Japonicum Cells Research Articles

Immunological and Biochemical Evidence that Blepharismin is not a Prosthetic Group

A polyclonal, multispecific antiserum was raised against a whole 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate-extract of nonirradiated Blepharisma japonicum cells. It was used to reveal the composition of solutions that were hypothesized to contain the photoreceptor of the ciliate. A Bio-Gel A 1.5 m fine column chromatography of the extract allowed recovery of a single elution peak isolated by recording the 580 nm light absorbance. Fused-rocket immunoelectrophoresis of this material revealed a large number of > 300 kDa coeluted proteins. Blepharismin-rich material with a molecular mass of approximately 50 kDa, consisting of at least nine proteins was obtained when the same extract underwent preparative isoelectric focusing before column chromatography separation. Purification of the pigment obtained from light-exposed cells gave blepharismin-rich material with a molecular weight of approximately 200 kDa. Comparison of the materials obtained under the same conditions, either from the dark-kept or light-irradiated cells, by means of pore-gradient electrophoresis confirmed that proteins present in the two preparations were different. It revealed only a very small amount, if any, of proteins in the chromatography fractions with the highest absorbance at 600 nm. Results are discussed on the basis of the hypothesis that a specific blepharismin-binding protein does not exist in the protozoan.

Genistein preincubation of Bradyrhizobium japonicum cells improves strain competitiveness under greenhouse, but not field conditions.

In the soybean-B. japonicum symbiosis, genistein has been identified as one of the major compounds in soybean seed and root exudates responsible for inducing expression of the B. japonicum nodYABC operon. In this study, we have tested the possibility that genistein treatment prior to inoculation can increase the competitiveness of the treated B. japonicum strain under both greenhouse and field conditions. Two mutants of the two B. japonicum strains each with a different antibiotic resistant marker were selected. They were tested with one or the other treated with genistein. The results showed genistein treated mutants had higher levels of nodule occupancy than the untreated mutant or parent strain under greenhouse conditions. Mutants from 532C had higher nodule occupancies than mutants from USDA110, especially at 15 °C. In the more complex field environment, genistein treated mutants formed fewer nodules than the untreated mutants. The contradictory results of strain competitiveness for greenhouse and field experiments are discussed.

Possible determinants of rhizosphere competence of bacteria

Because extensive colonization of the rhizosphere is important to obtain the benefits of bacterial inoculation, a study was conducted to determine possible traits that are important for rhizosphere competence of bacteria. The final population sizes of 19 bacteria introduced into the rhizosphere of soybeans grown in nonsterile soil differed markedly. The ability to colonize the rhizosphere was not correlated with the capacity of low or high densities of these bacteria to adhere to the roots. The growth rates of the 19 bacteria in liquid culture or of eight of the bacteria in sterile rhizosphere soil were also not correlated with the extent of growth in the nonsterile rhizosphere. Tests with small inocula of three bacteria with different rates of growth in the sterile rhizosphere showed no relationship between those growth rates and the extent of colonization of the nonsterile rhizosphere by these organisms. The extent of colonization of the nonsterile rhizosphere by test bacteria was not correlated with their densities in the rhizosphere containing large numbers of protozoa. Additions of a readily-available carbon source to soil receiving a small number of Bradyrhizobium japonicum cells provided no evidence that the concentration of available carbon limited the colonizing ability of this bacterium. With five of seven test bacteria, the maximum population density reached in the rhizosphere depended on the size of the initial population in soil. The results suggest that the ability of bacteria to survive in large numbers in soil is a major determinant of their success in subsequent colonization of the rhizosphere.

Role of Hydroxylysine on the Nodulation of Root Nodule Bacteria

The growth of Bradyrhizobium japonicum as well as Rhizobium leguminosarum bv. phaseoli growing in minimal medium was repressed by the addition of hydroxylysine (Hyl), although the sensitivity of the former to Hyl seemed to be lower than that of the latter. The nodulation efficiency of both Glycine max (L.) inoculated with B. japonicum cells and Phaseolus vulgaris (L.) inoculated with R. leguminosarum bv. phaseoli cells was reduced in the presence of Hyl, concomitantly with the decrease in the elongation of roots. Besides, the Hyl contents in the seed (seedling) exudates tended to increase when the host plants were inoculated with an unfavorable strain for their nodulation. These results suggest that the Hyl plays a role in the effective symbiotic relationship by regulating the growth of the root nodule bacteria on the root surface and/or the elongation of the host plant’s roots.

