Lipopolysaccharides of Rhizobium

Hot phenol-water extractions were carried out of cells from 12 strains of the fast-growing rhizobia Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii and Rhizobium meliloti. Purified lipopolysaccharide preparations contain neutral sugars, hexosamines, 2-keto-3-deoxyoctonate and uronic acids. Glucose, galactose, mannose, rhamnose and fucose are present in the majority of the LPS-preparations, but in varying proportions. Heptose was only found in some of them. O-methylated sugars are present in small amounts is most preparations, the kind of sugar being characteristic for lipopolysaccharides from different species. The lipid A part of lipopolysaccharides from all strains examined has identical patterns of fatty acids, namely -OH-C_14:0, -OH-C_15:0 (anteiso branched), -OH-C_16:0 and -OH-C_18:0. Comparison of the total compositions of Rhizobium lipopolysaccharides shows many differences among different species as among strains of a single species. Nearly identical lipopolysaccharide compositions also exist among certain strains, which constitute the same chemotype and which are also immunologically related. In view of a possible role of surface carbohydrates of Rhizobium in the root nodule symbiosis, the specificity of the binding of legume lectins with exo- and lipopolysaccharides of Rhizobium is discussed.Non-Standard Abbreviations LPS lipopolysaccharide(s) - EPS exopolysaccharides(s) - cetavlon cetyltrimethylammoniumbromide - KDO 2-keto-3-deoxyoctonate - ECL equivalent chain length Part II on Surface Carbohydrates of Rhizobium     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Comparison between Rhizobium galegae and Rhizobium meliloti plasmid contents

Plasmid profiles of two strains of a newly classified rhizobial species- Rhizobium galegae -were compared with the profiles of several strains of another fast-growing Rhizobium species- Rhizobium meliloti .
The existence of a plasmid DNA band with a lower electrophoretic mobility than the R. meliloti megaplasmid band was demonstrated in the two R. galegae strains by a modified horizontal Eckhardt method. Thus R. galegae species contain giant plasmid(s) larger than the R. meliloti 1000 MD megaplasmids, previously considered to be the largest plasmids in the Rhizobiaceae family.
In one of the R. galegae strains an additional middle-size plasmid only a little smaller than 140 MD pRme41a of R. meliloti 41 was observed.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Rhizobium inoculation of legumes

The large potential market for Rhizobium inoculum, both in the USA and elsewhere, is stimulating research into the basic biology of Rhizobium—legume interaction. The application of genetic engineering techniques promises to produce rapid improvement in Rhizobium inoculum strains. At present, however, identification of those traits that will enhance inoculum performance is difficult.     

Rhizobium goes genomic

A report from the Rhizobium Functional Genomics Workshop, Sevilla, Spain, 15-16 September 2000.     

Improvement of Rhizobium Inoculants

A practical approach was used to develop a Rhizobium (Bradyrhizobium) japonicum inoculant that increases soybean (Glycine max (L.) Merr.) yield in fields with indigenous Rhizobium populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (P < 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective Rhizobium inoculants for any legume in any geographical area.     

Metabolism of Rhizobium Bacteroids

Nitrogen fixation within legume nodules results from a complex metabolic exchange between bacteria of the family Rhizobiaciae and the plant host. Carbon is supplied to the differentiated bacterial cells, termed bacteroids, in the form of dicarboxylic acids to fuel nitrogen fixation. In exchange, fixed nitrogen is transferred to the plant. Both the bacteroid and the plant-derived peribacteroid membrane tightly regulate the exchange of metabolites. In the bacteroid oxidation of dicarboxylic acids via the TCA cycle occurs in an oxygenlimited environment. This restricts the TCA cycle at key points, such as the 2-oxoglutarate dehydrogenase complex, and requires that inputs of carbon and reductant are balanced with outputs from the TCA cycle. This may be achieved by metabolism through accessory pathways that can remove intermediates, reductant, or ATP from the cycle. These include synthesis of the carbon polymers PHB and glycogen and bypass pathways such as the recently identified 2-oxoglutarate decarboxylase reaction in soybean bacteroids. Recent labeling data have shown that bacteroids synthesize and secrete amino acids, which has led to controversy over the role of amino acids in nodule metabolism. Here we review bacteroid carbon metabolism in detail, evaluate the labeling studies that relate to amino acid metabolism by bacteroids, and place the work in context with the genome sequences of Mesorhizobium loti and Sinorhizobium meliloti. We also consider a wider range of metabolic pathways that are probably of great importance to rhizobia in the rhizosphere, during nodule initiation, infection thread development, and bacteroid development. Referee: Dr. Robert Ludwig, Department of Molecular, Celluar, and Developmental Biology, Sinheimer Laboratories, University of California, Santa Cruz, CA 95064     

Improvement of Rhizobium inoculants

[This corrects the article on p. 865 in vol. 55.].     

