Fast-growing Rhizobia Research Articles

Rhizobia in tropical legumes: Ineffective nodulation of Arachis hypogaea L. By fast-growing strains

Members of the legume tribe Aeschynomeneae are thought to be nodulated by a different process to that which occurs in most other legumes. Rather than invade root-hairs, infective rhizobia directly enter the inter-ccllular spaces of the roots where they are engulfed by the cortical cells. Once within the cells, the rhizobia divideand the bacteroid-containing cells themselves undergo mitosis. Division of both bacleroids and their host-cells is characteristic of this nodulation process. As a prelude to studying the molecular genetics of the rhizobial component in such symbioses, we sought a fast-growing, plasmid-bearing Rhizobium that would nodulate a group of plants which normally favour slow-growing Bradyrhizobium sp. A collection of 27 fast-growing tropical rhizobia was used to screen the nodulation capacity of Arachis hypogaea cv. NC2. Seventy percent of the isolates were able to ineffectively nodulate groundnuts growing in “Leonard” jars. Four of these were chosen to inoculate six different A. hypogaea cultivars. Only one strain, Rhizobium sp. NGR234, was able to nodulate all six varieties. Nodules produced by Rhizobium sp. NGR234 on the different cultivars were compared with nodules that developed following infection with Bradyrhizobium sp. Apart from the fact that bacteroids induced by Rhizobium sp. NGR234 were polymorphic while those developing after infection by Bradyrhizobium sp. were spherical, the organisation of nodules produced by both groups of bacteria on all varieties of plants was similar. NGR234 multiplication in the host cytoplasm and the absence of infection threads suggest that fast-growing rhizobia also enter the roots via the direct inter-cellular route.

Physiological and biochemical characteristics of a fast-growing strain of lupin rhizobia isolated from the sonoran desert

An effective, fast-growing strain of lupin rhizobia was isolated from a species of Lupinus native to the Sonoran Desert near San Felipe, Baja, Mexico (generation time, 3.6 h). Bacteria isolated from the roots of lupins are normally slow growing, however. Lupin 43 was fast-growing, possessed multiple flagella and produced acid in a denned medium. In comparison to a slow-growing lupin strain. Nitragin 96AII; Lupin 43 had a low intrinsic resistance to antibiotics and was able to utilize a wider range of C and N-sources. When compared to a fast-growing strain of Rhizobium meliloti, also isolated from the Sonoran Desert, Lupin 43 had a slightly faster growth rate, was less resistant to antibiotics and produced alkaline end products when asparagine was supplied as N-source. Based on these characteristics. Lupin 43 was more similar to other fast-growing rhizobia than the slow growing lupin rhizobia.

Rhizobium nodulation gene nodD as a determinant of host specificity

The symbiosis between bacteria of the genus Rhizobium and their leguminous host plants results in the formation of root nodules in a species-specific way, in that a particular bacterial species can nodulate only a limited number of host species. In fast-growing Rhizobium species many nodulation (nod) genes, including the functional interchangeable or common nod genes nodA,B,C,I,J and nodD and the host-specificity genes nodE.F, are localized on large Sym (for symbiosis) plasmids1–5. On the Rhizobium meliloti Sym plasmid three nodD genes have been localized. Two of these, nodD1 and nodD2, are required for efficient nodulation of the host plant (ref 6 and M. Honma, personal communication). Exudates of leguminous plants induce the expression of several Sym-plasmid-localized nod operons7–10, a process in which the constitutively expressed nodD product is supposed to act as a positive regulator8,9. The inducing compounds found in these exudates have been identified as flavones, flavanones or closely related compounds11–14. We report here that the nodD products of the various fast-growing Rhizobium species differ from each other in that they confer different responsiveness, in a species-specific way, to different sets of flavonoids and exudates. Moreover, in one case, the nodD gene was shown to be a determinant of host-specific nodulation.

Characterisation of glycerophosphorylated cyclic β-1,2-glucans from a fast-growing Rhizobium species

A family of cyclic β-1,2-glucans, substituted with sn-glycerol-1-phosphate at the C6 position of some of the glucose residues, has been found in the culture supernatant of the fast-growing, broad host range Rhizobium species, NGR234. The dynamic behaviour of the molecules, studied by 13C nuclear magnetic resonance spectroscopy, showed that they are disc-shaped with significant, but limited internal motion.