Action Spectra for UVB Impacts on Blepharisma japonicum Motility and Photobehavior

Abstract— The ciliate Blepharisma japonicum was exposed to artificial polychromatic and monochromatic UV radiation to evaluate the relative roles of UVB (280–315 nm UV radiation) and UVA (315–400 nm UV radiation) in altering its motility and photobehavior and to determine absolute weighting coefficients for these effects in the UVB range. Under polychromatic UV irradiation B. japonicum cells showed a severe reduction of cell speed and of the capability to respond to light stimuli. At low doses, however, UV caused a significant increase in the average velocity of a cell population. The UVB exclusion experiments indicated that UVA does not significantly alter motility and photoresponsiveness. The increase and the subsequent decrease in cell velocity was observed also under monochromatic irradiation at 281, 290 and 300 nm, whereas at 310 nm cells swim faster up to the highest photon flux density used. The cell capability of reacting to photic stimuli, conversely, steadily declined with increasing photon flux density at all the tested UVB wavelengths. The action spectra for the alteration of cell velocity and the impairment of photoresponsiveness show that the lower the irradiation wavelength, the more remarkable are the UVB effects and suggest different targets for the increase and the decrease in cell velocity.

Action Spectra for UVB Impacts on Blepharisma japonicum Motility and Photobehavior

Abstract— The ciliate Blepharisma japonicum was exposed to artificial polychromatic and monochromatic UV radiation to evaluate the relative roles of UVB (280–315 nm UV radiation) and UVA (315–400 nm UV radiation) in altering its motility and photobehavior and to determine absolute weighting coefficients for these effects in the UVB range. Under polychromatic UV irradiation B. japonicum cells showed a severe reduction of cell speed and of the capability to respond to light stimuli. At low doses, however, UV caused a significant increase in the average velocity of a cell population. The UVB exclusion experiments indicated that UVA does not significantly alter motility and photoresponsiveness. The increase and the subsequent decrease in cell velocity was observed also under monochromatic irradiation at 281, 290 and 300 nm, whereas at 310 nm cells swim faster up to the highest photon flux density used. The cell capability of reacting to photic stimuli, conversely, steadily declined with increasing photon flux density at all the tested UVB wavelengths. The action spectra for the alteration of cell velocity and the impairment of photoresponsiveness show that the lower the irradiation wavelength, the more remarkable are the UVB effects and suggest different targets for the increase and the decrease in cell velocity.

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum.

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion. The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.

Resazurin reducing time as an indicator of Bradyrhizobium viable cell count

A method based on the use of resazurin (RSZ) is described to determine the number of viable Bradyrhizobium japonicum cells in culture medium. The observation of RSZ reduction can be done spectrophotometrically or visually. B. japonicum strains behaved differently when the reducing time was considered. This methodology can be used to determine the number of viable cells during the liquid culture stage of inoculant production.

Differences among Rhizobium meliloti and Bradyrhizobium japonicum strains in tolerance to desiccation and storage at different relative humidities

The behaviour of Rhizobium meliloti and Bradyrhizobium japonicum (three strains of each) during storage in atmospheres of controlled relative humidities (r.h.) offrom 3 to 83.5% at 30°C in the presence of air were investigated. Whatever the r.h. values imposed, the desiccation step did not significantly affect the survival rates except for B. japonicum G49. However, differences in behaviours and tolerances against protracted storage at the five r.h. values tested were noted between rhizobia and bradyrhizobia. Rhizobium meliloti strains were found more susceptible to storage at 3 or 83.5% r.h. than B. japonicum strains. Whatever the r.h. values, cell populations of B. japonicum declined at a rather constant rate and no surviving cells were observed after 100 days of storage. On the contrary, two step decreases were noted for R. meliloti stored at ⩽ 67.8% r.h. and appreciable survival rates were still obtained after 100 days of storage at 22, 43.6 or 67.8% r.h. Based on scanning electron microscope examinations, it was found that drying by itself did not morphologically affect brady(rhizobia) cells. The combined processes of drying and rehydration led to minor increases in cell volumes (for cells stored at 83.5% r.h.) and appearance of numerous granules and strands of exopolymers (for cells stored at 3% r.h.). After storage in their dried states, loss of infectiveness was observed for B. japonicum cells whereas R. meliloti cells maintained the same degree of symbiotic infectivity.

