Slow-growing Rhizobia Research Articles

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This “core” homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.

Nodulation of Lotus pedunculatus in acid rooting solution by fast- and slow-growing rhizobia

The nodulation of Lotus pedunculatus and the multiplication of three Rhizobium loti (fast-growing, acid-producing) and two Bradyrhizobium (slow-growing, alkali-producing) strains was investigated in acidified rooting solution. R. loti strains multiplied at pH 4.5 but Bradyrhizobium strains failed to multiply. No difference in growth rate between R. loti and Bradyrhizobium strains was apparent in rooting solution at pH 6.7. Similar responses to pH were observed in yeast extract-mannitol broth except that Bradyrhizobium strains multiplied more slowly than R. loti at pH 6.7. All strains nodulated L. pedunculatus growing in acid (pH 4.5) rooting solution when presented as single cultures. Following inoculation with 1:1 mixtures of R. loti and Bradyrhizobium strains, R. loti formed 93% of nodules at pH 4.5 and significantly fewer nodules (66%) at pH 6.7. These results demonstrate a competitive advantage for acid-tolerant strains over acid-sensitive strains in nodulation of their lost legume at pH 4.5.

Underexpression of Ap from R-Plasmids in Fast-Growing Rhizobium Species

The presence of the plasmid RP1 in the cells of Rhizobium leguminosarum strains Rld1, 300, and 248, R. phaseoli 1233, R. trifolii strains T1 and 6661, and R. meliloti 4013 was found to appreciably increase bacterial resistance toward kanamycin and tetracycline but not toward ampicillin. The presence of 16 other R-plasmids in R. leguminosarum was also found to either not increase or only marginally increase bacterial resistance toward ampicillin. It appears now that underexpression of the plasmid-specified ampicillin function is common to most fast- and slow-growing rhizobia.

Effect of phosphorus supply on periplasmic protein profiles in Rhizobia

Lysozyme/EDTA treatment of four fast-growing rhizobia released repeatable protein profiles after polyacrylamide slab gel electrophoresis. Similar treatment of slow-growing rhizobia failed to release such periplasmic proteins. For the four-fast-growing rhizobia, both P-repressible and P-inducible protein bands occurred. The only P-repressible protein identified was alkaline phosphatase, which showed strain differences in both electrophoretic mobility and activation by Mg2+. The derepression of the P-repressible periplasmic proteins in cowpea Rhizobium NGR234 correlated with derepression of both phosphate and glycerol 1-phosphate uptake.

Adsorption of Slow- and Fast-Growing Rhizobia to Soybean and Cowpea Roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10(4)Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27 degrees C) and substantially attenuated at both 4 and 37 degrees C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.

Growth and nutritional characteristics of cowpea rhizobia

Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO2 and H2 with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.

Exopolysaccharides and lipopolysaccharides from a fast-growing strain ofRhizobium japonicum(USDA191)

Analysis of the exopolysaccharides from Rhizobium japonicum USDA191 showed substantial amounts of mannose (22.5%) and uronic acids (13.4%). These sugars are normally absent in the fast growers like Rhizobium meliloti. In addition USDA191 contained pyruvate (5.1%), which is normally absent in slow-growing strains of Rhizobium japonicum. The heterogeneity in the exopolysaccharide composition of strain 191 indicates a closer similarity with the slow-growing Rhizobium japonicum. SDS-urea polyacrylamide gel analysis of the lipopolysaccharides also reflects this heterogeneity.

Exopolysaccharides and lipopolysaccharides from a fast-growing strain of Rhizobium japonicum (USDA191)

Analysis of the exopolysaccharides from Rhizobium japonicum USDA191 showed substantial amounts of mannose (22.5%) and uronic acids (13.4%). These sugars are normally absent in the fast growers like Rhizobium meliloti. In addition USDA191 contained pyruvate (5.1%), which is normally absent in slow-growing strains of Rhizobium japonicum. The heterogeneity in the exopolysaccharide composition of strain 191 indicates a closer similarity with the slow-growing Rhizobium japonicum. SDS-urea polyacrylamide gel analysis of the lipopolysaccharides also reflects this heterogeneity.

Biochemical Characterization of Fast- and Slow-Growing Rhizobia That Nodulate Soybeans

Fast-growing, acid-producing soybean rhizobia were examined to determine their biochemical relatedness to each other, to typical slow-growing Rhizobium japonicum strains, and to other fast-growing species of Rhizobium. Although both the fast- and slow-growing soybean rhizobia were positive for catalase, urease, oxidase, nitrate reductase, and penicillinase, the fast-growing strains grouped with other fast-growing species of Rhizobium in that they tolerated 2% NaCl, were capable of growth at pH 9.5, utilized a large variety of carbohydrates (notably disaccharides), and produced serum zones in litmus milk. In addition, these fast-growing strains were similar to other fast-growing species of Rhizobium in that they produced appreciable levels of β-galactosidase and nicotinamide adenine dinucleotide phosphate-linked 6-phosphogluconate dehydrogenase but had no detectable hydrogenase activity. The fast-growing soybean rhizobia share symbiotic host specificity with Bradyrhizobium japonicum, but appear to be related biochemically to the other fast-growing species of Rhizobium.