12] Site-directed Tn5 and transplacement mutagenesis: Methods to identify symbiotic nitrogen fixation genes in slow-growing Rhizobium

Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used in the laboratory to identify genes essential for symbiotic nitrogen fixation in slow-growing rhizobium species. This chapter has experimented with several methods for this kind of mutagenesis and will discuss the protocol that has worked most successfully in conjunction with these organisms. The chapters also describe a method for site-directed transplacement of in vitro altered DNA sequences that should be applicable to all gram-negative bacteria. Tn5 is stably maintained in the chromosome throughout the life cycle of the rhizobium cell in the nodules of the plant. Maintenance of an extrachromosomal plasmid, such as plasmid (pVK100) under these same nonselective conditions, however, is sometimes a problem. Moreover, false-positive complementation results can occur between two Tn5 insertions that are actually within the same transcriptional unit as a result of marker exchange between the homologous Rhizobium DNA contained on the plasmid and in the chromosome. Although the advancement of the genetics of the slow-growing Rhizobium species has preceded more slowly than that of the fast-growing Rhizobium species, the techniques for mutagenesis described in this chapter should facilitate progress in this field.

Metabolism of glucose and gluconate in fast- and slow-growing rhizobia

Enzymatic evidence was sought for the operation of pathways involved in glucose and gluconate catabolisms in fast- and slow-growing Rhizobium species including members of the cowpea group. Enzymes of the Entner-Doudoroff pathway, pentose phosphate pathway and tricarboxylic acid cycle were detected in fast-growing rhizobia but the pentose phosphate pathway was absent in slow-growers, regardless of the carbon source used. When analysed for enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways in glucose-grown cells, the pathways were found to operate simultaneously in rhizobia.

Rhizobium nod genes are involved in inducing an early nodulin gene

Rhizobium bacteria can invade the roots of leguminous plants and elicit the formation of root nodules. This process involves the expression of at least 20 nodule-specific genes encoded by the host plant, the so-called nodulin genes, which include the leghaemoglobin genes1–3. During development of the root nodules the nodulin genes are differentially expressed1. In fast-growing Rhizobium species several essential symbiotic genes are located on a large plasmid, and when fragments of the plasmid which contain the genes essential for nodulation (the nod region) are cloned and transferred to Rhizobium strains lacking the symbiotic plasmid (‘cured’ strains), the recipients regain the ability to form nodules. However, these nodules cannot fix nitrogen because essential nitrogen-fixation genes are absent4–6. We show here that transfer of the nod region (10 kilobases; kb) alone from a Rhizobium leguminosarum symbiotic plasmid into cured Rhizobium strains is sufficient to elicit nodulin gene expression in the host Pisum sativum. We also show that pea root nodules induced by an Agrobacterium strain containing the R. leguminosarum symbiotic plasmid express an early nodulin gene but no other nodulin genes. These results show that at least two signals are involved in the induction of expression of nodulin genes and the presence of the Rhizobium nodulation genes seems to be required to elicit the first signal.

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.

A note on physiological characteristics and genetic relatedness of a fast-growingRhizobiumfrom stem nodule ofAeschynomene asperaL

A fast-growing Rhizobium strain was isolated from a stem nodule of Aeschynomene aspera and was characterized by standard laboratory tests, nodulation, nutritional and carbohydrate utilization patterns. Its relationship to other members of Rhizobium was studied by DNA homology tests which suggest it cannot be classified as any known species of Rhizobium.

Periplasmic proteins ofRhizobium: Variation with growth conditions and use in strain identification

The pattern of periplasmic proteins released by lysozyme-EDTA treatment of fast-growing rhizobia was influenced by the growth phase, the pH of the medium and the carbon source. The pattern for a particular strain of Rhizobium grown under defined conditions was characteristic of that strain, and we suggest that it can be used as an adjunct to existing methods of strain identification.

Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation.

A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.

