Japonicum Cells Research Articles

Comparison of cell propagation methods for their effect on monoclonal antibody yield in fermentors

Four different systems for the propagation and maintenance of hybridoma cells in fermentors were compared to batch cultures for their effect on cell viability and monoclonal antibody production. For these studies cells were grown as either batch, fed-batch, semi-continuous, 2-stage, or perfusion types of cultures. The hybridoma line employed produced a monoclonal antibody reactive with a somatic antigen on Rhizobium japonicum cells. It was previously shown that final yields in batch cultures ranged between 100–200 μg/ml with an average production rate of 15 mg/l culture per day. Daily addition of fresh medium to batch cultures (fed-batch propagation) raised the titer to over 200 μg/ml and increased the productivity to about 27 mg/l per day. In the semi-continuous system, medium was periodically (twice daily) added to the culture and an equal amount of the culture was removed. With a replacement rate of 200 ml/l per day, cells were maintained at a level of 2.4 × 10 6/ml for over 2 months. The antibody titers averaged 170 μg/ml and the productivity was 34 mg/l per day. Further increase in productivity was achieved by applying a second stage to the semi-continuous fermentation where cells were periodically fed with glucose and glutamine. With this additional stage, an antibody titer of 310 μg/ml and a productivity of 62 mg/l per day, was obtained. A perfusion culturing method was found to be the most effective for production of McAb. A cylindrical filter with 5 μm openings was mounted around the stirring shaft of a 1 liter fermentor. Cells were grown on the outside of the filter with continuous feeding of growth medium while, at the same rate, cell-free supernatant liquid containing antibody was removed from the inner part of the filter. Cells were propagated in this system on a continuous basis for about a month. After 12 days the system reached steady-state conditions in which concentration of live cells, dead cells, glucose, lactic acid, ammonium and McAb were relatively constant. Average concentrations of 2.2 × 10 7 live cells/ml and 390 μg of antibody/ml, corresponding to a productivity of 660 mg/l per day, were achieved.

Kinetics of monoclonal antibody production in low serum growth medium

Factors affecting growth and monoclonal antibody production in vitro by a mouse-mouse hybridoma cell line have been investigated in a series of studies. The goal was to maximize antibody yields and demonstrate that antibodies can be produced efficiently on a large-scale in fermentors. This initial report describes (i) development of a radial immunodiffusion assay for accurate determination of antibody levels in culture, (ii) a culture medium formulation that allowed for reduction in the amount of fetal bovine serum required for good cell growth, and (iii) the kinetics of cell growth and monoclonal antibody production in low-serum media. The radial immunodiffusion assay, employing rabbit anti-mouse IgG antibodies in the immobile phase and the monoclonal antibody (an IgG2a to Rhizobium japonicum cells) as the antigen in the mobile phase, was more reproducible and reliable for determining antibody levels in culture broth than was an indirect enzyme-linked immunosorbent assay. Addition of 0.25% Primatone RL and 0.01%Pluronic F-68 to Dulbecco's modified Eagle medium allowed cells to adapt to growth in medium containing as little as 1% fetal bovine serum; without these additives, 5% serum was the lowest level attained. For the kinetic studies, cells were grown in the low-protein medium in 3 liter spinner flasks. Antibody production occured during the growth phase, however, significant amounts were also produced during later phases when the cells had stopped growing. Final titers were 100–200 μg/ml. It was concluded that maintenance of cell viability is more important than growth rate in production of antibody. This conclusion, confirmed in other studies, has developed into the major underlying strategy employed in subsequent investigations to maximize antibody production in stirred reactors.