In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated

In contrast to Klebsiella pneumoniae or fast-growing Rhizobium species, such as R. meliloti, where the nitrogenase structural genes are clustered in one operon (nifHDK), in slow-growing Rhizobium japonicum 110, nifH and nifDK are on separate operons.

Relationships Among Rhizobia from Native Australian Legumes

Isolates from 12 legumes at three sites in Victoria showed a wide range of morphological, cultural, symbiotic, and serological properties. Isolates from Acacia longifolia var. sophorae and Kennedia prostrata were fast growing but nodulated ineffectively Macroptilium atropurpureum and all native legumes except Swainsonia lessertiifolia. Isolates from S. lessertiifolia showed anomalous properties intermediate between fast- and slow-growing rhizobia. All isolates from the other two sites were slow-growing "cowpea" rhizobia. Symbiotic effectiveness was usually poor, and there was no relationship between effectiveness and host taxonomy or serological affinities of the isolates. This is the first report of fast-growing rhizobia from temperate Australian woody legumes and the first report of the symbiotic effectiveness of native Australian legumes with indigenous rhizobia.

Competition between fast- and slow-growing tropical legume rhizobia for nodulation ofVigna unguiculata

The competition between slow- and fast-growing types of rhizobia for nodulation of cowpeas was studied using modified Leonard-jars. Rhizosphere populations of Rhizobium, mixed and pure populations, were examined on cowpea plants grown in large glass tubes. At 25°C and at a low level of inoculation, the fast-growing strain, NGR 234, dominated the rhizosphere but at 30°C, the slow-growing strain CB 756 dominated or was equally represented depending on its initial level in the mixed inoculum. At 25°C/23°C (day/night), the fast-growing strain from Lablab (NGR 234) was a superior competitor for nodule sites compared with three slow-growing strains of Rhizobium. When plants were grown at 30°/26°C, the slow-growing rhizobia were the better competitors for host nodulation. Fast-growing strains from Mimosa and Leucaena were poor competiors at both temperatures. The results were influenced by the proportions of the competing strains, as well as the total numbers of rhizobia, in the mixed inoculum. Other factors, including ‘host preferences’, host compatibility and time taken for nodule initiation, also contributed to the relative success of competing strains. The results reflect the persistent isolation in tropical regions of slow-growing cowpea-type of rhizobia from most tropical legumes.

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the “core” T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the “core” T DNA.

Correlation between rifampicin-resistance of slow-growing Rhizobium strains and their ability to express nitrogenase activity in culture

Correlation between rifampicin-resistance of slow-growing Rhizobium strains and their ability to express nitrogenase activity in culture

An immunological comparison of nitrogenase proteins of fast and slow growing rhizobia

The enzyme complex nitrogenase, which catalyzes nitrogen reduction consists of two component proteins [ 1,2]: component I, a molybdenum and iron containing protein comprising two types of subunits and component II, a dimer iron-sulfur protein with identical subunits. The actual nitrogen reduction takes place on component I and this component is called dinitrogenase; component II which supplies component I with electrons is called dinitrogenase reductase [3]. The nitrogenase complex shows a remarkable structural similarity [2] among widely divergent organisms. The genus Rhizobium can be divided into two groups, viz. slow and fast-growing species [4,5]. These two groups differ in several characteristics. The fastgrowing rhizobia have a generation time of 2-4 h, are acid producers, have laterally arranged flagella and do not reduce nitrogen in the free-living form [4,5]. The slow-growing rhizobia have a generation time of 6-8 h, are alkali producers, have polar or subpolar flagella and reduce nitrogen in free-living and symbiotic form. Furthermore there are differences in the metabolic pathways and DNA base ratios. The results of Ruvkun and Ausubel [6] on the sequence homology between the nifgenes of a fast and a slow-growing Rhizobium pointed to a rather restricted homology. This result implies that a substantial difference may exist between nitrogenase proteins of fast and slow-growing rhizobia. In this paper we report on the immunological comparison of component I proteins of fast and slowgrowing rhizobia by the use of radioimmunoassays. We have used this technique because comparative immunological analyses of proteins from different organisms have been useful in asserting taxonomic relationships [7,8] and only small amounts of proteins are required in this procedure.