Carbohydrate binding activities of Bradyrhizobium japonicum: IV. Effect of lactose and flavones on the expression of the lectin, BJ38.

BJ38 is a galactose/lactose-specific lectin (M(r) approximately 38,000) found at one pole of Bradyrhizobium japonicum. It has been implicated in mediating the adhesion of the bacteria to soybean roots, leading to the establishment of a nitrogen-fixing symbiosis. When the ligand lactose is added to cultures of the bacteria for at least 1 h prior to harvesting the cells for BJ38 isolation, the yield of the protein was found to be elevated in a dose-dependent fashion. Half maximal stimulation was observed at approximately 50 microM; the effect was saturated at approximately 1 mM, where a 10-fold higher yield of BJ38 was obtained. Saccharides with a lower affinity for BJ38 than lactose yielded a correspondingly smaller induction effect when compared at a concentration of 1 mM. The higher level of BJ38 induced by lactose is also manifested by an elevated amount of BJ38 detectable at the cell surface and by a higher number of B. japonicum cells adsorbed onto soybean cells. Surprisingly, the induction of BJ38 expression seen with lactose was also observed with certain, but not all, flavonoids that induce the nod genes of the bacteria; genistein mimicked the induction observed with lactose, whereas luteolin failed to stimulate BJ38 production.

In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110.

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.

Acid—base and redox properties of Blepharismin photoreceptor pigments. A pulse radiolysis and flash photolysis study

Laser flash photolysis spectra of chromophore extracts from Blepharisma japonicum cells have been measured. The variation of the triplet state spectra with pH is reported and the related pK a , values of 2–3 for deprotonation of the excited triplet state was determined. The anionic Blepharismin triplet state does not appear to readily undergo one-electron reduction by mild reducing agents, but undergoes oxidation by benzoquinone. Pulse radiolysis has been used to independently determine the spectra of the one-electron reduced and one-electron-oxidised forms of Blepharismin.

Carbohydrate binding activities of Bradyrhizobium japonicum: unipolar localization of the lectin BJ38 on the bacterial cell surface.

A polyclonal antiserum generated against the Bradyrhizobium japonicum lectin BJ38 was characterized to be specifically directed against the protein. Treatment of B. japonicum cells with this antiserum and subsequent visualization with transmission electron microscopy and both conventional and confocal fluorescence microscopy revealed BJ38 at only one pole of the bacterium. BJ38 appeared to be organized in a tuft-like mass, separated from the bacterial outer membrane. BJ38 localization was coincident with the attachment site for (i) homotypic agglutination to other B. japonicum cells, (ii) adhesion to the cultured soybean cell line SB-1, and (iii) adsorption to Sepharose beads covalently derivatized with lactose. In contrast, the plant lectin soybean agglutinin labeled the bacteria at the pole distant from the bacterial attachment site. These results indicate that the topological distribution of BJ38 is consistent with a suggested role for this bacterial lectin in the polar binding of B. japonicum to other cells and surfaces.

Physicochemical and D-galactose-mediated interactions in the attachment of Bradyrhizobium japonicum to roots of Glycine max

The roles of electrostatic, hydrophobic, and lectin-mediated interactions in the attachment of Bradyrhizobium japonicum to soybean (Glycine max [L.] Merr.) seedling roots were examined. Cell suspensions (104 cfu/mL) of B. japonicum strain USDA 110 were exposed to excised root segments during 2-h adhesion assays. The presence of the specific hapten for the soybean seed lectin, the sugar N-acetyl-D-galactosamine (GalNac), did not inhibit attachment, but D-galactose did (29% of untreated control). Some D-galactose inhibition (69% of control) was observed when the bacteria were preincubated in the presence of the sugar before the attachment assay but not when the roots were preincubated. Attachment was pH dependent, reaching maximum values between pH 6.0 and 6.5. The polycations DEAE-dextran and poly-L-lysine stimulated adhesion 12- and 5-fold to excised root segments, respectively; DEAE-dextran caused a 7-fold attachment stimulation to intact seedling roots. Segregation of bacterial cells into hydrophobic and nonhydrophobic fractions by preexposure to polystyrene did not affect attachment to roots, nor did the presence of two detergents, sodium dodecyl sulfate and Tween 80. These results indicate that hydrophobic interactions are not involved in the attachment, but anionic repulsion may limit attachment between B. japonicum cells and G. max roots (both surfaces are negatively charged at the assay medium pH of 6.3). The attachment is not via the GalNac-specific soybean lectin but apparently is mediated by a D-galactose-binding moiety located on the bacterial cell surface.Key words: bradyrhizobia, attachment, bacterial lectins, polycations

