Pleomorphic Forms of Bradyrhizobium Japonicum H

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 produced 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. mM Tricine {N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-gly- To determine the carbon substrate(s) and the concentra- cine}, pH 8.0; Sigma Chemical Co., St. Louis, Mo.} 100 mM tion required to produce pleomorphic forms in free-living KCl, 0.5 mM CoCl2, and 25 mM fructose-1,6-bisphosphate; cells, we grew B. japonicum on various carbon substrates, and for the ED enzyme, 40 mM Tricine (pH 8.0) and 50 mM including sugars, amino acids, and dicarboxylates (Table 1). 6-phosphogluconate. This method effectively blanks out The sugars supported growth at all concentrations tested but background activity caused by endogenous NADH oxidase. did not induce pleomorphism up to 60 mM. The four-carbon 668 REDING AND LEPO APPL. ENVIRON. MICROBIOL.

TABLE 1. Growth substrate and pleomorphism of B. japonicum 1-110 Growth/pleomorphism" at substrate concn (mM) of: Carbon substrate 10 20 30 40 50 60 Glucose G/- G/- GI- GI- GI- GI- Fructose G/- G/- GI- GI- GI- GI- L-Arabinose G/- G/- G/- GI- GI- GI- Gluconate G/- G/- GI- GI+ GI+ GI+ + Pyruvate G/- G/- GI- GI+ GI+ + Gl++++ Glutamate G/- G/+ G/- GI- GI+ GI+ + Citrate G/- GI- G/+ GI++++ GI++++G+++++ o-Ketoglutarate G/- GI- GI+ Gl++++ Gl++++ Gl++++ Succinate G/+ GI+ + G/+ + + G/++++ N/- N/- Fumarate G/+ GI+ + GI+++ GI++++ N/- N/- Malate G/- GI+ GI+ + G/+++ N/- N/- Oxalacetate G/- GI+ N/- N/- N/- N/- Malonate G/- GI- GI++ G/++++ GI++++ a Symbols: G, growth on indicated substrate; N, no growth on indicated substrate; +, <10% pleomorphism; + +, 11 to 30% pleomorphism; + + +, 31 to 50% pleomorphism; + + + +, >50% pleomorphism.

dicarboxylates, succinate and fumarate, were the only sub- morpholino)propanesulfonic acid] serving as the buffer. The strates that induced pleomorphic cells at 10 mM. Further- sodium concentration was adjusted over a range of 10 to 90 more, at 40 mM, these compounds produced pleomorphism mM (supplied as NaCl). Over 90% of the cells became in approximately 90% of the total cells. Higher concentra- pleomorphic when cultured in medium containing 40 mM tions of succinate or fumarate completely inhibited growth. succinate regardless of the sodium concentration. In cultures Growth on malate also generated pleomorphic cells but less containing 40 mM sodium gluconate, which usually does not efficiently than did growth on succinate or fumarate. Other generate pleomorphic cells, 60% of the bacteria became dicarboxylates, ot-ketoglutarate and malonate, and a tricar- pleomorphic when the sodium concentration was increased boxylate, citrate, also generated pleomorphic cells but only to 90 mM. However, cells cultured on 40 mM L-arabinose at concentrations of 30 mM or higher. Gluconate, pyruvate, produced no pleomorphic forms even in the presence of 90 and glutamate were poor inducers of pleomorphic cells, mM NaCl. Pleomorphism could be induced over a pH range requiring 50 mM before more than 10% of the cells became of 6.0 to 9.0 (data not shown). pleomorphic. In analogous experiments, similar results were Enzyme profiles. Because the Embden-Meyerhoff-Parnas obtained for B. japonicum strains C33, PJ18, USDA 136, and pathway, the ED pathway, and the tricarboxylic acid cycle USDA L-110 (data not shown). exist in most rhizobia and bradyrhizobia (39), we chose to The issue of whether the induction of pleomorphic cells is examine the initial enzymes of the Embden-Meyerhoff- a physiological-genetic effect or a physical-chemical effect Parnas pathway, the enzymes of the ED pathway, succinate was addressed. Finan et al. (5) have suggested that succinate dehydrogenase and hexose diphosphatase. Gluconate or chelates the divalent cations required for normal cell devel- succinate was used as the