BIOLOGICAL AND AGRONOMIC ASPECTS OF LEGUME INOCULATION IN ISRAEL

Inoculants prepared for farm use consist of cultures of rhizobial strains selected for their invasiveness and effectivity in greenhouse and field trials. The acetylene reduction assay, hemoglobin content of nodules and plant nitrogen content were used for evaluation of nitrogen fixation intensity. An autoclaved local enriched peat constituted a very suitable substrate for all fast-growing rhizobia, but was often toxic to slow-growing ones. This was overcome by culturing the slow-growing rhizobia together with fast-growing (non-infective), slime-producing bacteria. Chemically treated and fragile seeds, e.g. common bean or peanut, were inoculated by applying the bacterial suspension directly into the planting furrows. For soils not bearing local specific rhizobia, relatively small amounts of inoculant (100–500 g/ha) were sufficient for adequate nodulation. However, to overcome competition of native rhizobial population, it was often necessary to use greatly increased amounts of inoculant. The effect of irrigation regimes commonly used in Israel on success of inoculation, rate of nodulation, and plant vegetative and reproductive development was investigated. In nitrogen-poor soils, symbiotic nitrogen fixation could not only substitute for mineral nitrogen, but even provide higher yields than nitrogen fertilization. A high content of available soil nitrogen may lead to serious difficulties in legume inoculation practice. Ways of overcoming these difficulties were examined. In certain soils fertilization with adequate amounts of phosphorus was found to be a prerequisite for proper nodulation and symbiotic nitrogen fixation. In calcareous and alkaline soils, application of iron chelate was necessary for N 2 fixation to occur.

Nodulation Efficiency of Legume Inoculation as Determined by Intrinsic Antibiotic Resistance

Patterns of intrinsic resistance and susceptibility to different levels of antibiotics were determined for strains of both fast- and slow-growing rhizobia. These patterns were stable to plant passage when they were used to identify Rhizobium strains in nodule suspensions or nodule isolates. The method of identification by intrinsic resistance and susceptibility patterns was reliable for identifying strains in field nodules when strains were first isolated from the nodules to provide a standard inoculum size and then typed on antibiotic-containing media. Other patterns of resistance were encountered during identification of field isolates; these patterns may have resulted from acquired resistance to certain antibiotics or from mixed infections of the nodules. The occurrence of resistance patterns identical to those of inoculant strains among native strains was directly related to the size of the soil population. High strain recovery was associated directly with high rates of inoculation.

Tetramethylthiuram disulfide (TMTD) resistance in slow-growing rhizobia

Different strains of Rhizobium (isolates from Glycine max, Phaseolus mungo and Vigna unguiculuta) adapted to tetramethylthiuram disulfide (thiram, TMTD) 30 μg ml −1). The number of transfers (5–19) and days of incubation (20–95) during which different strains of Rhizobium developed resistance varied. The results of reversion experiments show that the developed resistance was stable. The rates of growth was faster in resistant strains but their final cell numbers were less than those of sensitive strains. Dehydrogenase activity decreased with the development of resistance except in strain D-338. With the development of resistance total lipids and phospholipids increased, glycolipids decreased and neutral lipids varied. The presence of compounds stimulating lipid production in cells increased the total lipids of the strains and their resistance to TMTD.

Rhizobia in tropical legumes—XIII. Biochemical basis of acid and alkali reactions

Three fast-growing rhizobia ( Rhizobium meliloti isolated from Medicago saliva, R. trifolii from Trifolium subterraneum, and Rhizohium sp. from Leucaena leucocephala) and three slow-growing rhizobia R. japonicum from Glycine max, Rhizobium spp from Centrosema pubescens and Crotolaria anagyroides) were grown in defined media. The mean generation times of the fast-growing and slowgrowing strains were 3.8 h and 8.6 h respectively. Slow-growing organisms raised the initial pH of the defined medium while the fast-growing organisms lowered it. Rates of oxygen consumption tended to be higher in the slow-growing organisms. UMKL 19 (isolated from L. leucocephala) possessed all the normal reactions of fast-growing rhizobia but had a single sub-polar flagellum, similar to the three slow-growing strains studied. Certain combinations of amino acids and sugars (e.g. glutamine and galactose) induced an acidic reaction in the fast-growing organisms while the slow-growing ones changed the media to alkaline. Fast-growing organisms utilized more galactose for growth compared to slow-growing ones. Both types of organisms synthesized and released a wide range of amino acids into the medium. We suggest that pH changes produced by rhizobia growing on yeast-extract mannitol media are caused by the preferential utilization of sugars by fast-growing organisms and nitrogenous compounds by slow-growing ones.

Intérêt de la platine goniométrique pour l'étude d'analogues biologiques de cristaux-liquides à structure cholestérique

Click to increase image sizeClick to decrease image sizeSummaryA transmission electron microscope fitted with a goniometric stage provides the opportunity for studying the handedness or chirality of the fibers in biological analogues of liquid-crystals of the cholesteric type. Depending on the texture of the liquid-crystal one may use the phenomenon of inversion of arcs or the shift of dotted areas. These two phenomenons appear on the screen of the electron-microscope when the sections containing the twisted materials are suitably tilted.From the study of the nucleoids in bacteroids of slow-growing Rhizobium we can demonstrate that bacterial nucleoids in general are biological analogues of liquid-crystals of the cholesteric type in which the DNA fibers show a left handedness.Some cells of Vicia tetrasperma and Vicia hirsuta root nodules contain in their vacuoles many birefringent inclusions which are identical to a cholesteric mesomorph having a λ2+ texture in the terminology used by KLEMAN and FRIEDEL. With the preceding technique it appears that the fibers in the inclusions have a right handedness. This type of handedness is probably the first one reported in cholesterics.