The Expression in Soil Bacreria of Symbiotic Genes from Rhizobium Leguminosarum biovar trifolii

By Michael Fenton (1994)

Dept of Microbiology and Genetics, Massey University, Palmerston North, NEW ZEALAND


Rhizobium leguminosarum biovar trifolii strain ICMP2163::Tn5 was able to spontaneously transfer its pSym to the non-nodulating Rhizobium loti soil isolate NR40 in sterile soil microcosms containing Ramiha hill soil or Ashurst silt loam soil at pH 6.0 or higher. In sterile soil microcosms at pH 6.0 containing sterile ryegrass or white clover plants the frequency of NR40 transconjugants was higher than in microcosms containing soil alone. The survival of the parent strains decreased in soil with a pH of 5.5 or less, and no transconjugant NR40 bacteria were detectable.

Southern blots of the genomic digests probed with nodA DNA confirmed that transconjugant NR40 contained symbiotic genes. On artificial media strain ICMP2163::Tn5 transferred its symbiotic plasmid, by conjugation, to Sphingobacterium multivorum, an organism that can be found in soil. The transconjugant bacteria were able to nodulate white clover seedlings but were unable to fix nitrogen.

Microscopic examination revealed that the root nodule structure, and bacteroid formation, were abnormal. The bacteria occupying the nodules were isolated and the total DNA extracted. The partial 16S RNA gene sequence from a transconjugant derived from a nodule was shown to be identical with that of the recipient S. multivorum. Southern blots of the genomic digests probed with nodA DNA confirmed that the transconjugant contained symbiotic genes.

A Caulobacter sewage isolate was also able to induce a tumour- like growth on white clover seedlings after receiving the pPN1 co-integrate plasmid from E.coli strain PN200. Eckhardt gel analysis confirmed that the transconjugant Caulobacter carried the R68.45:pSym co-integrate plasmid. Bacteroids were absent but Caulobacter cells were found in the outer two or three layers of the growth and the plant cells in this region had degenerated.

Sequence data was obtained for a 260 bp fragment of the 16S rRNA gene from Sphingobacterium multivorum and Caulobacter crescentus corresponding to postions 44 to 360 on the Escherichia coli genome. A distance matrix was constructed showing the relationship between S. multivorum, C. crescentus, Rhizobium, and related bacteria and neighbor-joining was used to construct a tree.

From the tree given it is concluded that the ability to carry or express symbiotic genes is not dependant on having a phylogenetic relationship with Rhizobium.


1. The significance of the genus Rhizobium.

Pasture growth is limited by the quantity of fixed nitrogen available when other soil nutrient deficiencies have been corrected by top-dressing. Nitrogen fixation is the process by which atmospheric nitrogen gas is made available for incorporation into organic compounds. Only certain bacteria are capable of carrying out this process, the genus Rhizobium being the most common (Raven et al., 1981). Members of this genus are Gram negative aerobic rods that occur free-living in soil or as micro-symbionts in root nodules of leguminous plants (Jordan, 1984). Rhizobia in root nodules are estimated to carry out 50-70% of the world biological nitrogen fixation (Quispel, 1974), reducing approximately 20 million tonnes of atmospheric nitrogen to ammonia (Beringer et al.,1980). Biological nitrogen fixation is of particular importance to New Zealand agriculture, providing 1 million tonnes of nitrogen annually (Ball and Field, 1985). Compared to the 26,373 tonnes (Douglas and Cochrane, 1989) of nitrogenous fertiliser used by New Zealand farmers this is more than 97% our annual requirements.

Although this process is free, self-sustaining and non-polluting, it does not necessarily operate with optimum efficiency. Although New Zealand pasture soils contain high numbers (e.g. 10,000 to 1 million per gram of soil) of indigenous clover rhizobia (Bonash and MacFarlane, 1987) the introduction of superior nitrogen fixing strains is still considered an important management practice. However, the inoculant strains may be prone to loss of symbiotic traits such as infectiveness and effectiveness (O'Hara, 1985), and may not be competitive with the indigenous strains already present in the soil (Rhys and Bonish, 1984). The recommended inoculum for white clover consists of a mixture of three strains of Rhizobium leguminosarum biovar trifolii which includes strain ICMP2163, ICMP2666, and ICMP2668. Stock cultures are maintained by the Plant Diseases Division of the Crown Research Institute, Auckland (Bianchin, 1989).