Intra- and Intergeneric Similarities between the Ribosomal Ribonucleic Acid Cistrons of Rhizobium and Bradyrhizobium Species and Some Related Bacteria

14C-labeled ribosomal ribonucleic acids (rRNAs) were prepared from Bradyrhizobium japonicum NZP 5549T(T = type strain) and Rhodopseudomonas palustris DSM 130, and [3H]rRNAs were prepared from Rhizobium meliloti NZP 4009 and Rhizobium loti NZP 2037. Labeled rRNA from Agrobacterium tumefaciens ICPB TT111 (a member of Agrobacterium cluster 2) was also used. These rRNAs were hybridized under optimal conditions with filter-fixed deoxyribonucleic acids (DNAs) from 24 strains of rhizobia representing nine previously identified DNA-DNA homology groups and with DNAs from various other organisms. Each hybrid was described by the following two parameters: the temperature at which 50% of the hybrid was denatured (Tm(e) ) and percentage of rRNA binding. From rRNA similarity maps and a Tm(e) dendrogram the following conclusions were drawn. (i) There are at least three genetically distinct groups in the present genus Rhizobium. Group I includes Rhizobium meliloti, Rhizobium fredii, and Rhizobium leguminosarum. Group II is represented by Rhizobium loti, and group III includes rhizobia isolated from Galega officinalis and Galega orientalis (goatsrue.). (ii) The rRNA cistrons of group I strains resemble those of Agrobacterium cluster 2 strains. (iii) The rRNA cistrons of Rhizobium fredii resemble those of Rhizobium meliloti more closely than the rRNA cistrons of Rhizobium leguminosarum or Agrobacterium cluster 2 do. (iv) The rRNA cistrons of Rhizobium loti resemble the rRNA cistrons of Rhizobium meliloti less than the rRNA cistrons of strains belonging to either Agrobacterium cluster 1 or Agrobacterium cluster 2. (v) The rRNA cistrons of Galega rhizobia resemble the rRNA cistrons of Rhizobium meliloti about as much as the rRNA cistrons of Agrobacterium cluster 1 and resemble the rRNA cistrons of Rhizobium loti less. (vi) The rRNA cistrons of Rhizobium and Bradyrhizobium resemble one another much less than do the rRNA cistrons of Rhizobium and other genera presently included in the family Rhizobiaceae (Agrobacterium and Phyllobacterium). (vii) Bradyrhizobium strains belonging to four DNA-DNA homology groups all contain rRNA cistrons which closely resembled the rRNA cistrons of Bradyrhizobium japonicum NZP 5549T. (viii) B. japonicum contains rRNA cistrons which have diverged from the rRNA cistrons of Rhodopseudomonas palustris to about the same extent as the rRNA cistrons of Rhizobium group I and Agrobacterium; on this basis we suggest that the photosynthetic ancestor of Bradyrhizobium may have been a rhodopseudomonad and that a phylogenetic classification should group these genera together. (ix) Stemnodulating strain ORS 571, from Sesbania rostrata belongs to the Rhodopseudomonas palustris rRNA branch; it is quite distinct from B. japonicum and Rhodopseudomonas palustris. Two representative strains which nodulate roots belong in the group containing the fast-growing rhizobia.

Sequence of Events during the Infection of the Tropical Legume Macroptilium atropurpureum Urb. by the Broad-host-range, fast-growing Rhizobium ANU240

Summary The infection of the tropical legume Macroptilium atropurpureum Urb. by the broad-host-range, fast-growing Rhizobium ANU240 was studied using a spot-inoculation technique which localised infection events to a Rhizobium attached to the root surface within minutes of inoculation. By 5–6 h after inoculation the affected hairs grew asymmetrically due to the effect of the bacteria. By 9–11 h the hairs contacted their own basal part or neighbouring cell and continued to grow along the epidermal surface; the outer wall of the infected cells thickened and appeared brown under bright field microscopy. Between 12 and 20h after inoculation Rhizobium colonized the small region between the epidermis and the overlying hair wall; during this time, the hair wall microfibrils became loosened. Between 20 and 22 h, the Rhizobium entered an interfacial zone between the hair wall and plasmalemma. By 22–24h tip growth ceased, infection thread synthesis commenced, and subepidermal cell nucleoli became enlarged. Between 24 and 40 h infection threads ramified the hair cell, and sub-epidermal and cortical cell division commenced and formed a focus of cytoplasmic-rich cells immediately below the infection site. Between 42 and 52 h, threads oriented towards the cortical tissue, penetrating and ramifying through the focus of cells.

