Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation

We show that expression of common nodulation genes in Rhizobium meliloti is under positive as well as negative control. A repressor protein was found to be involved in the negative control of nod gene expression. Whereas the activator NodD protein binds to the conserved cis-regulatory element (nod-box) required for coordinated regulation of nod genes, the repressor binds to the overlapping nodD1 and nodA promoters, at the RNA polymerase binding site. A model depicting the possible interaction of the plant-derived nod gene inducer (luteolin), the NodD and the repressor with the nod promoter elements is presented. Mutants lacking the repressor exhibited delayed nodulation phenotype, indicating that fine tuning of nod gene expression is required for optimal nodulation of the plant host.     

Characterization of Astragalus sinicus Rhizobia by Restriction Fragment Length Polymorphism Analysis of Chromosomal and Nodulation Genes Regions

Two hundred and four isolates of rhizobia were sampled from root nodules of Astragalus sinicus grown in rice fields of six southern provinces of China. Genotypic diversity was determined by Southern hybridization using nodDBC genes as a probe, restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S-23S rDNA intergenic spacers (IGS), and plasmid profile. Our results show that rhizobia associated with A. sinicus were very diverse, and 10 genotypes were resolved within the previously identified dominant 16S rDNA type. Diversity levels varied greatly between different geographical locations. The same nod gene genotypes were harbored by distinct chromosomal types, suggesting that lateral plasmid transfer occurred during the evolution process. Received: 14 June 1999 / Accepted: 20 July 1999     

NoIR controls expression of the Rhizobium meliloti nodulation genes involved in the core Nod factor synthesis

The synthesis of Rhizobium meliloti Nod signal molecules, encoded by the nod gene products, is finely regulated. A negative control of plasmid-borne nod gene expression is provided by the NoIR repressor encoded by the chromosomal noIR gene. NoIR was previously shown to downregulate the expression of the activator nodD1 gene and the common nodABC operon by binding to an overlapping region of the two promoters adjacent to the n1 nod-box (Kondorosi et al., 1989). We demonstrate here that NoIR also controls the expression of two additional genes, nodD2 and nodM, but does not directly regulate the expression of the host-specific nod genes located downstream of the n2, n3 and n5 nod-boxes. Thus, the nod genes are differentially regulated by NoIR and only those providing common nodulation functions, by determining the synthesis of the core Nod factor structure, are subjected to this negative regulation. Furthermore, NoIR has a strong negative effect on the production of Nod metabolites, the level of which may serve as a fine-tuning mechanism for optimal nodulation, specific to host-plant genotypes. In addition, it elicits preferential synthesis of Nod factors carrying unsaturated C₁₆ fatty acids. Expression of noIR was high both in the free-living bacterium and in the bacteroid and it was downregulated by its own product and by the nod gene inducer luteolin.     

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S–23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Chemotaxis of Rhizobium meliloti towards Nodulation Gene-Inducing Compounds from Alfalfa Roots

Luteolin, a flavone present in seed exudates of alfalfa, induces nodulation genes (nod) in Rhizobium meliloti and also serves as a biochemically specific chemoattractant for the bacterium. The present work shows that R. meliloti RCR2011 is capable of very similar chemotactic responses towards 4′,7-dihydroxyflavone, 4′,7-Dihydroxyflavanone, and 4,4′-dihydroxy-2-methoxychalcone, the three principal nod gene inducers secreted by alfalfa roots. Chemotactic responses to the root-secreted nod inducers in capillary assays were usually two- to four-fold above background and, for the flavone and flavonone, occurred at concentrations lower than those required for half-maximal induction of the nodABC genes. Complementation experiments indicated that the lack of chemotactic responsiveness to luteolin seen in nodD1 and nodA mutants of R. meliloti was not due to mutations in the nod genes, as previously thought. Thus, while nod gene induction and flavonoid chemotaxis have the same biochemical specificity, these two functions appear to have independent receptors or transduction pathways. The wild-type strain was found to suffer selective, spontaneous loss of chemotaxis towards flavonoids during laboratory subculture.     

Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt

The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.     

Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis

Background and aims

Legumes of the genus Lessertia have recently been introduced to Australia in an attempt to increase the range of forage species available in Australian farming systems capable of dealing with a changing climate. This study assessed the diversity and the nodulation ability of a collection of Lessertia root nodule bacteria isolated from different agro-climatic areas of the Eastern and Western Capes of South Africa.