growth substrate. opment. Therefore, we grew cells in broth cultures of We found that extracts of B. japonicum 1-110 cultured on HUM-succinate (40 mM) containing different concentrations 20 mM gluconate contained glucokinase, fructokinase, glu- of Mg2" (MgSO4 7H2O) and Ca2+ (CaCl2- 2H20). Higher conokinase, glucose-6-phosphate dehydrogenase, the ED concentrations of these divalent cations in the media allowed enzyme, and succinate dehydrogenase but lacked fructose- cells to divide for a longer period of time (Fig. 2). Neverthe- 1,6-bisphosphate aldolase and hexose diphosphatase (Table less, at the stationary phase of growth, such cultures con- 2). Comparable levels of sugar-catabolizing enzymes and the tained about 90% pleomorphic cells, although mid-log-phase gluconeogenic enzymes, hexose diphosphatase and fructose- cultures contained less than 10% pleomorphic cells (data not 1,6-bisphosphate aldolase, were found in bacteria cultured shown). on succinate. We could not detect fructokinase in cells Possible chelation effects were also tested by growing the grown on succinate; however, fructokinase was found in bacteria in HUM-L-arabinose (20 mM) broth containing very low amounts when cells were cultured on gluconate different concentrations of EDTA. The bacteria grew in the (Table 2) or fructose (data not shown). Under similar condi- presence of 2 mM EDTA but were unable to grow in 4 mM tions, B. japonicum USDA 136 expressed sugar-catabolizing EDTA (data not shown). However, no pleomorphic forms enzymes at levels comparable to those of strain 1-110 (data were found in cultures containing 2 or 4 mM EDTA. not shown for strain USDA 136). Furthermore, when we cultured B. japonicum 1-110 on 20 Active transport of glucose and succinate. We cultured B. mM L-arabinose in the presence of 40 mM itaconic acid (a japonicum 1-110 on defined media and examined the ability succinate analog that does not support growth), the cells did of the cells to transport glucose and succinate. Glucose not become pleomorphic. We also found that L-arabinose did transport was highest in cells grown on 20 mM glucose. Cells not prevent the induction of pleomorphism if the bradyrhizo- cultured on 20 mM glucose plus 10 mM succinate trans- bia were grown in media that also contain succinate (data not ported glucose at 0.7 nmol/min per mg of whole-cell protein, shown). a rate half that seen with glucose cultures, which was 1.4 Because we supplied succinate in the growth medium as nmol/min per mg of whole-cell protein. In cultures grown on disodium succinate, the possible effects of sodium concen- 40 mM succinate or 40 mM succinate plus 20 mM glucose tration on cell morphology were tested. Succinate was (both are conditions that induce pleomorphism), no glucose supplied as free succinic acid, and the pH of the medium was uptake could be detected. adjusted by using NH40H, with 40 mM MOPS [3-(N- These bacteria were also tested for ability to transport VOL. 55, 1989 DICARBOXYLATE-INDUCED PLEOMORPHISM OF B. JAPONICUM 669

2.0 This morphological transformation could not be prevented by increasing the magnesium or calcium concentration in the medium, nor could it be produced solely by growing the bacteria in the presence of EDTA; therefore, the chelation of divalent cations alone is not capable of producing pleomorphism. Magnesium and calcium have similar effects in some fast-growing rhizobia. Urban and Dazzo (42) reported that in media containing 16.6 mM succinate, R. trifolii continue to divide if 40 mM MgSO4 and 46 mM CaCl2 are added, E although pleomorphic cells are still formed. 