A Rapid Regulatory Response Governing Nodulation in Soybean

The number of nodules which develop on the primary root of soybean seedlings (Glycine max L. Merr) after inoculation with Rhizobium japonicum is substantially diminished in the region of the root developmentally 10 to 15 hours younger than the region maximally susceptible to nodulation at the time of inoculation. This rapid inhibition of nodulation has been investigated by inoculating soybean seedlings with rhizobia at two different times, 15 hours apart. Living R. japonicum cells, but not heterologous rhizobia or UV-killed cells of the homologous bacterium, were capable of eliciting the rapid inhibitory response. Nodulation responses to varying inoculum concentrations showed that bacterial dosages could be superoptimal, resulting in reduced nodulation and reduced inhibition of nodulation. When suspensions of R. japonicum were dripped uniformly onto the root surfaces, the degree of inhibition of nodulation in developmentally younger regions of the root was correlated with the number of nodules formed in the older and initially most susceptible region of the root. Nodulation in the developmentally younger region of the root, however, was affected very little if the first inoculum was restricted to contact with root cells in the region initially most susceptible to nodulation. The rapid regulatory response may be an important factor contributing to the clustering of nodules in the crown region of soybean roots in field-grown plants and the sparse nodulation commonly observed in younger regions of the root.

Spot inoculation of soybean roots withRhizobium japonicum

A method has been developed to inoculate soybean roots in the zone of susceptible root cells with nanoliter droplets containing the symbiont,Rhizobium japonicum. The position of inoculation is marked by means of ion exchange beads which adhere to the root surface. InoculatedR. japonicum cells from a nanoliter droplet spread over an area of approximately 35 host epidermal cells and have a high probability (∼ 80%) of inducing nodule formation at exactly the point of inoculation. The spot inoculation method is highly efficient in terms of the number of bacteria required to achieve a given probability of nodule formation. The method greatly facilitates ultrastructural analysis of the infection process and is useful for certain physiological studies of infection.

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 μg ml(-1). Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30-70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.

RNA polymerase from Rhizobium japonicum.

DNA-dependent RNA polymerase (EC 2.7.7.6) from Rhizobium japonicum was purified. The subunit structure was found to be beta beta' alpha 2 alpha, with the following apparent molecular weights determined by electrophoresis: Mr (beta and beta') 150,000 each, Mr (sigma) 96,000, Mr (alpha) 40,000, Mr (holoenzyme) 490,000, Mr (core enzyme) 380,000. The recovery of sigma was 28%. RNA polymerase from aerobically grown R. japonicum cells and from nitrogen-fixing cells have the same electrophoretic properties suggesting that no chemical modification of the enzyme occurs when cells undergo this metabolic differentiation. The enzyme is Mg2+-dependent, rifampicin-sensitive, and has optimal activity at alkaline pH (8--10) and at 35--40 degrees C. It binds strongly to bacteriophage T7 promoters, weakly to antibiotic resistance genes, and not at all to cloned R. japonicum nif DNA. Preliminary in vitro transcription experiments, including nif DNA as template, revealed that additional factors may be required for selective transcription from promoters.

Cytochrome patterns in Rhizobium japonicum cells grown under chemolithotrophic conditions

The addition of H2 to suspensions of chemolithotrophically grown Rhizobium japonicum strikingly increased the rates of reduction of cytochromes of the b and c-types. There was no effect of H2, however, on the cytochrome reduction rates of suspensions of heterotrophically cultured cells which do not show hydrogenase activity. An additional component whose difference spectrum appears to be similar to that of b-type cytochromes was found in chemolithotrophically grown R. japonicum. The behavior of this additional component in the presence and absence of H2 and KCN was investigated and it is concluded that it is involved in H2 metabolism.

Development of nodules of Glycine max infected with an ineffective strain of Rhizobium japonicum

