The 10th Nitrogen Cycle Meeting 2004

Plasmid replicons of Rhizobium

L.C. Crossman


Rhizobium spp. are found in soil. They are both free-living and found symbiotically associated with the nodules of leguminous plants. Traditionally, studies have focused on the association of these organisms with plants in nitrogen-fixing nodules, since this is regarded as the most important role of these bacteria in the environment. Rhizobium sp. are known to possess several replicons. Some, like the Rhizobium etli symbiotic plasmid p42d and the plasmid pNGR234b of Rhizobium NGR234, have been sequenced and characterized. The plasmids from these organisms are the focus of this short review.

  • nitrogen fixation
  • nodule
  • plasmid
  • Rhizobium
  • sequencing
  • symbiosis

Rhizobium sp. strain NGR234

This unusual Rhizobium strain forms nitrogen-fixing symbiotic root nodules with many different leguminous plants. The unusual broad specificity of the interactions of the strain with plant roots has made this strain the focus of several studies. The plasmid pNGR234a carries many of the specificity determinants necessary for nitrogen fixation and strains cured of pNGR234a were unable to fix nitrogen [1]. However, several of the fix genes are located on the chromosome in this strain [2]. In related species, such as Bradyrhizobium, the genes involved in nitrogen fixation and symbiosis are located on the chromosome in ‘symbiotic islands’ [3]. Specified on the plasmid are nod factors, short N-acetylglucosamine factors that trigger nodulation responses in plants. The response to the production of these factors may allow nodulation or not, in various plants [4]. pNGR234a encodes a type III secretion system, which may be employed for delivering nodulation factors. This has led to the suggestion that symbiosis and pathogenesis share significant similarities.

This replicon is 536165 bp and 58.5% G+C (guanine+cytosine), although there are several regions of differing G+C content. Genes which have a lower G+C content include those for nod factor and polysaccharide biosynthesis (at 45–55% G+C). Approximately 12% of the plasmid encodes functions involved in nitrogen fixation and nodulation. An additional 10% encodes transposase and IS (insertion sequence) related elements, with approx. 18.4% having functions associated with transport [5].

The sister replicon pNGR234b has now been partially sequenced from this strain [6]. Sequencing 257655 bp of contig 1 and 234455 bp of contig 2 revealed a 61.8% G+C content. Genes for the production of a type IV pilus are present that are described as potentially important in adhesion, biofilm formation, conjugative DNA transfer, infection by phage and motility. There are several genes that play a part in resistance and detoxification such as CopC copper resistance protein and heat-shock proteins. In addition, there are approx. 58 coding sequences involved in transcriptional regulation from the LysR, GntR and TetR families. The density of transporter proteins is similar to that of pNGR234a. This plasmid has not yet been cured, and this finding taken together with the fact that there are several pathways for amino acid and cofactor biosynthesis as well as a 30 S ribosomal protein gene may indicate that the plasmid is essential. A large gene cluster synthesizing exopolysaccharide is present. The genes exs and exo encode biosynthesis of low molecular weight exopolysaccharides, which are vital for the invasion of nodules [7]. Unexpected findings are cell wall biosynthesis proteins, polygalacturonases and pectinases similar to those of the plant pathogenic Erwinia chrysanthemi. There may be a role for these proteins in the infection process. Similarly there are closely related genes involved in iron transport and metabolism as well as genes probably involved in the modification/degradation of cell walls.

Symbiotic plasmids of Rhizobium etli

R. etli plasmid p42d from R. etli CFN42 has been fully analysed [8]. This plasmid is 371255 bp with an average G+C content of 58.1% (Table 1). In all, six plasmids are carried by this strain. P42d is the equivalent of pNGR234a, carries the nod, nif and fix genes, and was originally designated pSymA in Sinorhizobium meliloti 1021 [9]. The nod genes are present in a cluster of 16 kb; in contrast the nif and fix genes are clustered in five distinct regions. There are three clusters containing the nifH nitrogenase reductase component gene, which also includes nifDE genes for dinitrogenase. One such cluster contains a nifE pseudogene, the second cluster contains a nifD pseudogene and the last cluster is the intact nifHDKENX. The fixNOQP genes encode a cytochrome oxidase, fixGHIS encodes a cationic pump and fixABCX encodes a flavoprotein, all of which are required for nitrogen fixation. In the root nodule environment, this oxidase provides the energy required, whereas the flavoprotein directs the flow of electrons to the oxidase. Not all fix genes are present on p42d, some are present on another R. etli replicon, p42f, and some are present in more than one copy. Nod boxes are found in the upstream regions of nod genes, which bind nodD transcriptional regulator and are known to regulate genes in response to root exudates. Other genes involved in the assembly of the complex nitrogenase protein are present, but notably absent is the nifV gene, which is implicated in biosynthesis of the metal cofactor for nitrogenase. In addition to the well-characterized regulators of nitrogen fixation, there are 12 other predicted regulators. Genes for both type III and type IV secretion systems are present, as are tra genes involved in specifying the conjugal pilus and cytochrome P450. There are genes encoding polysaccharide biosynthesis, electron transport and plasmid-maintenance. A large number of ISs-/transposon-related sequences are present, several of which represent partial and pseudogenes. Analysis of the ISs by Gonzalez et al. [8] indicates that the genes involved in symbiosis have flanking IS elements and may represent a mobile symbiotic island. There is also some evidence of plasmid rearrangements due to repeat sequences; amplifications, inversions and deletions were also detected. Relatively few rhizobial sym plasmids are transmissable in laboratory media, although sequence data derived from genome sequencing projects suggest that the replicons possess an origin of transfer (oriT or mob) and are mobilizable. A recent study of mob regions in R. etli has detected functional mob regions in both the major nitrogen-fixation plasmids p42d and p42a [10].

View this table:
Table 1 Summary of the major characteristics of the sequenced Rhizobium sp. replicons

When sequences of p42d are compared with other α-proteobacteria, S. meliloti and Mesorhizobium loti shared 51 and 45% orthologues respectively. In general, genes involved in symbiosis were the most conserved. Members of the α-proteobacteria such as Caulobacter crescentus and Agrobacterium tumefaciens share from 25 to 32% of the gene orthologues on p42d, whereas plant pathogens such as Ralstonia solanacearum share 31%. The least number of orthologues are found in human pathogenic bacteria with small genomes. Genes involved in nitrogen fixation show little synteny with those of other nitrogen-fixing bacteria.

Analysis of another strain, R. etli CE3, has detected a single copy of catalase, katG and its regulator oxyR located on a plasmid which also encodes several fix genes. These genes are involved in resistance to peroxide species. It is known that these genes are not essential for symbiosis [11].

The genome of R. leguminosarum biovar viciae is currently being sequenced and analysed at the Wellcome Trust Sanger Institute ( It remains to be seen whether the genes on the R. leguminosarum replicons are syntenic with those of its relatives or whether this genome holds some major surprises in store.


The author thanks J. Parkhill for helpful comments and advice.


  • The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).

Abbreviations: IS, insertion sequence


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