Competition of Rhizobium japonicum Strains in Early Stages of Soybean Nodulation

The effects of preexposure of soybean (Glycine max L. Merrill) roots to Rhizobium japonicum strains and subsequent establishment of other strains in the nodules were investigated by using combinations of effective strains (USDA 110 and USDA 138) and effective-ineffective strains (USDA 110 and SM-5). Strain USDA 110 was a better competitor than either USDA 138 or SM-5 on cultivars Lee and Peking. However, when either of the two less-competitive strains was inoculated into 2-day-old seedlings before USDA 110 was, their nodule occupancy increased significantly on both cultivars. With USDA 138 as the primary inoculum and USDA 110 delayed for 6, 48, and 168 h, the incidence of USDA 138 nodules increased on cultivar Peking from 6% (at zero time) to 28, 70, and 82% and on cultivar Lee from 17% (at zero time) to 32, 88, and 95% for the three time delays, respectively. Preexposure of 2-week-old roots of cultivar Lee to USDA 138 had essentially the same effect: the incidence of USDA 138 nodules increased from 23% at zero time to 89 and 97% when USDA 110 was delayed for 24 and 72 h, respectively. When the ineffective strain SM-5 was used as the primary inoculum, followed by USDA 110 72 h later, the percentage of nodules containing SM-5 increased from 7 to 76%. These results indicate that the early events in the nodulation process of soybeans are perhaps the most critical for competition among R. japonicum strains.     

Immunofluorescent polar tips of Rhizobium japonicum: possible site of attachment or lectin binding.

Rhizobium japonicum USDA 31 demonstrated marked polarity by binding homologous fluorescent antibody (FA) heavily on one end of the cell. FA prepared against R. japonicum strains 110 and 138, and against R. trifolii TA1 cross-reacted with strain 31 only in the polar tip region. No polar immunofluorescing tips could be seen with FA against two other strains of R. japonicum or with those against several unrelated microorganisms. Common antigens localized only in a polar region were seen in many rhizobia stained with R. japonicum 31 FA: 22 of 23 strains of R. japonicum, 10 of 17 strains of R. trifolii, 3 of 7 strains of R. melitolii, 3 of 6 strains of R. phaseoli, and 3 of 9 strains of R. leguminosarum had some cells with detectable polar tips. The proportion of R. japonicum 31 cells with polar tips was high throughout the growth cycle. Polar tip staining was not affected by drastic cell treatments. A function was proposed for the polar tip region as a site for attachment. R. japonicum 31 cells attached to each other in a tip-to-tip fashion and endwise to fungal hyphae with the polar tip in contact with the hyphal wall. Binding of fluorescein isothiocyanate-labeled soybean lectin to certain strains of R. japonicum gave additional evidence of polarity. Polar binding of both antibody and lectin may provide insights into relationships between rhizobia and roots of host legumes.     

N2 fixation by soybean-Bradyrhizobium combinations under acidity,low P and high Al stresses

Five strains of Bradyrhizobium japonicum (USDA 6, 110, 122, 138, and 143) were screened in cell culture for tolerance to acidity (pH 4.2, 4.4, and 4.6) and Al (0, 3, 4, 5, and 6 mg L^–1) under low P conditions. Each strain was later grown in association with seven soybean [Glycine max. (L) Merr.] cultivars which were also screened for tolerance to the same stresses in nutrient culture to determine which soybean-Bradyrhizobium combinations would establish the most effective symbiotic N₂ fixing relationships. Results indicated that strains USDA 110 and 6 were more tolerant than USDA 122, 138 and 143 with USDA 110 being the most tolerant. Acidity appeared to be the more severe stress; but even when strains showed tolerance to the stresses, cell numbers were significantly reduced. This suggests that colonization of soils and soybean roots can be adversely affected under similar conditions in the field which may result in reduced nodulation. The strains found to be more tolerant to the stresses were more effective N₂ fixers in symbiosis with all soybean cultivars, with USDA 110 being definitely superior. The association between the more tolerant strains and cultivars had the largest nitrogenase activity. Further studies on the inclusion of tolerant Bradyrhizobium strains in inoculum used on tolerant soybean cultivars in the field are warranted.     

Evidence from Internally Transcribed Spacer Sequence Analysis of Soybean Strains that Extant Bradyrhizobium spp. Are Likely the Products of Reticulate Evolutionary Events