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the 'core' T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the 'core' T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.

Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod-) or unable to fix nitrogen (Fix_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.

Conservation of leghemoglobin heterogeneity and structures in cultivated and wild soybean

The leghemoglobins from a genetically diverse selection of 69 cultivated soybean (Glycine max (L.) Merr.) cultivars and plant introductions and 18 wild soybean (Glycine soja Sieb. & Zucc.) plant introductions all consist of the same set of major leghemoglobins (a, c1, c2, c3), as determined by analytical isoelectric focusing. The conservation of both leghemoglobin heterogeneity and also all four major leghemoglobin structures provides strong circumstantial evidence that leghemoglobin heterogeneity is functional. Glycine max and G. soja produced the same leghemoglobins in the presence of Bradyrhizobium japonicum (Kirchner) Jordan and in the presence of fast-growing Rhizobium japonicum.

Inhibition of growth of Rhizobium japonicum by cyclic GMP

Exogenous cyclic guanosine-3',5'-monophosphate (cGMP) inhibited the growth of Rhizobium japonicum at less than 100 microM. Other nucleotides, including cyclic AMP, cyclic IMP, and cyclic CMP, had no inhibitory effect even at higher concentrations nor was the inhibition by cGMP reversed by cyclic AMP. The inhibitory effect was independent of the carbon and nitrogen source(s) used. cGMP did not inhibit the growth of any other species of bacterium tested, including several fast-growing Rhizobium species. The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition. Subsequently, growth resumed at a slower rate. Resumption of growth was not due to destruction of the nucleotide. Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions. No effect of cGMP on the derepression of respiratory nitrate reductase was observed.

Concurrent evolution of nitrogenase genes and 16S rRNA in Rhizobium species and other nitrogen fixing bacteria

It was known that nitrogenase genes and proteins are well conserved even though they are present in a large variety of phylogenetically diverse nitrogen fixing bacteria. This has lead to the speculation, among others, that nitrogen fixation (nif) genes were spread by lateral gene transfer relatively late in evolution. Here we report an attempt to test this hypothesis. We had previously established the complete nucleotide sequences of the three nitrogenase genes from Bradyrhizobium japonicum, and have now analyzed their homologies (or the amino acid sequence homologies of their gene products) with corresponding genes (and proteins) from other nitrogen fixing bacteria. There was a considerable sequence conservation which certainly reflects the strict structural requirements of the nitrogenase iron-sulfur proteins for catalytic functioning. Despite this, the sequences were divergent enough to classify them into an evolutionary scheme that was conceptually not different from the phylogenetic positions, based on 16S rRNA homology, of the species or genera harboring these genes. Only the relation of nif genes of slow-growing rhizobia (to which B. japonicum belongs) and fast-growing rhizobia was unexpectedly distant. We have, therefore, performed oligonucleotide cataloguing of their 16S rRNA, and found that there was indeed only a similarity of S AB=0.53 between fast- and slowgrowing rhizobia. In conclusion, the results suggest that nif genes may have evolved to a large degree in a similar fashion as the bacteria which carry them. This interpretation would speak against the idea of a recent lateral distribution of nif genes among microorganisms.

Alteration of the Effective Nodulation Properties of a Fast-growing Broad Host Range Rhizobium due to Changes in Exopolysaccharide Synthesis

Summary Strain NGR234 is a broad host range Rhizobium which can effectively nodulate a broad spectrum of legumes. Ninety mutants with altered exopolysaccharide production were isolated after strain NGR234 was subjected to transposon Tn5 mutagenesis. These mutants were classified on the basis of their physiological properties into 9 groups. Their symbiotic porperties were tested on four legumes which form spherical (determinate) nodules ( Macroptilium atropurpureum, Desmodium intortum, Desmodium uncinatum and Lablab purpureus ) and on the legume Leucaena leucocephala which forms cylindrical (indeterminate) nodules. On these plants strain NGR234 forms nitrogen-fixing nodules (Nod + Fix + ). The results from the testing of the various mucoid-defective mutants on these plants show that it is possible to alter the synthesis of the surface polysaccharides of strain NGR234 and produce a narrow host range Nod + Fix + Rhizobium .