Fast-growing Rhizobium Species Research Articles

Structural identification of the lipo-chitin oligosaccharide nodulation signals of Rhizobium loti.

Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R. loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium loti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.

Genomic Heterogeneity among French Rhizobium Strains Isolated from Phaseolus vulgaris L.

Levels of DNA relatedness between strains isolated from root nodules of Phaseolus vulgaris and reference strains of different Rhizobium species were determined by performing DNA-DNA hybridization experiments (S1 nuclease method). The nine strains examined were members of three genomic groups previously delineated by a restriction fragment length polymorphism analysis among strains isolated from P. vulgaris at different sites in France. In agreement with the results of the restriction fragment length polymorphism analysis, three genomic species were found. We confirmed that one of these species corresponded to Rhizobium leguminosarum since the strain examined was 100% related to the type strain of this species. The other two species were new genomic species which were less than 21% related to reference strains belonging to other Rhizobium species, including Rhizobium etli and Rhizobium tropici, and were 18% related to each other. As determined by an analysis of partial 16S ribosomal DNA sequences, each of the genomic species was found to belong to a lineage independent from the lineages of previously described Rhizobium species. Nevertheless, they were included in the group formed by the fast-growing Rhizobium species. Both genomic species 1 and genomic species 2 contained a majority of strains which were capable of nodulating both P. vulgaris and Leucaena leucocephala, like R. tropici. However, they also contained strains with a nodulation phenotype restricted to P. vulgaris, like R. leguminosarum bv. phaseoli and R. etli bv. phaseoli. Our data are the first evidence that in Europe species other than R. leguminosarum nodulate P. vulgaris.

Partial purification, stereospecificity and stoichiometry of three dehalogenases from aRhizobiumspecies

A fast-growing species of Rhizobium that utilized 2,2-dichloropropionate (2,2DCP) and d,l-2-chloropropionate (dl-2CP) as sole sources of carbon and energy was shown to contain three inducible dehalogenases. These enzymes differed in their substrate specificities: dehalogenase II degraded 2,2DCP, d- and l-2CP, monochloroacetate (MCA) and dichloroacetate (DCA) whilst dehalogenase I showed activity only towards l-2CP and DCA. Dehalogenase III liberated halide from d-2CP and MCA. This is the first report of a dehalogenase acting solely on the d-isomer of a haloalkanoate. All three dehalogenases inverted the isomeric configuration during dehalogenation, forming d(−) and l(+) lactate from l- and d-2CP, respectively.

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.

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.

Isolation and partial characterisation of dehalogenase-deficient mutants of aRhizobiumsp.

A fast-growing species of Rhizobium that can utilise 2,2-dichloropropionate and 2-chloropropionate as sole sources of carbon and energy was mutated to give strains which had lost this ability. Such mutants lacked two inducible dehalogenase activities that were present in the parent organism. Secondary mutants which had regained the ability to grow on 2,2-dichloropropionate (2,2DCP) and/or 2-chloropropionate (2CP) were isolated from the original mutants. Some were constitutive for dehalogenase I and did not form dehalogenase II, some were constitutive for dehalogenase II and did not form dehalogenase I, whilst others, like the parent organism, formed both dehalogenases inducibly. A possible model for the control of dehalogenase synthesis is proposed.

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.

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.

Species-specific, symbiotic plasmid-located repeated DNA sequences in Rhizobium trifolii

A repeated DNA sequence has been characterized in the clover symbiont, Rhizobium trifolii. Analysis of three copies of this repeated sequence revealed that it constitutes a reiteration of the nifHDK promoter region and, in some copies, an additional reiteration of the N-terminal end of the nifH gene. This sequence, as exemplified by the nifHDK promoter region, is highly conserved within all the geographically-distinct isolates of R. trifolii examined, and is located exclusively on the Sym (symbiotic) plasmid. The R. trifolii repeated sequences (designated RtRS) were shown by DNA hybridization analysis to be specific for R. trifolii and not to hybridize to DNA of any other fastgrowing Rhizobium species examined. Based on the observed species-specificity and Sym-plasmid location of these sequences, as well as the available genetic evidence, we propose a model in which the expression of symbiotic genes is host-specifically activated via these species-specific repeated (promoter) sequences. The results presented indicate that the RtRS sequences can be used as a molecular probe for both species and strain identification and should facilitate the molecular taxonomy of Rhizobium.