Bacteroids in ineffective (nitrogenase negative) nodules of Glycine max, infected with Rhizobium japonicum 61-A-24, as compared to those in effective nodules are characterized by reduced specific activities of alanine dehydrogenase to 15%, of 3-hydroxybutyrate dehydrogenase to 50%, and an increase of glutamine synthetase to 400%. In the plant cytoplasm of ineffective nodules, glutamine synthetase activity is reduced to 10-30%, glutamate dehydrogenase to 50-70%, and the aspartate aminotransferase and alanine aminotransferase are enhanced to 120-200%, depending on the age of the nodules. The total pool of soluble amino acids is reduced to 52 μmol per g nodule fresh weight, as compared to 186 μmol in effective nodules, with a replacement of asparagine (42 mol% of the amino acids) by an unknown amino compound. This compound is absent in nitrogenase, repressed and derepressed, free-living Rhizobium japonicum cells and in the uninfected root tissue. In nitrogenase derepressed, as compared to the repressed free-living cells of Rhizobium japonicum 61-A-101, arginine shows the most obvious change with a reduction to less than one tenth. The ultrastructure of the ineffective nodule is different from the effective organ even in the early stages. The membrane envelopes of the infection vacuoles are decomposing in heavily infected cells within 18 to 20 d after infection. In lightly infected cells very large vacuoles develop with only a few bacteroids inside. No close associations of cristae-rich mitochondria with amyloplasts are observed as in effective nodules. The uninfected cells keep their large starch granules even 40 d after infection. Some poly-β-hydroxybutyrate accumulation in the bacteroids is observed but only in the early stages, and it is almost absent in old nodules (40 d). At this age the infected cells are obviously compressed by uninfected cells, whereas in effective nodules with nitrogenase activity and leghaemoglobin formation, the infected cells have a much higher osmotic pressure than the neighbouring uninfected cells.

Hydrogen (H2) Evolution by Rhizobia after Synergetic Culture with Soybean Cell Suspensions

Rhizobium japonicum cells were grown in liquid suspension cultures and separated from soybean plant cells by two to three bacterial membrane filters. Under these conditions, the plant cells elaborated materials into the medium which aided in the expression of a major rhizobial phenotype, namely, nitrogenase activity (acetylene reduction). The evolution of H(2) was also measured and this activity relative to acetylene reduction, was influenced by: (a) O(2); (b) the quantity of conditioned plant medium; and (c) ammonia. It is concluded that plant substances are of major importance in the H(2) evolution and nitrogenase activities of free-living rhizobia in suspension cultures.

Differentiation of Rhizobium japonicum, I. enzymatic comparison of nitrogenase repressed and derepressed free living cells and of bacteroids.

Derepressed free living cells of Rhizobium japonicum strain 61-A-101 with leucine as single nitrogen source develop a maximum nitrogenase activity of 180 nmol C2H4.mg protein -1.H-1 in liquid culture under 2% 2% O2 in the gas phase. Only 10% of this activity is found with no oxygen in the gas phase during a 90 min incubation period. The maximum activity under 2% oxygen in the gas phase is unaffected by addition of 1-100 mM NH+4 and by addition of low concentrations of glutamine (0.36-1.44 mM). Specific activities of alanine dehydrogenase (E.C. 1.4.1.1.) asparatate aminotransferase (E.C. 2.6.1.1.) and, with much lower activities, of GOGAT (E.C. 1.4.1.13) in nitrogenase active free living cells are more similar to bacteroids than to nitrogenase repressed free living cells from liquid culture. The activities in nitrogenase repressed cells were about 50% lower. Glutamine synthetase (E.C. 6.3.1.2.) activity in bacteroids and in nitrogenase active cells were also similar, but only about 25-30% of that found in nitrogenase repressed Rhizobium japonicum cells.

Immunological evidence for the capability of free-living Rhizobium japonicum to synthesize a portion of a nitrogenase component

Immunodiffusion tests conducted under aerobic conditions demonstrated that cross-reactive material to antiserum prepared against the MoFe protein component of nitrogenase from soybean nodule bacteroids was detectable in extracts of free-living Rhizobium japonicum cells cultured in a standard medium under: aerobic conditions; aerobic conditions with nitrate; aerobic conditions with ammonia; anaerobic conditions with nitrate; and anaerobic conditions with nitrate and ammonia. The most intense precipitin bands resulted from cross-section of the antiserum with extracts of cells cultured anaerobically with nitrate or anaerobically with ammonia and nitrate. Immunodiffusion experiments with crude bacteroid extract and purified MoFe protein revealed a greater number of precipitin bands in tests conducted under aerobic conditions than those conducted under anaerobic conditions. These results indicate that some of the cross-reactive material observed under aerobic conditions resulted from breakdown of the MoFe protein. Bacteroid extracts of nodules from plants supplied with ammonia exhibited only a trace of nitrogenase activity. The addition of an excess of the Fe protein component of nitrogenase, however, resulted in a 270-fold enhancement of activity indicating the presence of active MoFe protein in these extracts.Our experiments together with results published elsewhere provide evidence that the genetic information for synthesis of a part of the MoFe component of nitrogenase is carried by Rhizobium.