0 The sodium concentration or the pH of the medium does 0.4 not seem relevant to this morphological transformation. Enzyme profiles. Gluconate is metabolized primarily by the ED pathway (20); therefore, enzymes of this pathway should be present in extracts of gluconate-grown cells. was it is capable of generating either 0 Succinate used because . rod-shaped or pleomorphic forms, depending on its concentration in the medium (Table 1). Furthermore, in Rhizobium sp. strain 32H1, succinate represses the synthesis of sugar- catabolizing enzymes (39). Succinate-grown cells of cowpea Rhizobium sp. strain NGR 234 resemble bacteroids in that they lack fructokinase and show very low levels of other sugar-catabolizing en- co L B zymes (34). Bacteroids of B. japonicum USDA 110 lack the ED enzyme (the coupled activity of 6-phosphogluconate dehydratase plus 2-keto-3-deoxy-6-phosphogluconate aldolase), NADP-6-phosphogluconate dehydrogenase, and fructokinase, although other sugar-catabolizing enzymes are present (28, 32). Our results are similar to those reported by Mulongoy and Elkan (25), who used yeast extract mannitol-HEPES-mor- 0 20 40 60 80 pholineethanesulfonic acid (MES) as the culture medium. We found that the gluconate-grown cells, used as a positive TIM1E (hours) control, contained the enzymes of the ED pathway, as did FIG. 2. Effect of divalent cations on growth on succinate. Cul- the cells grown 10 or 40 mM succinate. tures were inoculated from a washed suspension of early-log-phase Two different serogroups (USDA 110 and USDA 136) of cells grown on 20 mM gluconate. (A) Cultures containing 40 mM B. japonicum showed similar enzyme profiles; thus, this succinate, 1 mM CaCl2, plus MgSO4 at concentrations of 1 (0), 10 pattern was not confined to a single strain and serogroup. (*), and 40 (U) mM. (B) Cultures containing 40 mM succinate, 4 However, a much broader study would be necessary to mM CaCI2, and MgSO4 at concentrations of 1 (LO), 10 (+), and 40(E) further assess the prevalence of the pattern among the rather mM. heterogeneous bradyrhizobia. Possibly, when B. japonicum I-110 was grown on 40 mM succinate. Transport was highest in cells cultured on 10 mM succinate, growth was inhibited before the bacteria had succinate or 20 mM glucose. When the bradyrhizobia were adequate time to repress the synthesis of sugar-catabolizing grown in medium containing 40 mM succinate, which generated 90%opleomorphic cells, succinate transport (deter- TABLE 2. Specific enzyme activities of B. japonicum 1-110 mined with 1-mmn assays) was 4 nmol/min per mg of protein. grown on different substrates This is a very low rate compared with that of rod-shaped cells produced by growth on 10 mM succinate or 20 mM Sp acta (nmol of substrate oxidized/ glucose, which was 51 or 64 nmollmin per mg of whole-cell min per mg of protein) Enzyme protein, respectively. When we grew the bacteria in 40 mM 20 mM Succinate succinate with 40 mM MgSO4 and 4 mM CaCi2, the increase gluconate 10 mM 40 mM in Mg2+Ca24and had no effect on glucose or succinate transport (data not shown). However, increasing the con- Glucokinase 6.22 9.86 9.06 centration of these divalent cations does enhance succinate Fructokinase 0.50 0 0 transport in B. japonicum grown on 15 mM succinate (24). Fructose-1,6-bisphosphate 0 2.38 3.84 aldolase DISCUSSION Gluconokinase 4.19 3.49 2.91 Glucose-6-phosphate 14.65 15.49 25.16 The induction of pleomorphism appears to be a substrate- dehydrogenase dependent physiological-genetic phenomenon, with succi- ED enzymeb 5.36 14.34 9.09 nate and fumarate being the best substrates for inducing the Hexose diphosphatase 0 1.56 2.11 transformation. Interestingly, strains of R. meliloti that lack Succinate dehydrogenase 10.37 4.04 2.93 succinate to dehydrogenase but have the ability transport a Mean of at least two experiments. succinate fail to produce pleomorphic forms when grown in b Combined activity of 6-phosphogluconate dehydratase and 2-keto-3- the plant or in media containing succinate (8). deoxy-6-phosphogluconate aldolase. 670 REDING AND LEPO APPL. ENVIRON. MICROBIOL.