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

Five specific transposon-induced nodulation defective (Nod(-)) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod(-) mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod(-) on all their known plant hosts, (b), they could not induce root hair curling (Hac(-)) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.

Conservation of nif- and species-specific domains within repeated promoter sequences from fast-growing Rhizobium species.

In the fast-growing Rhizobium species, repeated DNA sequences, which include the promoter region of the nif HDK operon have been described. These repeated sequences are promoters which specifically activate transcription in the endosymbiotic state. Hybridization analysis of these sequences from R. trifolii has revealed that they may be involved in the species-specific activation of the various genes whose transcription they promote. Comparative analysis of various copies of these repeated sequences, from R. trifolii (the clover symbiont) and R. meliloti (the alfalfa symbiont), reveals the presence of domains of intra- and interspecific conservation within the promoter regions. We suggest that these promoter elements represent sites which are involved in the species-specific and general, nif-specific activation of Rhizobium symbiotic genes.

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.

Structure determination of carbohydrates and glycosphingolipids by fast atom bombardment mass spectrometry

We have used fast atom bombardment mass spectrometry to aid the structural analysis of blood group glycosphingolipids, carbohydrates excreted by gangliosidosis patients, glucans from fast growing Rhizobium species and a variety of plant cell wall oligosaccharides. Samples have been examined in their native form or as acetyl, permethyl, methoxime or pentafluorobenzyloxime derivatives. Negative FAB spectra of permethylated carbohydrates have been obtained for the first time by salt dosing. High sensitivity for permethylated samples has been achieved in the positive mode by ammonium cationisation. Spectra up to m/z 6620 are reported for permethylated glucans.

Growth-Related Substituent Changes in Exopolysaccharides of Fast-Growing Rhizobia

Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species.

THE GENETIC DETERMINATION OF HOST RANGE IN THE RHIZOBIACEAE

The family Rhizobiaceae includes bacteria which form nitrogen-fixing root nodules on the roots of leguminous plants (the genus Rhizobium ) and bacteria which form tumours on dicotyledenous plants (the genus Agrobacterium). For both genera classification at the species level is determined by plant interactions: strains of Agrobacterium which induce root proliferation (hairy root disease) are included in the species A. rhizogenes , those which form tumours are classified as A. tumefaciens and those which elicit no plant response as A. radiobacter. For Rhizobium , speciation is determined primarily by the range of legume hosts which are nodulated. Strains of Rhizobium and Agrobacterium contain large amounts of extrachromosomal DNA (plasmids). Host recognition and tumour-forming properties are determined by these plasmids, which can be transferred between different species and also between Agrobacterium and Rhizobium. Such transfer experiments have shown that for Agrobacterium and some fast-growing species of Rhizobium the plant-interaction characteristics of strains and hence the criterion for their taxonomie status is plasmid mediated. Whether or not host range properties are readily exchanged between these bacteria in nature needs to be determined.

Antibiotic sensitivity in Rhizobium and Agrobacterium

Summary The resistance of 81 Rhizobium strains and 11 Agrobacterium strains to 16 antibiotics was investigated. Most strains were resistant even to high concentrations of lincomycin. All the strains of the species Rhizobium meliloti, Rhizobium phaseoli, Rhizobium japonicum and the genus Agrobacterium reacted relatively homogeneously to all the antibiotics, unlike Rhizobium trifolii, Rhizobium leguminosarum and Rhizobium spp. The majority of the fast-growing species of Rhizobium tested was found to be resistant to > 32 μg/ml penicillin and to Rhizobium were susceptible to penicillin concentrations 1 μg/ml. The majority of the Agrobacterium strains tested were resistant to streptomycin concentrations > 32 μg/ml, allowing them to be distinguished from strains of R. meliloti which were rarely resistant to such high concentrations.

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.