The internally transcribed spacer (ITS) sequences of several members within each of 17 soybean bradyrhizobial serogroups were determined to establish whether the regions within all members of each serogroup were identical. The rationale was to provide a sequence-based alternative to serology. The objective also was to link the extensive older literature on soybean symbiosis based on serology with ITS sequence data for more recent isolates from both soybean and other legumes nodulated by rhizobia within the genus Bradyrhizobium. With the exception of serogroup 31 and 110 strains, sequence identity was established within each serogroup. Variation ranged from 0 to 23 nucleotides among serogroup 31 strains, and the regions in the type strains USDA 31 (serogroup 31) and USDA 130 (serogroup 130) were identical. Sequence identity was established among most strains within serogroup 110. The exceptions were USDA 452 and USDA 456, which had ITS sequences that were identical with those of the serotype 124 strain, USDA 124. Perhaps this would imply that USDA 452, USDA 456, and serogroup 31 strains are members of rhizobial lineages resulting from genetic exchange and homologous recombination events. This conclusion would be supported by the construction of a phylogenetic network from the ITS sequence alignment implying that the genomes of extant members of the genus Bradyrhizobium are likely the products of reticulate evolutionary events. A pairwise homoplasy index (phi or Φ_w) test was used to obtain further evidence for recombination. The ITS sequences of USDA 110 and USDA 124 were more divergent (53 nucleotides) than this region between the type strain Bradyrhizobium japonicum USDA 6^T and the proposed species Bradyrhizobium yuanmingense (28 nucleotides) and Bradyrhizobium liaoningense (48 nucleotides). Therefore, support for assigning discrete species boundaries among these three proposed species appears limited, considering the evidence for recombination, the narrow divergence of the ITS sequence, and their relative placement on the phylogenetic network.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.     

豌豆根瘤菌与新疆中华银瘤菌原生质体的属间隔合研究

以青霉素和氯霉素分别作为Rhizobium leguminosoum USDA2370和Sinorhizobium xinjiangnesis CCBAU110)的抗药性标记。利用原生质体融合技术,成功地获得了USDA2370和CCBAU110的属间隔合菌株。该融合菌株可分别在双亲寄主植物上结瘤。融合菌株在细菌形态、大小、菌落特征及蛋白质电泳图谱上与亲本菌株均有所不同。融合菌株与USDA23703的DNA同源性为56．6％,而与CCBAU110的DNA同源性为10．2％。     

Symbiotic Effectiveness and Host-Strain Interactions of Rhizobium fredii USDA 191 on Different Soybean Cultivars

Nodulation, acetylene reduction activity, dry matter accumulation, and total nitrogen accumulation by nodulated plants growing in a nitrogen-free culture system were used to compare the symbiotic effectiveness of the fast-growing Rhizobium fredii USDA 191 with that of the slow-growing Bradyrhizobium japonicum USDA 110 in symbiosis with five soybean (Glycine max (L.) Merr.) cultivars. Measurement of the amount of nitrogen accumulated during a 20-day period of vegetative growth (28 to 48 days after transplanting) showed that USDA 110 fixed 3.7, 39.1, 4.6, and 57.3 times more N₂ than did USDA 191 with cultivars Pickett 71, Harosoy 63, Lee, and Ransom as host plants, respectively. With the unimproved Peking cultivar as the host plant, USDA 191 fixed 3.3 times more N₂ than did the USDA 110 during the 20-day period. The superior N₂ fixation capability of USDA 110 with the four North American cultivars as hosts resulted primarily from higher nitrogenase activity per unit nodule mass (specific acetylene reduction activity) and higher nodule mass per plant. The higher N₂-fixation capability of USDA 191 with the Peking cultivar as host resulted primarily from higher nodule mass per plant, which was associated with higher nodule numbers. There was significant variation in the N₂-fixation capabilities of the four North American cultivar-USDA 191 symbioses. Pickett 71 and Lee cultivars fixed significantly more N₂ in symbiosis with USDA 191 than did the Harosoy 63 and Ransom cultivars. This quantitative variation in N₂-fixation capability suggests that the total incompatibility (effectiveness of nodulation and efficiency of N₂ fixation) of host soybean plants and R. fredii strains is regulated by more than one host plant gene. These results indicate that it would not be prudent to introduce R. fredii strains into North American agricultural systems until more efficient N₂-fixing symbioses between North American cultivars and these fast-growing strains can be developed. When inoculum containing equal numbers of USDA 191 and of strain USDA 110 was applied to the unimproved Peking cultivar in Perlite pot culture, 85% of the 160 nodules tested were occupied by USDA 191. With Lee and Ransom cultivars, 99 and 85% of 140 and 96 nodules tested, respectively, were occupied by USDA 110.     

Transposon mutagenesis of Rhizobium japonicum

Summary We report here successful mutagenesis with Transposon Tn5 of three slow-growing strains of Rhizobium japonicum USDA 122, 61A76, USDA 74 and one fast-growing strain, USDA 191. Strains were chosen as representatives of different DNA homology and serogroups of this divergent species, which effectively nodulate North American soybean cultivars. The source of Tn5 was the suicide plasmid pGS9, which possesses broad host range N-type transfer genes in a narrow host range p15A replicon. The selection of Tn5 mutants was facilitated by the expression of the Tn5 encoded streptomycin gene in R. japonicum. Kanamycin and streptomycin resistant colonies appeared from interspecific crosses with E. coli at optimal frequencies of 10^-6 for R. japonicum USDA 61A76 and USDA 191 and 5x10^-7 for R. japonicum USDA 122 and USDA 74. Altogether, 6550 Tn5 mutants were isolated in USDA 122 and 61A76, and a small number from USDA 74 and USDA 191. Colony hybridization showed that all tested mutants of 61A76 and USDA 122 contained Tn5. Physical analysis of total DNAs from representative numbers of USDA 122, 61A76 and USDA 191 mutants revealed that each of them carried one copy of the transposon integrated randomly in the genome. This was also true for most USDA 74 mutants. Screening of mutants for auxotrophy showed frequencies of 0.2% for USDA 122 and 0.08% for 61A76. Several symbiotically defective mutants were identified on plants, Glycine soja and G. max.     

