Viability of Rhizobium bacteroids.

Bacteroids prepared from nodules of soybean and bean were tested for viability. Contrary to the prevailing view that bacteroids are nonviable, it was found that bacteroids averaged 90% viability, irrespective of Rhizobium strain, nodule age, or nodule environment.     

Cupriavidus taiwanensis bacteroids in Mimosa pudica Indeterminate nodules are not terminally differentiated

The beta-rhizobium Cupriavidus taiwanensis forms indeterminate nodules on Mimosa pudica. C. taiwanensis bacteroids resemble free-living bacteria in terms of genomic DNA content, cell size, membrane permeability, and viability, in contrast to bacteroids in indeterminate nodules of the galegoid clade. Bacteroid differentiation is thus unrelated to nodule ontogeny.     

DNA synthesis and fragmentation in bacteroids during Astragalus sinicus root nodule development

Histo- and cytochemical techniques were used to study the DNA replication and fragmentation patterns in bacteroids formed by Mesorhizobium huakuii subsp. rengei in nodules of Astragalus sinicus. DNA replication was detected by the incorporation of 5-bromo deoxy-uridine. Signals denoting DNA synthesis were observed in plant nuclei within the nodule meristem and in bacteroids near the meristem. The TUNEL (TdT-mediated dUTP nick-end labeling) assay was used to measure DNA fragmentation. In nutrient-depleted 1-mpi (month(s) post inoculation) nodule sections, some bacteroids were in vacuoles, and DNA fragmentation signals were observed only in such bacteroids. In contrast, 1-mpi nodule sections without nutrient depletion showed neither bacteroid localization in vacuoles nor DNA fragmentation signals. The bacteroid translocation into vacuoles upon nutrient starvation might results from autophagy of the plant. In 2-mpi nodule sections, bacteroids with DNA fragmentation signals appeared within the cytoplasm of some nodule cells in the senescence zone.     

Poly-beta-hydroxybutyrate Utilization by Soybean (Glycine max Merr.) Nodules and Assessment of Its Role in Maintenance of Nitrogenase Activity

Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-β-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer. Changes in poly-β-hydroxybutyrate content and in activities of nitrogenase, β-hydroxybutyrate dehydrogenase, and isocitrate lyase in nodule bacteroids were investigated under conditions in which the supply of carbohydrate from the soybean plants was interrupted. The poly-β-hydroxybutyrate content of bacteroids did not decrease appreciably until the carbohydrate supply from the host plants was limited by incubation of excised nodules, incubation of plants in the dark, or by senescence of the host plant. Isocitrate lyase activity in bacteroids was not detected until poly-β-hydroxybutyrate utilization appeared to begin. The presence of a supply of poly-β-hydroxybutyrate in nodule bacteroids was not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant. An unusually high activity of β-hydroxybutyrate dehydrogenase was observed in bacteroid extracts but no significant change in the activity of this enzyme was observed as a result of apparent utilization of poly-β-hydroxybutyrate by nodule bacteroids.     

Effect of water stress on the reduction of nitrate and nitrite by soybean nodules

The effects of water stress on nitrate reductase and nitrite reductase activities in symbiotic nodules were examined in field-grown soybean plants (Glycine max L Merr. cv Clark). The in vitro assays of enzyme activity indicated that the nodule cytosol and bacteroids contained both nitrate reductase and nitrite reductase activities. The reduction of nitrate in bacteroids increased significantly as nodule water potential declined from −0.6 to −1.4 megapascals, and then decreased when −1.8 megapascals water potential was reached. On the contrary, the reduction of nitrate in nodule cytosol was inhibited as water stress progressed. Increases in water stress intensity also caused a significant inhibition in nitrite reductase activities of bacteroids and nodule cytosol within soybean nodules. The results show that nitrate reduction occurred both in the cytosol and bacteroids of water-stressed soybean nodules. The reduction of nitrate functioned at different physiological modes in these two fractions.     

Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.     

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts

Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.     

PHB颗粒在红豆草根瘤细菌发育中的动态变化

红豆草根瘤胞间隙和侵入线中另有个别细菌含有PHB颗粒,而且数量很少,一个细菌通常仅有一个。随着细菌被从侵入线中释放到寄主细胞中,这些PHB颗粒立即消失。幼龄细菌不含PHB颗粒,成熟细菌一般也不含这种内含物。当细菌衰老时,它们又再度出现,并大量增加,而后很快减少,直至完全消失。从未发现这种颗粒存在于解体细菌中,尽管它们处于各种不同的解体状态。PHB颗粒在细菌发育中的变化表明,它的多少不仅与根瘤细菌发育密切有关,而且也受制于根瘤品种。     