2. The Biology of Nitrogen Fixation.

Rhizobium bacteria are able to invade the root hairs of leguminous plants via an infection thread formed by the plant cells. The plant cells then respond by undergoing rapid cortical division to form either a tumour or a refined structure called a nodule. The genetic requirements for nodulation are divided between the Rhizobium bacteria and the host plant. Both contain genes that are only expressed in the presence of the other. The process is reviewed in Djordjevic et al. (1987). Flavanoids, excreted by the plant, activate nodulation (nod) genes carried by the bacteria. The nodAB genes on the Rhizobium symbiotic plasmid may produce a low molecular weight substance that induces plant cell divsion (John et al., 1988). Attachment of the bacterial cell to the root hair is proposed to be mediated by binding to lectin. Rhizobia appear to attach in an end-on fashion followed by involution of the plant cell wall to form an infection thread. As the infection thread grows through 3 to 6 layers of root outer cortex cells, meristematic activity is initiated in a small group of root cortical cells directly in front of the tip of the infection thread. Growth of the infection thread continues into this meristematic region where rhizobia are released into the inner most cells, where the bacteria continue to divide until the cytoplasm is filled with bacteroids (Robertson and Farnden, 1980).

The nodules formed on clover are called indeterminate nodules. The infection threads continue to penetrate the plant cortical cells in the nodule meristem, providing a continuous release of rhizobia into the plant cells as the nodule increases in size (Beringer et al., 1979). In the process of nodule development, the bacteria undergo morphological and physiological changes that lead to the formation of bacteroids (Irigoyen et al., 1990). Free living rhizobia are not capable of fixing atmospheric nitrogen as oxygen inactivates the nitrogenase enzyme that converts nitrogen to ammonia and blocks the transcription of nitrogenase genes. The atmosphere in the nodule environment is micro-aerophilic due to high concentrations of the plant protein leghaemoglobin. This protein plays a role in the transport of oxygen by maintaining a sufficiently high pO2 in the plant cytoplasm for oxidative phosphorylation, while providing a sustained low level of oxygen to the bacteroids (Verna and Long, 1983).

In this environment, bacteroids are able to supply the plant with ammonia which is assimilated into glutamate, glutamine and other translocatable products. In return, the bacteria is supplied with an abundance of carbon compounds such as sugars, and is provided with a protected environment from the outside world. An ineffective nodule which is not able to fix nitrogen may be formed if the plant is infected by a Rhizobium strain with a mutation in the nitrogen fixing (fix) genes.

3. Taxonomy of Rhizobium.

Until recently, the rhizobia that infect beans, peas, and clovers were clustered in a single species, Rhizobium leguminosarum (Jordan, 1984), which had three biovars; Rhizobium leguminosarum bv phaseoli, Rhizobium leguminosarum bv viceae, and Rhizobium leguminosarum bv trifolii. The artificial nature of this simplistic classification scheme is becoming more evident as knowledge is acquired and new species discovered. Currently three species, Rhizobium leguminosarum bv phaseoli, R. etli bv phaseoli, and R. tropici, two new Rhizobium genomic species, and other unclassified genotypes have been isolated from nodules of Phaseolus vulgaris (Laguerre et al., 1994).

It appears that there may be a greater diversity of bacteria capable of nodulating legumes than was previously recognised (Laguerre et al., 1994). Within R. leguminosarum biovar trifolii there is considerable phenotypic variability (Dughri and Bottomley, 1984; Harrison et al., 1987), reflected by the genetic diversity observed (Jarvis et al., 1980; Crow et al., 1981). Jarvis et al. (1980) compared reference DNA from clover inoculant strains NZP561 and TAI with DNAs from 18 other R. leguminosarum bv trifolii strains. The range of DNA-relatedness and DTm(e) values with strains NZP561 and TAI was 61 - 91% and 0 - 8.2oC and 49 - 94% and 1.3 - 7.0oC respectively. DTm(e) is a statistic which expresses the base sequence homology in the fraction of DNA which hybridises. Each 1oC represents a 1% miss-match in the hybridising sequences (Jarvis et al., 1991). The values quoted extend well beyond the phylogenetic limits for a bacterial species as proposed by Wayne et al., (1987). It is concluded that, Rhizobium leguminosarum bv trifolii may not be a single species but a group of inter-related species capable of expressing the appropriate symbiotic genes.