Competitiveness of indigenous populations of Bradyrhizobium japonicum serocluster 123 as determined using a root-tip marking procedure in growth pouches

Soil Bradyrhizobium populations limit nodule occupancy of soybean by symbiotically-superior inoculant strains throughout much of the American midwest. In this study, the competitiveness of indigenous populations of B. japonicum serocluster 123 from Waukegan and Webster soils was evaluated in growth pouches using a root-tip marking procedure. The native rhizobia were from soils incubated 0–8 h in soybean root exudate (SRE) or plant nutrient solution (PNS) prior to inoculation. Populations of serocluster 123 strains in soil and nodule occupancy by these strains were assessed using fluorescent antibodies prepared against B. japonicum USDA 123. There were no significant differences in populations that came from SRE or PNS incubated soils: both populations increased in number over the incubation period. Nodule occupancy by both populations in growth pouches was similar to that previously encountered in field studies with these two soils. With the Waukegan soil, the serocluster 123 population dominated nodulation forming 69 and 62% of taproot nodules above and below the root tip mark, respectively. However, for the more alkaline Webster soil, serocluster 123 strains were much less competitive, producing only 9 and 13%, respectively, of the nodules formed above and below the root tip mark. In growth pouches, soil populations of bradyrhizobia from the Webster soil produced significantly more nodules than those from the Waukegan soil, but both strains and a pure culture of USDA 110 had a similar distribution of nodules.     

Effects of Bradyrhizobium japonicum and Soybean (Glycine max (L.) Merr.) Phosphorus Nutrition on Nodulation and Dinitrogen Fixation

Cells of Bradyrhizobium japonicum were grown in media containing either 1.0 mM or 0.5 μM phosphorus. In growth pouch experiments, infection of the primary root of soybean (Glycine max (L.) Merr.) by B. japonicum USDA 31, 110, and 142 was significantly delayed when P-limited cells were applied to the root. In a greenhouse experiment, B. japonicum USDA 31, 110, 122, and 142 grown with sufficient and limiting P were used to inoculate soybeans which were grown with either 5 μM or 1 mM P nutrient solution. P-limited cells of USDA 31 and 110 formed significantly fewer nodules than did P-sufficient cells, but P-limited cells of USDA 122 and 142 formed more nodules than P-sufficient cells. The increase in nodule number by P-limited cells of USDA 142 resulted in significant increases in both nodule mass and shoot total N. In plants grown with 1 mM P, inoculation with P-limited cells of USDA 110 resulted in lower total and specific nitrogenase activities than did inoculation with P-sufficient cells. Nodule numbers, shoot dry weights, and total N and P were all higher in plants grown with 1 mM P, and plants inoculated with USDA 31 grew poorly relative to plants receiving strains USDA 110, 122, and 142. Although the effects of soybean P nutrition were more obvious than those of B. japonicum P nutrition, we feel that it is important to develop an awareness of the behavior of the bacterial symbiont under conditions of nutrient limitation similar to those found in many soils.     

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.     

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.     

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.     

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N₂O) reductase (N₂OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N₂O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N₂O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N₂OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N₂OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.     

The use of transposons for the mutagenesis of R. phaseoli and R. japonicum

Plasmid pSUp2011 has been used to transfer transposon Tn5 into the cells of R. japonicum 110 and R. phaseoli 693. Transposition of Tn5 into the chromosomes of R. japonicum and R. phaseoli has been demonstrated,resulting in isolation of auxotrophic and symbiotic mutants of both strains. The frequencies of selected auxotrophic mutations have reached 4% in R. japonicum 110 and 0.6% in R. phaseoli 693. Streptomycin resistance gene locating on Tn5 has been found to be phenotypically expressed in R. japonicum 110 and R. phaseoli 693 cells.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

The occurrence of hopanoid lipids in Bradyrhizobium bacteria

Abstract Lipid extraction procedures followed by GLC and GLC-MS analysis were used to investigate the triterpenoid content in Bradyrhizobium and Rhizobium bacteria. Unlike the tested strains of Rhizobium bacteria, a range of triterpenoids e.g., squalene and different classes of hopanoid derivatives were detected in bacteria from all Bradyrhizobium strains investigated (different strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii as well as Bradyrhizobium sp.). Furthermore, related compounds were identified from some hopanoid lipids (e.g., diplopterol) that carried an additional methyl group in their molecular structure. The hopanoid content was high in some strains and accounted for more than 40% of the total lipid fraction (e.g., in strains Bradyrhizobium japonicum USDA 110 and USDA 31), while other strains contained only about a tenth of that amount (e.g., Bradyrhizobium japonicum ATCC 10324 and Bradyrhizobium sp. ( Lupinus ) ATCC 10319).     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.