Denitrification in Rhizobium

Thirty-three strains of Rhizobium were examined for their reduction of nitrate under anoxic conditions. Three patterns of dissimilatory nitrate reduction were observed: (1) reduction to N2O and N2 (denitrification), (2) reduction to and subsequent accumulation of NO2- (nitrate respiration), (3) no reduction. Strains of R. japonicum and the cowpea miscellany displayed all three types, while strains of R. leguminosarum, R. phaseoli, and R. trifolii did not reduce nitrate by dissimilatory means. The production and subsequent metabolism of N2O was considerably different among the denitrifying strains: in some instances, N2O was a transient intermediate, while in others, it continued to accumulate during the incubation period.     

Quorum-sensing in Rhizobium

Quorum-sensing signals are found in many species of legume-nodulating rhizobia. In a well-characterized strain of R. leguminosarum biovar viciae, a variety of autoinducers are synthesised, and all have been identified as N-acyl-homoserine lactones. One of these N-acyl-homoserine lactones, is N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone, previously known as small bacteriocin, which inhibits the growth of several R. leguminosarum strains. The cinRI locus is responsible for the production of small bacteriocin. CinR induces cinI in response to the AHL made by CinI, thus forming a positive autoregulatory induction loop. A complex cascade of quorum-sensing loops was characterized, in which the cinIR locus appears to be the master control for three other AHL-dependent quorum-sensing control systems. These systems include the raiI/raiR, traI/triR and rhiI/rhiR. Other rhizobial strains appear to share some of these quorum sensing loci, but not all loci are found in all strains. Small bacteriocin along with the other N-acyl-homoserine lactones produced by these three AHL-based control systems regulate (i) growth inhibition of sensitive strains, (ii) transfer of the symbiotic plasmid pRL1JI, and (iii) expression of the rhizosphere-expressed (rhi) genes that influence nodulation. Some of the genes regulated by these systems have been identified. While the functions of some, such as the trb operon regulated by triR are clear, several of the regulated genes have no homologues of known function. It is anticipated that several other genes regulated by these systems have yet to be identified. Therefore, despite the regulation of one of the most complex quorum-sensing cascade being understood, several of the functions regulated by the quorum-sensing genes remain to be elucidated. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pigmented variants of Rhizobium

Summary Non-infective pigmented variants of Rhizobium have been isolated from several cultures after treatment with antibiotics, heat and U.V. irradiation. These variants tolerated much higher doses of heat, U.V. and certain antibiotics than the wild type strains from which they originated. Some of the pigmented variants produced reverse mutants on subculture which had lost their pigment but recovered their infectivity. These reverse mutants also lost their extreme U.V.-resistance. However, many pigmented mutants were very stable and reverted only after U.V. irradiation.This investigation shows that pigmentation in Rhizobium and loss of ability to form nodules on the roots of leguminous plants are genetically linked and may be pleiotropic effects. So far, more than 10 pigmented variants have been isolated, none of them was infective, while reverse mutants which had lost their pigment always gave rise to nodules.     

Behaviour of a sym plasmid from Rhizobium 'hedysari' in different Rhizobium species

Abstract The symbiotic plasmid pRHc1J of Rhizobium 'hedysari' has been transferred to different Rhizobium species. It expression and incompatibility with the recipient resident plasmids as well as the effect of the host plants on the selection of Rhizobium symbiotic information has been studied. When the symbiotic plasmid pRHc1J was transferred to Nod⁺Fix⁺ Rhizobium species, it underwent specific deletions, either spontaneously or after the passage of transconjugants through plants, leading to the loss of some essential nod genes.     

Expression of Rhizobium meliloti nod genes in Rhizobium and Agrobacterium backgrounds.

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.     

Transposition of Tn1831 to sym plasmids of Rhizobium leguminosarum and Rhizobium trifolii

Abstract All transposon-induced symbiotic mutants of Rhizobium described so far have been obtained using Tn 5 , which codes for kanamycin resistance (Km^R). To enable genetic complementation studies, we tried to find an effective transposon carrying another resistance marker. We report here a method for the apparent random transposition in Rhizobium of Tn 1831 , which codes for resistance against spectinomycin (Sp), streptomycin (Sm) and mercury chloride. When the suicide plasmid pMP12 (RP4::Tn 1831 , Km::Mu) was transferred to Rhizobium , in almost all cases the exconjugants harbour a deleted transfer-deficient R plasmid. From this deleted R plasmid transposition occurred to self-transmissible Sym-plasmids of R. leguminosarum and R. trifolii . Using this method a number of Tn 1831 -induced symbiotic mutants of pRL1JI were isolated.     

Tryptophan auxotrophs of Rhizobium japonicum

Eleven tryptophan-requiring mutants of Rhizobium japonicum I-110 ARS were isolated after nitrous acid mutagenesis and fell into five groups based on characterization by supplementation with intermediates and enzyme assays.