Normally the primary isolation of Rhizobium strains is from nodulated legumes (Schofield et al., 1987; Vincent, 1970; Young, 1985) and this has made it difficult to define phylogenetic relationships with other bacteria in the soil. However, the ability to nodulate leguminous plants is regarded as the characteristic function of the genus Rhizobium with nitrogen fixation a normal but not essential consequence of nodulation (Jordan, 1984). The nodulation and nitrogen fixation genes are usually located on a symbiotic plasmid (pSym), that encodes distinct nodulation specificities (Johnston et al., 1978; Hirsch et al., 1980). The plasmid may be lost under certain environmental conditions, so that soil bacteria lacking this plasmid cannot be classified as rhizobia although they may be able to express the symbiotic genes. Strains of bacteria exist that fail to satisfy Jordan's definition but are clearly rhizobia lacking the symbiotic plasmid (Scott and Ronson, 1982; Soberon-Chavez and Najera, 1988; Segovia et al, 1991).

Another difficulty arises from the ability of the symbiotic plasmid to be transferred from one strain of Rhizobium to another. This may change the strains host specificity or lead to the loss of the ability to nodulate. It has been shown that pSym genes can be expressed to a limited degree in Agrobacterium species (Hooykass et al. 1981; Kondorosi et al., 1982; O'Connell et al., 1987), Pseudomonas aeruginosa and Lignobacter species (Plazinski and Rolfe, 1985). Jarvis et al. (1989) suggested that Rhizobium classification should be defined in terms of DNA-DNA or rRNA-DNA homology to accepted reference bacteria. In addition, it may be useful to use the 16S ribosomal DNA sequence to determine what is a 'true' rhizobia. PCR-RFLP analysis has been described as a rapid method for the identification of nodule isolates and new taxa (Laguerre et al., 1994). The use the fatty acid composition profiles has also been described as another reliable means of rapid identification (Jarvis and Tighe, 1994).

4. The Symbiotic Plasmid.

The nodulation and nitrogen fixation genes are usually located on large (>100 kb) symbiotic plasmids (pSym or Sym plasmid), some of which can be transferred to other bacteria via conjugation (Djordjevic et al., 1983; Johnston et al., 1978). There is evidence that pSym transfer occurs in natural field populations., Schofield et al., (1987) studied 16 soil isolates of Rhizobium leguminosarum and observed similar Sym plasmids in different host chromosomal backgrounds and different Sym plasmids in similar host chromosomal backgrounds, as well as the presence of a putative recombinant Sym plasmid. Jarvis et al., (1985) reported the isolation of soil bacteria that showed DNA homology to Rhizobium leguminosarum but were unable to nodulate white clover. Transconjugation experiments with the co-integrate plasmid pPN1 (Scott and Ronson, 1982) showed that these bacteria could express symbiotic genes from clover rhizobia. Plasmid transfer in non-sterile soil has been demonstrated between Rhizobium fredii and a pSym cured Rhizobium leguminosarum (Kinkle and Schmidt, 1991) and between Rhizobium leguminosarum and Enterobacter (Dohler and Klingmuller, 1988).

Indigenous soil bacteria, including native rhizobia, are well adapted to survive in the absence of a host plant. Potential competitors may not initially be able to nodulate crop plants but may be enabled to by obtaining the appropriate symbiotic plamid (Dowling and Broughton, 1986). If complemented by a Sym plasmid from an introduced Rhizobium strain, the indigenous soil bacteria will compete for nodulation sites and may form the majority of nodules on the host plant (Meade et al., 1985; Weaver & Frederick, 1974a, 1974b). The inoculant strain may need to be supplied at 1000X the level of the indigenous Rhizobium population in order to form 50% of the nodules. For the inoculation industry this may yield unexpected benefits if it were possible to isolate indigenous soil bacteria able to nodulate and fix nitrogen better than the commercial Rhizobium inoculant. However, it becomes a problem when the indigenous soil bacteria form ineffective nodules incapable of nitrogen fixation. In this instance, increasing the inoculum added to the soil is simply adding more DNA for the competitors to pick up. There may also be important consequences for the release of genetically engineered micro-organism.