Adsorption and selection of rhizobia with ion-exchange papers

Ion exchange papers were used to study the adsorption of 32P-labelled rhizobia on defined surfaces. Two strains of Rhizobium japonicum and one each of R. leguminosarum and R. lupini were compared with Escherichia coli and Bacillus subtilis. The ratio of adsorption to strong and to weak acid papers/strong and weak basic papers was consistantly higher for all rhizobial strains compared to the other bacteria. The process of desorption by increasing the ion-concentration causes about 35% desorption between 0.02 and 0.1 M MgCl2, however, an increase to 1 M does not desorb more labelled Rhizobium japonicum or E. coli cells. The ratio of adsorbed cpm to colony formers, desorbed by 0.1 M NaCl was similar with Rhizobium japonicum for all six ion exchange papers. For E. coli this ratio varied widely for the different papers. The selection of Rhizobium against a more closely related bacterium by this adsorption/desorption procedure was demonstrated with mixed cultures of Rhizobium japonicum and Chromobacterium violaceum giving a more than 80 fold enrichment of the former. Rhizobium japonicum cells, ad/desorbed from all ion exchange papers kept their infectivity and formed nodules on Glycine max with an activity of 20-40 nM C2H4-hr(-1)-mg nodule(-1). A desorption of Rhizobium japonicum from soybean roots also occurred by increasing the ion concentration. 2-3 times as many cells were removed in this way compared to washing with water.

Effect of Inoculum Size on Nodulation of Glycine max (L.) Merril, Variety Ford1

AbstractA criterion for determining adequacy of inoculation of Glycine max by Rhizobium japonicum is needed for testing of commercial inoculants and for determining adequacy of nodulation. This study was undertaken to determine the inoculum level needed for maximum nitrogen fixation of Glycine max and to find a qualitative nodulation pattern that would be useful in recognizing good nodulation. To accomplish this end, soybeans were inoculated and grown in pots containing sand or soil in the greenhouse. Inoculation levels up to 2.4 ✕ 105 and 1.4 ✕ 107 Rhizobium japonicum cells per seed were used for soil‐ and sand‐grown plants respectively. Taproot nodulation was still increasing at the highest inoculum level in soil and sand, but lateral root nodulation had leveled off. Maximum total nitrogen fixed in plant tops was achieved at inoculation levels of 2 ✕ 103 and 1 ✕ 105 cells per seed for soil‐ and sand‐grown plants, respectively. At these inoculation levels at least four taproot nodules were formed. Results suggest that taproot nodulation of soybeans is a qualitative characteristic that may be useful in determining the adequacy of nodulation and may indicate abundance of rhizobia for a soybean variety.

PREPARATION AND SOME PROPERTIES OF A SOLUBLE NITRATE REDUCTASE FROM RHIZOBIUM JAPONICUM.

A method was developed for the preparation of a soluble nitrate reductase extract of Rhizobium japonicum cells derived from pure cultures of the organisms or from soy-bean nodules. In the procedure the bacterial suspension, glass beads and small quantities of sodium hydrosulfite and benzylviologen were sealed in a Tygon plastic tube. The cells were broken by a series of grinding and freezing and thawing operations. The cell-free extract was collected by centrifugation. The soluble enzyme preparation is extremely susceptible to oxidation. It was necessary to maintain completely reduced conditions throughout the purification procedures and the experimental operations in which properties of the enzyme were investigated. A simple assay procedure was developed in which benzylviologen (reduced by sodium hydrosulfite) was employed as the electron donor. Since excess reducing agent was present in assay tubes, assays could be conducted under aerobic conditions and still maintain reduced conditions in the reaction mixtures. The excess sodium hydrosulfite was oxidized at the end of the assay periods by vigorously shaking each tube in air. After this the nitrite in each reaction mixture was determined by a colorimetric procedure. The soluble enzyme was purified about 11-fold by (NH4)2SO4 fractionation and chromatography on a calcium phosphate column. Attempts to purify extracts containing sodium hydrosulfite but lacking benzylviologen were unsuccessful. NADH2 and succinate, which were effective electron donors for a particulate enzyme preparation from R. japonicum, were completely ineffective donors for nitrate reduction with the soluble preparation. A series of compounds were tested in an attempt to find a naturally occuring electron donor, but reduced benzyl-or methyl-viologen were the only compounds that functioned with the soluble enzyme. The effects of pH, substrate concentration, and various inhibitors on the activity of the enzyme, were investigated.