Bacteroid distributions in alfalfa nodules upon dark-induced senescence and subsequent partial rejuvenation

Photosynthate availability directly controls the maturation, senescence and distribution of bacteroids (inoculum Rhizobium meliloti 102 F28) in alfalfa ( Medicago saliva L. cv. Buffalo) nodules. Mature, dinitrogen-fixing bacteroids were located principally in the middle section (region) of 6- to 8-week-old nodules in light-grown alfalfa plants. Upon dark treatment of the plants, bacteroids in the middle region of a nodule were induced to senescence while those in the tip region began to mature faster. Senescence and deterioration of bacteroids in the basal region of a nodule also were more advanced in the dark-treated plants. Sugar supplied exogenously during the dark period retarded the senescence process. Exposure of the dark-treated plants to light partially restored nitrogenase activity. The distribution of bacteroids in the rejuvenated nodules was similar to that of the light-grown plants.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules.

Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.     

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.     

Site of natural N enrichment of soybean nodules

Soybean (Glycine max L. Merrill) nodules are usually more enriched in ¹⁵N than other tissues. We show that both bacteroids and nodule cortex are considerably more enriched in ¹⁵N than nodule cytosol, with bacteroids being slightly more enriched than the cortex. Hence, ¹⁵N enrichment occurs in cells of both plant and bacterial origin.     

Co-localisation of membranes and actin in growing infected cells of Glycine max root nodules

The root nodule of Glycine max (L.) Merr. is almost spherical at maturity, and its central tissue consists of infected cells filled with numerous symbiosomes containing bacteroids, interspersed with uninfected cells. During the growth of the nodule, the volume of each infected cell and the number of bacteroids per cell increases, and thus abundant membranes are required for the proliferation of symbiosomes. In expanding infected cells, there are areas adjacent to the nucleus that are devoid of bacteroids, but these areas are filled with numerous membranes and actin filaments, surrounded by endoplasmic reticulum membranes, indicating a perinuclear reservoir of newly formed membranes and a role for actin in delivering membranes to proliferating symbiosomes.     

Growth of nodule bacteria in soil]

As was found with the aid of capillary microscopy, the nodule bacteria of pea and lupine in soil are encountered as rods, bacteroids, and cocci. The rod-like cells form bacteroids not only in the nodules but also outside, in soil. The bacteroids are viable and later reproduce the coccoid cells (arthrospores). The rods and bacteroids prevail in soil during flowering of the legumes, and the cocci are predominant at the end of vegetation.     

The pea nodule environment restores the ability of a Rhizobium leguminosarum lipopolysaccharide acpXL mutant to add 27-hydroxyoctacosanoic acid to its lipid A

Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.     

A dual-targeted soybean protein is involved in Bradyrhizobium japonicum infection of soybean root hair and cortical cells

The symbiotic interaction between legumes and soil bacteria (e.g., soybean [Glycine max L.] and Bradyrhizobium japonicum]) leads to the development of a new root organ, the nodule, where bacteria differentiate into bacteroids that fix atmospheric nitrogen for assimilation by the plant host. In exchange, the host plant provides a steady carbon supply to the bacteroids. This carbon can be stored within the bacteroids in the form of poly-3-hydroxybutyrate granules. The formation of this symbiosis requires communication between both partners to regulate the balance between nitrogen fixation and carbon utilization. In the present study, we describe the soybean gene GmNMNa that is specifically expressed during the infection of soybean cells by B. japonicum. GmNMNa encodes a protein of unknown function. The GmNMNa protein was localized to the nucleolus and also to the mitochondria. Silencing of GmNMNa expression resulted in reduced nodulation, a reduction in the number of bacteroids per infected cell in the nodule, and a clear reduction in the accumulation of poly-3-hydroxybutyrate in the bacteroids. Our results highlight the role of the soybean GmNMNa gene in regulating symbiotic bacterial infection, potentially through the regulation of the accumulation of carbon reserves.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Soybean root nodule ultrastructure during dark-induced stress and recovery

Summary Root-nodules of soybean plants dark-stressed for 8 days and then allowed to recover for up to 17 days were examined by transmission electron microscopy. Control nodules possessed all the ultrastructural features characteristic of infected and uninfected nodule cells. Minimal changes in the appearance of host cells and bacteroids occurred during the first four days of dark stress. After 8 days of dark stress, damage was observed in the cellular and organelle membranes; however, very few changes were observed in the bacteroids. Nodule structure continued to degrade during the first two days of recovery after which time nodules either recovered or completely degraded. In the former case, structural integrity returned to all nodule cells. In the latter case all structural integrity of the host cell disappeared; however, bacteroids appeared intact suggesting that they remained viable.Published as Paper no. 7974, Journal Series, Nebraska Agriculture Research Division.