However, many factors influence the competitive ability of a Rhizobium strain, and any factor which adversely effects plant growth will also profoundly effect competition for nodulation. Phosphorous limitation has been shown to be exacerbated by low pH and the combination of low pH and phosporous levels can have a strong influence on competition for nodulation (Dowling and Broughton, 1986). Most soils in New Zealand are moderately acidic, having a pH between 5.0 and 6.5. It appears that an acidity of pH 5.8-6.0 is considered ideal for the legume to prevent aluminium and manganese toxicity, but the other partner in the symbiotic relationship appears to have been overlooked. Other environmental factors such as soil type, temperature, and moisture also affect the outcome of competition. Biological factors, such as bacteriophage effects, epiphtyic bacteria, mycorrhizal effects predation by protozoa should all be considered when applying laboratory results outside. It is concluded that symbiotic plasmid transfer occurs between Rhizobium strains and other bacteria in soil but the nature and diversity of the recipient remains unclear.


1. Conjugation in sterile soil microcosms.

1.1 Strains used :

Rhizobium leguminosarum bv trifolii strain ICMP2163 is used as a white clover inoculant strain in New Zealand. The symbiotic plasmid was labelled by insertion of the transposon Tn5 which confers neomycin resistance to its host (Rao et al., 1994). The Sym plasmid from strain ICMP2163::Tn5 used in this study was shown to be fully effective for strain PN165, a pSym cured derivative of strain ICMP2163. This demonstrated that the pSym was self-transmissible and that insertion of Tn5 did not affect the expression of nodulation genes. Our laboratory had been investigating the transfer of pSym from New Zealand inoculant strains to native non-nodulating soil isolates. The soil isolate NR40 was identified by its fatty acid profile, a method shown to be reliable (Jarvis and Tighe, 1994), as Rhizobium loti. Growth requirements, cell morphology and colony morphology of NR40 on TY agar are consistant with those of R.loti, however a detailed study comparable to Segovia et al., (1991) would confirm this identification. The observation that transconjugant NR40 forms ineffective nodules on white clover seedlings would not be surprising for R. loti transconjugants.

1.2 Factors affecting soil rhizobia.

A number of factors affect the survival of micro-organisms in soil. Investigators tend to concentrate on factors that lend themselves to study in the laboratory, such as temperature, pH, moisture content and organic matter content (reviewed by Dowling and Broughton, 1986). Rhizobium strains vary in their acid tolerance. In soil, pH not only directly affects the growth of micro-organisms, but also affects the solubility of many cations which may indirectly alter growth patterns. A strain of Rhizobium fredii did not to survive in soil below pH 5.25 (Richaume, 1989). Dughri and Bottomley (1984) were able to alter the outcome of competition between indigenous rhizobia in the soil by changing the acidity. Rhizobium leguminosarum bv trifolii strain ICMP2163::Tn5 was able to transfer its Tn5-marked symbiotic plasmid to the pSym deficient Rhizobium soil isolate NR40, in sterile soil at a pH greater than 5.5. NR40 was not able to incite root nodule formation but transconjugant bacteria were able to form ineffective nodules on white clover seedlings. The parent strains survived for a maximum of 21 days in soil at pH 5.5 or less. It is concluded that the the inoculant strain ICMP2163 would not be suitable for use in acidic soils. There was a significant increase in the frequency of plasmid transfer in the presence of ryegrass or white clover plants; from 1 X 10-6 to 3 X 10-6. This may be due to stimulating factors associated with the rhizosphere. Bacteria associated with the rhizosphere will have access to attachment sites, nutrients and minerals at high concentrations and as a consequence, will be metabolically more active than their free-living counterparts.