Methods

The diversity and phylogeny of 43 strains was determined via the partial sequencing of the dnaK, 16srRNA and nodA genes. A glasshouse experiment was undertaken to evaluate symbiotic relationships between six Lessertia species and 17 rhizobia strains.

Results

The dnaK and 16S rRNA genes of the majority of the strains clustered with the genus Mesorhizobium. The position of the strains at the intra-genus level was incongruent between phylogenies with few exceptions. The nodA genes from Lessertia spp. formed a cluster on their own, separate from the previously known Mesorhizobium nodA sequences. Strains showed differences in their nodulation and nitrogen fixation patterns that could be correlated with nodA gene phylogeny. L. diffusa, L. herbacea and L. excisa nodulated with nearly all the strains examined while L. capitata, L. incana and L. pauciflora were more stringent.

Conclusion

Root nodule bacteria from Lessertia spp. were identified mainly as Mesorhizobium spp. Their nodA genes were unique and correlated with the nodulation and nitrogen fixation patterns of the strains. There were marked differences in promiscuity within Lessertia spp. and within strains of root nodule bacteria.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.     

Strain-Specific Inhibition of nod Gene Induction in Bradyrhizobium japonicum by Flavonoid Compounds

A broad-host-range plasmid, pEA2-21, containing a Bradyrhizobium japonicum nodABC'-'lacZ translational fusion was used to identify strain-specific inhibitors of the genes required for soybean nodulation, the common nod genes. The responses of type strains of B. japonicum serogroups USDA 110, USDA 123, USDA 127, USDA 129, USDA 122, and USDA 138 to nod gene inhibitors were compared. Few compounds inhibited nod gene expression in B. japonicum USDA 110. In contrast, nod gene expression in strains belonging to several other serogroups was inhibited by most of the flavonoids tested. However, the application of two of these strain-specific compounds, chrysin and naringenin, had little effect on the pattern of competition between indigenous and inoculum strains of B. japonicum in greenhouse and field trials. Preliminary studies with radiolabeled chrysin and naringenin suggest that the different responses to nod gene inhibitors may be partly due to the degree to which plant flavonoids can be metabolized by each strain.     

Organization Pattern of Common nod Genes in Mesorhizobium ciceri Strain MC 18-7

The common nodulation genes (nod ABC) are normally present together as a single operon in most rhizobia; however, there are few exceptions. Fast growing Mesorhizobium ciceri strain MC 18-7 was examined for structural organization of the nodABC genes by PCR amplification. Results indicated that in Mesorhizobium ciceri strain MC 18-7 nodA and nodC genes are present together under same nod box, while nodB gene has a separate nod box present immediately upstream to it, just like in its close relative Mesorhizobium loti.     

Three Phylogenetic Groups of nodA and nifH Genes in Sinorhizobium and Mesorhizobium Isolates from Leguminous Trees Growing in Africa and Latin America

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009^T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609^T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.     

The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2. 0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.     

European Origin of Bradyrhizobium Populations Infecting Lupins and Serradella in Soils of Western Australia and South Africa

We applied a multilocus phylogenetic approach to elucidate the origin of serradella and lupin Bradyrhizobium strains that persist in soils of Western Australia and South Africa. The selected strains belonged to different randomly amplified polymorphic DNA (RAPD)-PCR clusters that were distinct from RAPD clusters of applied inoculant strains. Phylogenetic analyses were performed with nodulation genes (nodA, nodZ, nolL, noeI), housekeeping genes (dnaK, recA, glnII, atpD), and 16S-23S rRNA intergenic transcribed spacer sequences. Housekeeping gene phylogenies revealed that all serradella and Lupinus cosentinii isolates from Western Australia and three of five South African narrow-leaf lupin strains were intermingled with the strains of Bradyrhizobium canariense, forming a well supported branch on each of the trees. All nodA gene sequences of the lupin and serradella bradyrhizobia formed a single branch, referred to as clade II, together with the sequences of other lupin and serradella strains. Similar patterns were detected in nodZ and nolL trees. In contrast, nodA sequences of the strains isolated from native Australian legumes formed either a new branch called clade IV or belonged to clade I or III, whereas their nonsymbiotic genes grouped outside the B. canariense branch. These data suggest that the lupin and serradella strains, including the strains from uncultivated L.cosentinii plants, are descendants of strains that most likely were brought from Europe accidentally with lupin and serradella seeds. The observed dominance of B. canariense strains may be related to this species' adaptation to acid soils common in Western Australia and South Africa and, presumably, to their intrinsic ability to compete for nodulation of lupins and serradella.     