enzymes and dissipate them from the cell. To test this In summary, growth on succinate, as well as other dicar- possibility, we grew the bacteria in HUM-succinate (40 mM) boxylates, transformed free-living bradyrhizobia into cells in which MgSO4 and CaCl2 were increased to 40 and 4 mM, having a bacteroid-like morphology. In addition, these trans- respectively. Increasing the Mg2' and Ca2+ concentrations formed cells possessed a bacteroid-like physiology with in medium containing 40 mM succinate extended the growth respect to ability to transport glucose and succinate. These period (Fig. 2). However, the sugar-catabolizing enzymes pleomorphic cells also expressed an enzyme profile similar were still expressed, and repression did not occur (data not to that reported for symbiotic bacteroids except that the shown). Therefore, in contrast to reports on other rhizobial bacteroids lacked the ED enzyme, although this result is strains (34, 39), succinate did not repress the synthesis of questionable (see Discussion). sugar-catabolizing enzymes in B. japonicum 1-110. In regard to cell morphology, cells grown on 10 tnM ACKNOWLEDGMENTS succinate (rod shaped) express enzyme profiles similar to This work was supported by National Science Foundation grant those of cells cultured on 40 mM succinate (pleomorphic). BSR-8416289 to J.E.L., by National Science Foundation equipment Thus, enzyme expression in B. japonicum is dependent on grant PCM-8312915, and by funds provided by the Department of the carbon growth substrate and is independent of cell Biology and the Office of Research of The University of Mississippi. morphology. We thank Harold L. Drake for helpful discussion. Our results indicate that pleomorphic cells of B. japonicum 1-110 express enzyme profiles similar to those of sym- LITERATURE CITED biotic bacteroids except for the ED enzyme, which Reibach 1. Bach, M. K., W. E. Magee, and R. H. Burris. 1958. Translation and Streeter (28) find absent in extracts from bacteroids of B. of photosynthetic products to soybean nodules and their role in japonicum USDA 110 and USDA 138. Salminen and Streeter nitrogen fixation. Plant Physiol. 33:118-124. (32) also were unable to detect the ED enzyme in either 2. Birkenhead, K., S. S. Manian, and F. O'Gara. 1988. Dicarbox- bacteroids or cultured bacteria. The presence of the E-D ylic acid transport in Bradyrhizobium japonicum: use of Rhizo- enzyme in cultured rhizobia, including the strains used by bium meloliti dct gene(s) to enhance nitrogen fixation. J. Bac- teriol. 170:184-189. Streeter and co-workers, has been fully established by many 3. Bradford, M. M. 1976. A rapid and sensitive method for the scientists (11, 19, 20, 22, 23, 25, 31, 34, 37-39). Therefore, it quantification of microgram quantities of protein utilizing the is conceivable that the assay used by Streeter and co- principle of protein-dye binding. Anal. Biochem. 72:248-254. workers may not be valid for B. japonicum. They assayed 4. Dazzo, F. B., C. A. Napoli, and D. H. Hubbell. 1976. Adsorption the ED enzyme by monitoring the oxidation of NADH, using of bacteria to roots as related to host specificity in the Rhizo- lactate dehydrogenase. We were likewise unable to detect bium-clover symbiosis. Appl. Environ. Microbiol. 32:166-171. any ED enzyme activity by this assay. Our assay protocol 5. Finan, T. M., J. M. Wood, and D. C. Jordan. 1981. Succinate allows for detection of the ED enzyme. In light of this, the transport in Rhizobium leguminosarum. J. Bacteriol. 148:193- reported lack of the ED enzyme in bacteroids of B. japoni- 202. 6. Finan, T. M., J. M. Wood, and D. C. Jordan. 1983. Symbiotic cum is questionable. properties of C4-dicarboxylic acid transport mutants of Rhizo- Transport of glucose and succinate. In regard to carbon bium leguminosarum. J. Bacteriol. 