Overall, there will be mucher greater opportunity for genetic exchange to occur. In sterile Ramiha hill soil at 50% water holding capacity (pH 6.0) with clover seedlings present, the number of transconjugants present per gram of soil increased 10-fold over an 18 day period; from 4 CFU/g to 44 CFU/g. The significance of these results are two-fold. Firstly, Theis et al., (1919a,1991b) have shown that as few as 50 indigenous rhizobia per gram of soil eliminated the inoculum response to 1million to 10 million rhizobia per seed. The inoculant strain ICMP2163 has the potential to transfer its pSym to indigenous soil bacteria at a high enough frequency to eliminate future inoculum responses. Secondly, in order to get meaningful results, laboratory simulations should be as close to conditions in natural environments as possible. These experiments involved one potential recipient strain. In non-sterile soil there are a great number of species that could be involved, although there is no indication as to how many that may be. There appeared to be no significant difference in using Ramiha hill soil or Ashurst silt loam soil in the above mentioned experiments. This implies that what happens in one soil type may well occur in others. There may be no need to taylor bacteria for specific soil types if this is true. However, the experiments carried out so far are rather simplistic, looking at a few of the variables associated with soil. Experiments with non-sterile soil, in the manner of Kinkle and Schmit (1991), would be more convincing.

1.3 Significance of plamid transfer in soil.

Rhizobium strains lacking symbiotic plasmids in soil may act as biological sinks for the symbiotic plasmids from inoculant strains. Strains of bacteria exist that fail to satisfy Jordan's definition but are clearly rhizobia lacking the symbiotic plasmid (Scott and Ronson, 1982; Soberon-Chavez and Najera, 1988; Segovia et al, 1991). This could explain the temporal loss of inoculant strains in the field and the appearance of indigenous rhizobia where there was no evidence of previous Rhizobium populations (Roughley et al., 1976). It is now well established that self-transmissible symbiotic plasmids can be exchanged between strains of Rhizobium on artificial media and there is evidence that this exchange occurs in the natural field populations. Two independent studies, one involving Rhizobium leguminosarum bv trifolii (Schofield et al., 1987), and the other involving Rhizobium leguminosarum bv viceae (Young and Wexler, 1988), have reported that similar symbiotic plasmids could be found in genetically unrelated isolates. Kinkle and Schmit (1991) observed the transfer of the symbiotic plasmid pJB5JI between strains of Rhizobium in sterile and non-sterile soil. It is concluded that the improved use of Rhizobium seed inoculants will require further study of plasmid transfer mechanisms between the inoculant bacteria and the other soil bacteria.

2. Expression of Symbiotic Genes by Non-Rhizobium Species.

The recognition that Rhizobium strains lacking Sym plasmids exist in soil has meant that soil is often screened for new Rhizobium strains. Isolates are usually selected on the basis of having particular growth characteristics and colony morphology on solid media compared to known reference strains. It was in this manner that NR40 was isolated. It can take some time for an isolate to be completely characterised and accurately identified. One of the soil isolates used in our laboratory (NR64) was thought to be a Sphingobacterium (Bianchin, 1989) but was later shown to be a strain of Rhizobium. A strain of Sphingobacterium was obtained for furthur study. The approach used was to cross Rhizobium leguminosarum bv trifolii strain ICMP2163::Tn5 with a number of known non-Rhizobium strains. In this manner it was hoped to gain some insight as to the distance that symbiotic genes could travel.

2.1 Transfer of pSym to Sphingobacterium multivorum.

Sphingobacterium multivorum is an organism that can be found in soil. It is able to grow agar at 37 oC, and is sometimes isolated from clinical samples. As strain NZRM1228 was spontaneously resistant to neomycin it was not possible to use the Tn5 antibiotic resistance marker to select for transconjugants retaining pSym on artificial media. The symbiotic plasmid was lost from nodule isolates after 2 - 3 rounds of single colony purification. Luria Rif.Neo.Str agar ensured that only the Sphingobacterium would grow for use in plant inoculation tests. The use of white clover seedlings to select for transconjugants and the isolation of the bacteria from the root nodules was the most effective way to obtain enough bacteria for study. This suggests that as long as the appropriate selection pressure is applied the Sphingobacterium transconjugants would continue to nodulate other clover seedlings. Comparison of the electron micrographs in indicates that the nodule occupant of the MF100 plant was different from the Rhizobium donor strain. Total genomic digest profiles of the nodule isolate were the same as strain NZRM1228 and Southern blots probed with nodA DNA confirmed that the nodule isolate contained symbiotic genes. 16S rDNA sequence analysis identified the nodule isolate as Sphingobacterium multivorum recipient strain NZRM1228. This is the first report of the spontaneous transfer of the symbiotic plasmid from an inoculant strain of Rhizobium leguminosarum bv trifolii to Sphingobacterium multivorum.

