of changes substrates replaced the gluconate as indicated. The medium in carbon substrate metabolic capabilities. In general, sugar- was adjusted to pH 7.0 with 5 N NaOH or concentrated catabolizing pathways, e.g., Entner-Doudoroff (ED) and NH40H before autoclaving. Phosphates (10 mM NaPO4, pH Embden-Meyerhoff-Parnas, are shut down (28, 32, 34, 39). 7.0), iron-EDTA, and carbon substrates were autoclaved Moreover, free-living rhizobia actively transport glucose separately and added to the sterile salts-vitamin solution. (33, 38) and dicarboxylates (5, 16, 24), whereas bacteroids Solid medium contained 15 g of agar (Sigma Chemical Co., transport dicarboxylates but not sugars (6, 12, 29, 33). St. Louis, Mo.) per liter. This study further elucidates the role of dicarboxylates in Experimental cultures grown on solid media were incu- the Rhizobium-legume symbiosis. We have characterized bated at 29°C. Broth cultures in 20-mm test tubes were requirements for the induction of a bacteroid-like morphol- shaken at 29°C in a shaker-incubator (model G25; New ogy in free-living cells as well as the effects of factors such as Brunswick Scientific Co., Inc., Edison, N.J.). Growth was monitored by noting the optical density at 540 nm, using a Spectronic 501 spectrophotometer (Bausch & Lomb, Inc., * Corresponding author. Rochester, N.Y.). t Present address: Department of Microbiology, University of To determine cell morphology, heat-fixed smears were Georgia, Athens, GA 30602. prepared at the desired time, stained for 1 min with crystal t Present address: ECOGEN Inc., 2005 Cabot Boulevard West, violet, and viewed under bright-field oil immersion, using a Langhorne, PA 19047-1810. Nikon Optiphot microscope. 666 VOL. 55, 1989 DICARBOXYLATE-INDUCED PLEOMORPHISM OF B. JAPONICUM 667 FIG. 1. B. japonicum 1-110 grown on (A) 20 mM gluconate and (B) 40 mM succinate. Photomicrographs were taken with a phase-contrast Nikon Optiphot microscope (magnification, x4,000). Inoculation of cultures for enzyme and transport studies. All enzymes were assayed at 30°C in a quartz cuvette with Starter cultures grown on HUM-gluconate (20 mM) or a 1-cm light path. The change in absorbance was monitored HUM-L-arabinose (20 mM) were transferred to sterile cen- by using a Bausch & Lomb Spectronic 501 spectrophotom- trifuge tubes and centrifuged at 7,000 x g and 4°C for 10 min. eter; in each assay, minus-substrate controls were used. All The cells were washed twice with 10 mM phosphate-buffered commercial enzymes were purchased from Sigma. HUM salts, resuspended in the buffered salts, and used to Transport of glucose and succinate by whole cells. Cells inoculate experimental cultures for enzyme and transport from 500-ml liter cultures were collected by centrifugation, studies. washed twice with the uptake medium (HUM salts, NH4Cl, Protein determination. For the cell extract, protein was vitamins, and 10 mM NaPO4, pH 7.0), and suspended in 5 ml estimated by the dye-binding method of Bradford (3), with of uptake medium. The whole-cell suspension was diluted bovine serum albumin as a standard. For uptake assays, with uptake medium to contain 1 mg of protein per ml. Each whole cells and bovine serum albumin standards were first uptake assay required 2.5 ml of cell suspension. digested by a modification of the method of Stickland (36) in Glucose or succinate was added to the cell suspension at a which 1 ml of whole-cell suspension was heated to 100°C for final concentration of 2 mM. The assay was initiated by the 5 min in 3% NaOH. addition of [2,3-14C]glucose (0.13 to 0.28 ,uCi per assay Preparation of cell extract. Two liters of early-stationary- mixture) or [2,3-_4C]succinate (0.91 to 1.76 ,uCi per assay phase cells were harvested by centrifugation, washed twice mixture). The assay mixtures were shaken at 30°C in a water with 10 mM NaPO4-buffered HUM salts, suspended in 50 bath; 0.5-ml portions were removed when desired, vacuum mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesul- filtered through a 0.45-,um-pore-size nitrocellulose filters fonic acid; pH 7.5) with 0.2 mM dithiothreitol, and passed (TCM-450; Gelman Sciences, Inc., Ann Arbor, Mich.), and twice through a French press at 15,000 lb/in2. The crude washed with 10 ml of uptake medium without the carbon extract was then centrifuged at 44,000 x g for 20 min, and substrate. The filters were air dried, placed in 7-ml glass the supernatant fluid was assayed for the indicated enzymes. miniscintillation vials, and completely covered with 6 ml of Enzyme assays. Enzymes were assayed by published pro- Safety-Solve scintillation cocktail (Research Products Inter- cedures as follows: glucokinase (21), glucose-6-phosphate national Corp., Mount Prospect, Ill.). Radioactivity was dehydrogenase (21), fructokinase (10, 21), gluconokinase determined by using an LS6800 liquid scintillation counter (20), hexose diphosphatase (38), and succinate dehydroge- (Beckman Instruments, Inc., Fullerton, Calif.). The counts nase (15). from time zero were subtracted from each time reading to Fructose-1,6-bisphosphate aldolase and the ED enzyme correct for nonspecific binding of the substrate to the cells. were assayed by monitoring the reduction of NAD+ at 340 Transport rates were calculated from the linear portion of nm, using a glyceraldehyde-3-phosphate dehydrogenase- the curve. 3-phosphoglycerate phosphokinase-coupled assay system. The final reaction mixture contained 2.5 mM NaPO4, 0.2 RESULTS mM 3-NAD+, 1.66 mM ADP, and excess commercial glyc- Induction of pleomorphism. Figure 1A shows the typical eraldehyde-3-phosphate dehydrogenase-3-phosphoglycerate rod shape ofB. japonicum 1-110. The pleomorphic cells (Fig. phosphokinase in a total volume of 1 ml. For each assay, 1B) were induced within 38 h by 40 mM succinate and were additions were made to contain the following in the final re- typical of the altered morphology produced by several action mixture: for fructose-1,6-bisphosphate aldolase, 40 carboxylic acid substrates.