Stimulation of hydrogenase synthesis in free-livingBradyrhizobium japonicumby addition of nitrate and ammonium

Abstract Free-living cells as well as bacteroids of Bradyrhizobium japonicum are able to oxidize H2 evolved from nitrogenase reaction. Low partial pressure of O2 in the presence of H2 and low concentration of carbon substrates were considered to be required for the expression of the uptake hydrogenase (Hup) activity in free-living B. japonicum (Maier et al. 1978). However, the Hup activity reported by Maier et al. (1979) was lower than that of bacteroids (McCrae et al. 1978). When we examined the conditions of derepression for higher Hup activity in free-living B. japonicum cells, we observed that the addition of NH4C1 enhanced the Hup activity. Therefore, we decided to investigate the effects of nitrogen sources on the derepression of the Hup activity in free-living B. japonicum.

Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters.

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.

When Does the Self-Regulatory Response Elicited in Soybean Root after Inoculation Occur?

The inoculation of soybean (Glycine max L.) roots with Bradyrhizobium japonicum produces a regulatory response that inhibits nodulation in the younger regions of the roots. By exposing the soybean roots to live homologous bacteria for only a short period of time, the question of whether or not early interactions of rhizobia with root cells, prior to infection, elicit this regulatory response has been explored. B. japonicum cells mixed with infective bacteriophages were applied to the roots and then 6 or 24 hours later roots were again inoculated with phage-resistant rhizobia. Mixing of the rhizobia and bacteriophages caused bacterial lysis in 6 to 8 hours and allowed the bacteria to act as live symbionts on the root for only a few hours. However, the interaction of live homologous bacteria with the soybean roots for a few hours did not cause inhibition of nodulation in the younger regions of the roots. Results of these experiments indicate that the self-regulatory response in soybean is not rapidly produced by the early, pre-infection, interactions between rhizobia and the root cells.

Efficiency of Nodule Initiation in Cowpea and Soybean

When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts.

Response of soybean [ Glycine max (L.) merrill.] to Bradyrhizobium japonicum inoculation in three soils in ghana

Three antigenically distinguishable Bradyrhizobium japonicum streptomycin-resistant mutant strains (SRJ-S, S14-S and SX-S) were evaluated in three soil series in Ghana (Tikobo, pH 4.3; Amo, pH 5.4; Akuse, pH 6.1), for nodulating ability and competitiveness for nodule occupancy on soybean var. Williams. Few nodules developed on plants grown in the uninoculated soils. The most probable number counts on sand-grown soybean indicated 23, 280 and 330 native B. japonicum cells g −in the Tikobo, Akuse and Amo soils, respectively. Inoculation with single or mixed strain combinations of SRJ-S. S14-S and SX-S, at 10 5 cells g −1 soil greatly improved nodulation (>10-fold), N accretion (50–470%) and shoot dry weight (40–90%). When independently inoculated into soil, these strains formed between 75–100% of the soybean nodules, with no significant nodulation differences between any of the single strain inoculation treatments in each soil. However, strain identification from nodules formed by mixed strain inoculation treatments (in population ratios of 1:1 or 1:1:1) revealed marked variations in competitiveness between the three strains for nodule occupancy. Nodules formed by the most competitive strain, SRJ-S (obtained from Kenya), were also the most effective in N 3 fixation (as measured by the total N in plants). Of the locally isolated strains, S14-S was more competitive than SX-S. The nodulation, N content and dry matter yield results showed that, in general, mixed strain inoculation resulted in similar effects as those by the best individual in a particular strain-combination.