Diversification of Lupine Bradyrhizobium Strains: Evidence from Nodulation Gene Trees

Bradyrhizobium strains isolated in Europe from Genisteae and serradella legumes form a distinct lineage, designated clade II, on nodulation gene trees. Clade II bradyrhizobia appear to prevail also in the soils of Western Australia and South Africa following probably accidental introduction with seeds of their lupine and serradella hosts. Given this potential for dispersal, we investigated Bradyrhizobium isolates originating from a range of native New World lupines, based on phylogenetic analyses of nodulation (nodA, nodZ, noeI) and housekeeping (atpD, dnaK, glnII, recA) genes. The housekeeping gene trees revealed considerable diversity among lupine bradyrhizobia, with most isolates placed in the Bradyrhizobium japonicum lineage, while some European strains were closely related to Bradyrhizobium canariense. The nodA gene tree resolved seven strongly supported groups (clades I to VII) that correlated with strain geographical origins and to some extent with major Lupinus clades. All European strains were placed in clade II, whereas only a minority of New World strains was placed in this clade. This work, as well as our previous studies, suggests that clade II diversified predominately in the Old World, possibly in the Mediterranean. Most New World isolates formed subclade III.2, nested in a large “pantropical” clade III, which appears to be New World in origin, although it also includes strains originating from nonlupine legumes. Trees generated using nodZ and noeI gene sequences accorded well with the nodA tree, but evidence is presented that the noeI gene may not be required for nodulation of lupine and that loss of this gene is occurring.     

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Extended Region of Nodulation Genes in Rhizobium meliloti 1021. I. Phenotypes of Tn5 Insertion Mutants

Rhizobium meliloti Nod^- mutant WL131, a derivative of wild-type strain 102F51, was complemented by a clone bank of wild-type R. meliloti 1021 DNA, and clone pRmJT5 was recovered. Transfer of pRmJT5 conferred alfalfa nodulation on other Rhizobium species, indicating a role in host range determination for pRmJT5. Mutagenesis of pRmJT5 revealed several segments in which transposon insertion causes delay in nodulation, and/or marked reduction of the number of nodules formed on host alfalfa plants. The set of mutants indicated five regions in which nod genes are located; one mutant, nod-216, is located in a region not previously reported to encode a nodulation gene. Other mutant phenotypes correlated with the positions of open reading frames for nodH, nodF and nodE , and with a 2.2-kb EcoRI fragment. A mutant in nodG had no altered phenotype in this strain. One nodulation mutant was shown to be a large deletion of the common nod gene region. We present a discussion comparing the various studies made on this extended nod gene region.     

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132^T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132^Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132^T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132^T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.     

Bradyrhizobium canariense and Bradyrhizobium japonicum are the two dominant rhizobium species in root nodules of lupin and serradella plants growing in Europe

Forty three Bradyrhizobium strains isolated in Poland from root nodules of lupin species (Lupinus albus, L. angustifolius and L. luteus), and pink serradella (Ornithopus sativus) were examined based on phylogenetic analyses of three housekeeping (atpD, glnII and recA) and nodulation (nodA) gene sequences. Additionally, seven strains originating from root-nodules of yellow serradella (O. compressus) from Asinara Island (Italy) were included in this study. Phylogenetic trees revealed that 15 serradella strains, including all yellow serradella isolates, and six lupin strains grouped in Bradyrhizobium canariense (BC) clade, whereas eight strains from pink serradella and 15 lupin strains were assigned to Bradyrhizobium japonicum (BJ1). Apparently, these species are the two dominant groups in soils of central Europe, in the nodules of lupin and serradella plants. Only three strains belonged to other chromosomal lineages: one formed a cluster that was sister to B. canariense, one strain grouped outside the branch formed by B. japonicum super-group, and one strain occupied a distant position in the genus Bradyrhizobium, clustering with strains of the Rhodopseudomonas genus. All strains in nodulation nodA gene tree grouped in a cluster referred to as Clade II, which is in line with earlier data on this clade dominance among Bradyrhizobium strains in Europe. The nodA tree revealed four well-supported subgroups within Clade II (II.1-II.4). Interestingly, all B. canariense strains clustered in subgroup II.1 whereas B. japonicum strains dominated subgroups II.2-II.4.