2.2 Expression of pSym Genes in Caulobacter.

E. coli strain PN200, carrying the co-integrate plasmid pPN1, and Caulobacter strain MCDF23 was crossed in an alternative method to test the ability to express Sym plasmid genes. Scott and Ronson (1982) obtained the 770 Mda pPN1 by co-integrating the Sym plasmid from Rhizobium leguminosarum bv trifolii strain NZP514 (pRtr514) with the broad-host-range plasmid R68.45. The plasmid confers neomycin resistance to its host. Caulobacter belongs to the budding and prosthecate group of organisms and is found in soils and waterways. A transconjugant Caulobacter isolate MCDF100 containing the co-integrate plasmid pPN1 (Scott and Ronson, 1982) was able to induce a tumour-like growth within 12 days of inoculation onto sterile 3 day old white clover seedlings. Attempts to isolate the nodule occupants were unsuccessful. Examination by electron microscope showed that the growth did not appear to be invaded by bacteria. A few bacteria were found within the intercellular spaces of the outermost cells of the structure and the plant cells in this region had degenerated. Plazinski and Rolfe (1985) reported similar results with Pseudomonas strain PAO5. Expression of the Sym plasmid genes carried on pPN1 may have been affected by the RP4 tra genes in the R68.45 section of the co-integrate plasmid (Hynes and O'Connell, 1988). This is the first report of Caulobacter carrying symbiotic plasmid genes and causing tumour-like growths on white clover seedlings.

3. Consequences for Taxonomy.

The ability to nodulate leguminous plants is regarded as the characteristic function of the genus Rhizobium with nitrogen fixation a normal but not essential consequence of nodulation (Jordan, 1984). There are a number of Rhizobium species that carry the nodulation and nitrogen fixation genes on plasmids which may be transferred by conjugation. Soil bacteria other than rhizobia could be involved in the dissemination of symbiotic genes, perhaps acting as temporary hosts before passing the genes back to an appropriate Rhizobium strain. The existance of species of soil bacteria, outside of Rhizobium, capable of expressing Sym plasmid genes may have been overlooked because of the screening method or media used. Possible candidates are Agrobacterium (Hooykass et al. 1981; Kondorosi et al., 1982; O'Connell et al., 1987), Enterobacter (Dohler and Klingmuller, 1988), Pseudomonas and Lignobacter (Plazinski and Rolfe, 1985), Caulobacter, and Sphingobacterium multivorum. Caulobacter is distantly related to Rhizobium, and Sphingobacterium is the most distantly relaled. Schematic 2D models of the 16S RNA molecule indicate that Sphingobacterium is quite unrelated to Rhizobium. It is concluded that the ability to express or carry symbiotic plasmid genes is not dependant on having a phylogenetic relationship to Rhizobium. Clearly, it is undesirable for there to be this confusion in a classification scheme. A number of authors have suggested revised descriptions of the genus Rhizobium and Agrobacterium based on 16S rDNA sequences and DNA/DNA homology studies (Willems and Collins, 1993; Sawada et al., 1993; Yanagi and Yamasato, 1993).

4. Conclusion.

This study reports the isolation of a strain of Sphingobacterium multivorum that is able to nodulate white clover seedlings and would fit Jordan's definition of a Rhizobium species. It is concluded that taxonomic relationships based on characteristics carried on plasmids may not reflect real relationships amongst micro-organisms. It would be preferable to identify known Rhizobium species from their fatty acid profiles or specific DNA probes and define new species in terms of their 16S rRNA gene sequence and DNA-DNA relatedness with recognised reference strains. Further work needs to be done in looking at the factors affecting pSym transfer in soil, preferably using non-sterile soil. Non-Rhizobium strains should continue to be tested to see if they are able to receive the Sym plasmid from New Zealand inoculant strains. This may result in new inoculant strains becoming available with unique characteristics.


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