154:1403-1413. substrate uptake, free-living rhizobia actively transport glu- 7. Gardiol, A., A. Arias, C. Cervenansky, and G. Martinez-Drets. cose (33, 38) and succinate (5, 16, 24) into the cell. However, 1982. Succinate dehydrogenase mutant ofRhizobium meliloti. J. bacteroids of B. japonicum USDA 110 lack the ability to Bacteriol. 151:1621-1623. actively transport glucose, but they are able to actively 8. Gardiol, A., G. L. Truchet, and F. B. Dazzo. 1987. Requirement transport succinate and other dicarboxylates (6, 12, 29). The of succinate dehydrogenase activity for symbiotic bacteroid transport of glucose appears to be regulated by the growth differentation of Rhizobium meliloti in alfalfa nodules. Appl. substrate, since succinate-grown cells of cowpea Rhizobium Environ. Microbiol. 53:1947-1950. 1981. Oxidation of substrates by are transport and succi- 9. Glenn, A., and M. J. Dilworth. sp. strain 32H1 unable to glucose, isolated bacteroids and free-living cells of Rhizobium legumi- nate- or malate-grown cells of B. japonicum 1-110 show very nosarum. J. Gen. Microbiol. 126:243-247. low glucose uptake rates (33). 10. Glenn, A. R., R. Arwas, I. A. McKay, and M. J. Dilworth. 1984. When B. japonicum 1-110 was grown on 10 mM succinate Fructose metabolism in wild-type, fructokinase-negative and or 10 mM succinate plus 20 mM glucose, the rate of glucose revertant strains of Rhizobium leguminosarum. J. Gen. Micro- transport was one-half that seen in glucose-grown cells, biol. 130:231-237. which indicates that succinate represses glucose uptake. The 11. Glenn, A. R., I. A. MacKay, R. Arwas, and M. J. Dilworth. inability of our pleomorphic cells to transport glucose sug- 1984. Sugar metabolism and the symbiotic properties of carbo- gests that either (i) the higher concentration of succinate (40 hydrate mutants of Rhizobium leguminosarum. J. Gen. Micro- active transport of glucose or (ii) biol. 130:239-245. mM) completely repressed 12. Glenn, A. R., P. Poole, and J. Hudman. 1980. Succinate uptake since the pleomorphic cells also transported succinate at low by free-living and bacteroid forms of Rhizobium leguminosa- rates, these cells lost transport system proteins during the rum. J. Gen. Microbiol. 119:267-271. transition from a rod-shaped form to a bacteroid-like mor- 13. Gresshoff, P. M., and B. G. Rolfe. 1978. Viability of Rhizobium phology. In Bradyrhizobium sp. strain 32H1, growth on bacteroids isolated from soybean nodule protoplasts. Planta succinate also leads to similar swollen, bacteroid-like forms 142:329-333. which show an absence of several high-molecular-weight 14. Hardy, R. W. F., and U. D. Havelka. 1975. Photosynthate as a outer membrane polypeptides (27). major limitation to N2 fixation by field grown legumes with an Our pleomorphic bacteria generated by growth on 40 mM emphasis on soybeans, p. 421-439. In P. S. Nutman (ed.), succinate showed glucose and succinate transport capabili- International biological programme, symbiotic nitrogen fixation in plants, vol. 14. Cambridge University Press, New York. ties similar to those of symbiotic bacteroids. Both the 15. Hederstedt, L., E. Holmgren, and L. Rutberg. 1979. Character- pleomorphic cells and the bacteroids (29) lack the ability to ization of succinate dehydrogenase complex solubilized from transport glucose; in addition, the pleomorphic cells trans- the cytoplasmic membrane of Bacillus subtilis with the nonionic ported succinate at 4 nmol/min per mg of protein, and the detergent Triton X-100. J. Bacteriol. 138:370-376. bacteroids transport it at 3.7 nmol/min per mg of protein (29). 16. Hudman, J., and A. R. Glenn. 1980. Glucose uptake by free- VOL. 55, 1989 DICARBOXYLATE-INDUCED PLEOMORPHISM OF B. JAPONICUM 671

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