Heat Shock Tolerant Mutants of Rhizobium meliloti Forming Ineffective Nodules

Six heat shock tolerant mutants of Rhizobium meliloti Rmd201 were isolated through transposon Tn5 mutagenesis. The symbiotic assays of these mutants with alfalfa plants, showed four of these mutants to be affected in nitrogenase effectivity also. These four mutants could be classified into two separate complementation groups hssA and hssC through R-prime mediated merodiploid constructions. The hssC mutant Rmd1040 also showed poor interaction with phages indicating surface alterations. The results indicated possible involvement of these loci in symbiosis as well as heat shock response.     

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule Formation in Alfalfa

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in younger parts of alfalfa (Medicago sativum L.) root systems as a feedback response to development of the first nodules (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide synthesis can induce the formation of organized nodular structures (pseudonodules) on alfalfa roots but are defective in their ability to invade and multiply within host tissues. The formation of empty pseudonodules by exo mutants was found to elicit a feedback suppression of nodule formation similar to that elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of a split-root system 24 hours before inoculation of the second side with wild-type cells suppressed wild-type nodule formation on the second side in proportion to the extent of pseudonodule formation by the exo mutants. The formation of pseudonodules is thus sufficient to elicit systemic feedback control of nodulation in the host root system: infection thread development and internal proliferation of the bacteria are not required for elicitation of feedback. Pseudonodule formation by the exo mutants was found to be strongly suppressed in split-root systems by prior inoculation on the opposite side with the wild type. Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced redifferentiation of host root cells.     

Morphogenetic Rescue of Rhizobium meliloti Nodulation Mutants by trans-Zeatin Secretion

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.     

Analysis of Rhizobium meliloti Sym Mutants Obtained by Heat Treatment

Deletions in the pSym megaplasmid of Rhizobium meliloti were produced at a high frequency, and their lengths varied according to incubation temperature. Morphological differentiation into large and small colonies occurred after heat treatment. Small colonies elicited pseudonodules on alfalfa roots.     

Cooperative Action of Rhizobium meliloti Nodulation and Infection Mutants during the Process of Forming Mixed Infected Alfalfa Nodules

Alfalfa plants co-inoculated with Rhizobium meliloti nodulation (Nod-) and infection mutants deficient in exopolysaccharide production (Inf-EPS-) formed mixed infected nodules that were capable of fixing atmospheric nitrogen. The formation of infected nodules was dependent on close contact between the inoculation partners. When the partners were separated by a filter, empty Fix- nodules were formed, suggesting that infection thread formation in alfalfa is dependent on signals from the nodulation and infection genes. In mixed infected nodules, both nodulation and infection mutants colonized the plant cells and differentiated into bacteroids. The formation of bacteroids was not dependent on cell-to-cell contact between the mutants. Immunogold/silver staining revealed that the ratio of the two mutants varied considerably in colonized plant cells following mixed inoculation. The introduction of an additional nif/fix mutation into one of the inoculation partners did not abolish nitrogen fixation in mixed infected nodules. The expression of nif D::lacZ fusions additionally demonstrated that mutations in the nodulation and infection genes did not prevent the nif genes from being expressed in the mutant bacteroids.     

Sequential Analysis of the Organogenesis of Lucerne (Medicago sativa) Root Nodules Using Symbiotically-Defective Mutants of Rhizobium meliloti

Legume root-nodules are differentiated organs composed of peripheral tissue containing vascular bundles, and a central tissue in which are located the nitrogen-fixing bacteroids. The morphogenesis of these eukaryotic organs is induced by a prokaryotic organism, Rhizobium , which is amenable to genetic analysis. Inoculation of lucerne seedlings with leucine-requiring (Leu⁻) mutants of R. meliloti resulted in the formation of ineffective nodules. In these nodules, bacteria were not released from the infection threads into the host cytoplasm. When urea was provided as a nitrogen source to compensate for the defect in nitrogen fixation, the nodules became anatomically similar to those of effective nodules induced by the wild-type strain. The fact that these nodules were induced by bacteria which remained sequestered in infection threads indicates that nodule morphogenesis can be triggered from a distance. We hypothesize the existence of a bacterial nodule organogenesis-inducing principle (NOIP) which can cross the plant cell wall and plasmalemma.
In nitrogen-fixing nodules the central tissue exhibited a ploidy gradient, while in ineffective Leu⁻ nodules it was found to be monosomatic. The initiation of nodule formation is therefore independent of polyploidy. Supplying the defective plant-bacterial system with l -leucine or one of its precursors, α-ketoisovalerate or α-ketoisocaproate, caused the release of rhizobia into the plant cytoplasm and a restoration of nitrogen fixation. In the central tissue infected cells were polyploid and enlarged, and uninfected cells remained small and contained small nuclei. Therefore induction of differentiation of the central tissue requires the presence of bacteria in the cytoplasm. We hypothesize the role of a bacterial central tissue differentiation inducing principle (CTDIP) which cannot pass from cell to cell.     

Competitive Advantage Provided by Bacterial Motility in the Formation of Nodules by Rhizobium meliloti

The effect of motility on the competitive success of Rhizobium meliloti in nodule production was investigated. A motile strain formed more nodules than expected when mixed at various unfavorable ratios with either flagellated or nonflagellated nonmotile derivatives. We conclude that motility confers a selective advantage on rhizobia when competing with nonmotile strains.     

