SCOPE 44 - Introduction of Genetically Modified Organisms into the Environment

8.1 INTRODUCTION

Current progress on the knowledge of the genetic information involved in the symbiotic process will certainly provide in the near future genetically improved Rhizobium strains. If these bio-engineered bacteria are to be delivered into natural environments, the fate of these novel introductions should be carefully considered. Concerns have been raised mainly on the transfer of genetic information from introduced strains to pathogenic soil inhabitants or to strains that could eventually become pathogens, and also on the modification of the genetic information subsequent to the release of bacteria in the soil. It is imperative to learn the rules of the behaviour of introduced genetic information in natural environments, from the point of view of its stability and of the frequencies of exchange of information between different organisms.

Symbiotic nitrogen fixation is the main biological entrance of fixed nitrogen in the biosphere. This biological process is only performed by procaryotes, among these Rhizobium spp. have attracted special interest. Rhizobium strains have been used as inoculants in substitution of nitrogen fertilizer for a long time. In the following pages we will review several aspects of Rhizobium-legume symbiosis as well as some characteristics of the general organization and dynamics of the Rhizobium genome. We will also present an approach to evaluate the exchange of genetic information among strains in nature.

The bacteria of the genus Rhizobium form nitrogen-fixing nodules on the roots of legumes. These symbiotic interactions involve a differentiation process of both partners, the bacteria, and the plant which forms a new organ, the nodule. This process has been extensively studied in several legumes such as alfalfa, clover, pea, soybean, bean, and siratro. The establishment of the symbiosis includes several steps. The bacteria attach to the root hairs or to root cells that will become root hairs; Rhizobium cell surface polysaccharides have been considered to mediate this attachment. Rhizobia induce root hair curling and then a tubular structure of plant origin, the infection thread, is gradually formed, leading the bacteria to the cortical cells. There, the infection thread branches off and the bacteria are delivered into the plant cytoplasm surrounded by a membrane of host origin. The bacteria differentiate into bacteroids that fix nitrogen and that interact metabolically with the plant.

During symbiosis, specific sets of both bacteria and plant genes are expressed, establishing the basis for the differentiation process. Recent advances in molecular genetics have greatly contributed to our understanding of the Rhizobium-legume interaction. Several symbiotic genes have been identified and isolated; their expression under different conditions has also been studied.

Among others, the early nodulation genes (nod), host specificity genes (hsn), and nitrogen-fixation genes (nif) have been analysed (Long et al., 1982; Kondorosi et al., 1984; Downie et al., 1985; Schofield et al., 1984; Ramakrishnan et al., 1986; Lamb and Hennecke, 1986; Ruvkun and Ausubel, 1980; Hennecke, 1981; Corbin et al., 1982; Torok and Kondorosi, 1981; Ma et al., 1982; Scott et al., 1983, Quinto et al., 1982). In the near future a comprehensive scheme of the bacterial genes that participate in the symbiotic process will certainly emerge.

8.2 A DYNAMIC CONCEPT OF THE RHIZOBIUM GENOME

Our current model for the Rhizobium genome can be stated as follows. A single cell at a given time has a particular arrangement of genetic information. Two forces participate to increase the diversity of this arrangement of the genome in an ecological time scale: its internal dynamics and the exchange of genetic information with other bacterial strains in nature. The Rhizobium genome contains a large amount of repeated DNA sequences that might act as points for recombination resulting in genomic rearrangements. Since these rearrangements are frequently observed under laboratory conditions, a cell gives rise to a population of similar but not necessarily identical organisms. This means that even a single bacterial colony will present genetic heterogeneity. The collection of strains that exchange genetic information among them should be considered as a genomic pool equivalent to the concept of genospecies that Reanney (1978) introduced for bacterial populations.

8.3 REITERATED DNA SEQUENCES IN RHIZOBIUM

Repeated DNA sequences are a general characteristic of eukaryotic genomes, but in prokaryotes only in the case of the archaebacteria, Halobacterium halobium and Halobacterium volcanii, has DNA reiteration been reported as a common genomic feature (Sapienza and Doolittle, 1982).