Further studies on a particulate enzyme preparation from nodules of soybean plants

In previous publications (4, 6), the preparation and properties of a particulate enzyme complex from Rhizobiuin japonicum cells isolated from the nodules of soybean plants (Glycine max Merr.) have been described. It has been established that the particles catalyze the reduction of nitrate to nitrite andl that either DPNH or succinate serves as effective electron donor. Evidence indicates that the sequence of electron transport from DPNH involves an unidentified factor (replaced by menadione), a cytochrome, the Slater factor, nitrate reductase, and nitrate. When succinate is utilized, electron transfer appears to take place via succinic dehydrogenase, a cytochrome, and then a pathway from cytochronie to nitrate common with that utilized in reduced diphosphopyridine nucleotide (DPNH) oxidation. Investigations (5) on the possible physiological role of the nodule-nitrate reductase complex have provided considerable evidence that the enzyme activity, as measured by the rate of nitrate reduction, is correlated positively with the capacity, or with factors associated with the capacity, to fix atmospheric nitrogen. In general the hemoglobin content of nodules from plants subjected to various treatments and the nitrate reductase activity of Rhizobium japonicum cells obtained from nodules of the same plants are positively correlated. Since there is no evidence that nitrate is involved in the nitrogen fixing process, it has been postulated (4, 5) that the enzyme complex may be non-specific for oxidant and that some unidentified substance with the appropriate oxidation-reduction potential may serve as the natural oxidant under physiological conditions. In view of the physiological experiments reported (5) and the experimental evidence of Bergersen and Wilson (3) concerning the role of nodule hemoglobin in the nitrogen fixing process, the possibility has been considered that some form of nodule hemoglobin may serve as an electron acceptor for the complex. In this paper we report results of experiments in which oxygen, nodule hemiglobin, and nodule oxyhemoglobin have been studied as electron acceptors for the catalysis of succinate oxidation by the particulate enzyme complex isolated from nodule bacteriods.

Properties of a particulate nitrate reductase from the nodules of the soybean plant

A particulate fraction has been isolated from Rhizobium japonicum cells of soybean nodules which catalyzes the reduction of nitrate to nitrite with either DPNH or succinate. The properties of the complex have been studied with each of these electron donors. When succinate is used as an electron donor for the nitrate reductase complex, the system exhibits many of the properties of succinic dehydrogenase and succin-oxidase which have been reported. These include competitive inhibition by malonate and pyrophosphate, inhibition by fluoride and an activation by phosphate. The nitrate reductase complex, with either succinate or DPNH as the electron donor was inhibited by Antimycin A, dicumarol, DNP and p-chloromercuribenzoate. Inhibition by the latter compound was prevented by either glutathione or cysteine. No evidence has been obtained for the involvement of a flavin in electron transport from either DPNH or succinate to nitrate; however, it is concluded that the failure to demonstrate a flavin requirement is associated with the difficulties in dealing with the particulate system. From studies of the inhibition of the nodule nitrate reductase by metal chelating agents and from kinetic studies of the cyanide inactivation of the system it is concluded that metal ions are involved in electron transport at two cyanide-sensitive sites with either DPNH or succinate as the electron donor. When succinate is used as the electron donor for the complex it is suggested that succinic dehydrogenase, a cytochrome, an Antimycin A-sensitive site, two cyanide sensitive sites, and nitrate reductase are involved in the electron transport to nitrate. When DPNH is used as a source of electrons for the system, the evidence indicates involvement of a vitamin K, or a related quinone, a cytochrome, an Anti-mycin A-sensitive site, two cyanide-sensitive sites, and nitrate reductase.