Rhizobium meliloti Mutants Defective in Symbiotic Nitrogen Fixation Affect the Oxygen Gradient in Alfalfa (Medicago sativa) Root Nodules

Rhizobium meliloti bacteroids carrying mutations in either fdxNor fixX isolated from alfalfa root nodules were shown to containthe nitrogenase proteins NifH, NifD and NifK. In contrast toan in vitro system of N₂-fixation based on R. meliloti wild-typebacteroids, nitrogenase activity could not be restored in crudeextracts of these mutant bacteroids by the addition of an artificialelectron donor, indicating that the nitrogenase proteins werepresent but not functional. ESR-studies revealed that both mutantslacked the FeMo-cofactor of nitrogenase. To analyse the roleof free O₂ on the damage of the nitrogenase components and theFeMo-cofactor in these mutant bacteroids, microelectrode studiesof O₂ concentrations and gradients within alfalfa root noduleswere carried out. R. meliloti mutants defective in other genesnecessary for symbiotic N₂-fixation were also included in thisstudy. Four distinct types of O₂ gradients were defined by theapparent presence or absence of an O₂ diffusion barrier andby the minimum internal O₂ concentration. These data clearlydemonstrated the influence of the microsymbiont on the O₂ gradientswithin the nodules. Nodules induced by Rm0540, an R. melilotimutant with altered exopolysaccharide production, which is notable to infect plant cells, did not contain an O₂ diffusionbarrier. In contrast, nodules containing a mutant defectivein dicarboxylate transport (dctA-), produced an O₂ gradientsimilar to the wild-type. Microelectrode measurements revealedH₂ concentrations in alfalfa wild-type nodules comparable tosoyabean, whereas no hydrogen could be detected in nodules harbouringthe dctA mutant or any other mutant strain. Key words: Nitrogen fixation, Rhizobium meliloti bacteroids, ferredoxin-like proteins, microelectrode studies     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.     

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.     

Influence of Location, Host Cultivar, and Inoculation on the Composition of Naturalized Populations of Rhizobium meliloti in Medicago sativa Nodules

A phage typing system was used to evaluate the composition of indigenous populations of Rhizobium meliloti inhabiting nodules of Medicago sativa cultivars grown with and without inoculation at two field sites during 1983 and 1984. Soil at both locations contained established populations of R. meliloti at planting. Analysis of 1,920 nodule isolates revealed 55 unique phage types of indigenous R. meliloti at one site and 65 indigenous types at the other location. The distributions of phage types differed markedly between locations. At one site, the nodule population was dominated by two phage types; seven others occurred consistently but at lower frequency, and the remainder were encountered infrequently. No indigenous types predominated at the other location, although nine occurred more frequently than the remaining types. Indigenous R. meliloti predominated in nodules from inoculated plots at both sites, with inoculant recovery varying between 10 and 38% in each of two years. The frequency of occurrence of particular phage types at one location was significantly influenced by both M. sativa cultivar and inoculation. At this location, the interaction of cultivar and inoculation on the incidence of phage types suggests that the presence of an inoculant strain differentially affected nodule occupancy of M. sativa cultivars by members of the indigenous R. meliloti population. At both sites, the frequency of specific phage types differed between years. The data emphasize the importance of understanding the ecology and characteristics of indigenous Rhizobium populations as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations.     

lpsZ, a lipopolysaccharide gene involved in symbiosis of Rhizobium meliloti.

lpsZ+ is an allele that allows exo (exopolysaccharide-deficient) mutants of Rhizobium meliloti to invade nodules by modifying rhizobial lipopolysaccharide. We have cloned and sequenced the lpsZ gene. The predicted LpsZ protein has a molecular weight of 48,589 and is probably localized in the cytoplasm. A beta-glucuronidase fusion in the lpsZ gene indicates that lpsZ is not regulated by oxygen or nitrogen.     

Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).

For most samples, thin layer chromatography revealed major peaks of radioactivity at the R_F values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [³H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule.

     

Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E. Kondorosi, C.E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures. The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix- ). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.     

Regulation of Nodulation by Rhizobium meliloti 102F15 on Its Mutant Which Forms an Unusually High Number of Nodules on Alfalfa

A mutant (WL3A150) of Rhizobium meliloti 102F51 that elicits an unusually high number of nodules on its host, alfalfa (Medicago sativa), supports the idea that the host may rely on early bacteroid development in the nodule or on metabolites produced in the infection thread as one of the signals to control further nodulation. This mutant was initially isolated because of its Fix⁻ phenotype. It consistently formed many more nodules than all the other Fix⁻ mutants isolated from strain 102F51 (a total of 11 mutants). Nodules formed by this mutant were small and white and were indistinguishable in appearance from nodules formed by the other Fix⁻ mutants. An ultrastructural study of the nodules, however, showed that this mutant, although forming numerous infection threads, failed to develop into bacteroids. The ability of the mutant to form an unusually high number of nodules coulde be suppressed in a time-dependent manner by the presence of the wild type.     

Structural Studies on New,Non-reducing Oligosaccharides Produced by Rhizobium meliloti J7017

Three non-reducing oligosaccharides were isolated from the fraction of cyclic (1→2)-β-d-glucan of Rhizobium meliloti J7017 by reversed-phase chromatography and paper chromatography. Methylation and ¹H-NMR analyses indicated that they were α-d-glucopyranosyl α-kojitrioside, α-d-glucopyranosyl α-kojitetraoside, and α-d-glucopyranosyl α-kojipentaoside.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.