However, we have detected a large amount of repeated DNA elements in Rhizobium phaseoli, the symbiont of the common bean plant Phaseolus vulgaris, in Rhizobium meliloti, the symbiont of alfalfa, and also in Agrobacterium tumefaciens, a plant pathogen taxonomically closely related to Rhizobium (Flores et al., 1987). We calculate about two hundred repeated DNA families in the genome of some strains of R. phaseoli. The family size was usually small, from two to five elements, but some have more than ten elements. Rhizobium and Agrobacterium contain large plasmids in addition to the chromosome. The analysis of two R. phaseoli strains indicates that DNA reiteration is not confined to a particular replicon but is a property of the whole genome.

The nature of some of the repeated elements has been established in various Rhizobium strains. The nitrogenase structural genes are reiterated in Rhizobium phaseoli (Quinto et al., 1985), in Rhizobium fredii, symbiont of some varieties of soybean (Prakash and Atherly, 1984), in the broad host range strain ANU240 (Morrison et al., 1983), in strain ORS571 that produces stem and root nodules in Sesbania (Norel et al., 1985; Donald et al., 1986), and in strains originally isolated from different species of Phaseolus and from Pachyrhizus erosus (Martinez et al., 1985).

Other examples of repeated DNA sequences in Rhizobium and Bradyrhizobium (slow-growing rhizobia) strains include insertion sequences (Kaluza et al., 1985; Ruvkun et al., 1982), an early nodulation gene (nodD) (Gottfert et al., 1986), and nif and nod regulatory regions (Better et al., 1983; Rostas et al., 1986).

8.4 GENOMIC REARRANGEMENTS IN RHIZOBIUM

It is a common observation that Rhizobium strains can generate variability in regard to colony morphology and symbiotic properties. This has led to certain practices diminishing the possibility of losing some important characteristics. The preservation of strains and their use for inoculation are usually performed without the isolation of single colonies. Strains that are supposed to be homogeneous are commonly subjected to symbiotic cycles in order to select for clones presenting the desired properties.

Futhermore, several reports indicate that when exposed to certain stress conditions or to genetic manipulations, Rhizobium can present genomic rearrangements (Berry and Atherly, 1984; Christensen anhd Schubert, 1983; Djordjevic et al., 1982; Kaluza et al., 1985; Soberón-Chávez et al., 1986; Wang et al., 1986; Zurkowski, 1982). We have analysed genomic rearrangements that occur at high frequency under commonly used laboratory conditions that are not considered to cause stress in bacterial populations. The experimental approach was similar to that reported by Sapienza et al. (1982) demonstrating the presence of frequent genomic rearrangements in the archaebacteria H. halobium and H. volcanii. Descendants of a single cell were analysed in regard to the hybridization patterns obtained using as probes recombinant plasmids that reveal repeated DNA sequences. Differences in the hybridization pattern indicate that a genomic rearrangement occurred from the time the culture was a single cell to the time of harvesting individual colonies derived from such a cell. With some of the hybridization probes used, genomic rearrangements were observed at a frequency of 1-5% in 20 generations (Flores et al., 1988).

The mechanisms responsible for the genomic rearrangements observed are not known at the present time. Homologous recombination between different elements of reiterated DNA families could be involved as well as the transposition of insertion sequences. Regardless of the underlying mechanisms and of the actual frequency of genomic rearrangements, our data support the hypothesis that Rhizobium strains present internal genomic dynamics that are continually generating subpopulations of similar but not identical organisms.

8.5 COMPARTMENTALIZATION OF THE RHIZOBIUM GENOME

The Rhizobium genome is partitioned into different compartments, the chromosome, and several plasmids of high molecular weight on the order of 10²-10³ kilobase pairs. A usual finding in Rhizobium strains of different host specificities is that the nitrogenase structural genes and the nodulation genes are found on a single large plasmid, the symbiotic (sym) plasmid (Hombrecher et al., 1981, Prakash et al., 1981; Rosenberg et al., 1981). Of particular interest is the fact that host-specific determinants are, as well, present in the symbiotic plasmid. In the laboratory, plasmids can be transferred among Rhizobium strains with different specificities and it is frequently observed that strains acquire the specificity of the corresponding sym plasmid (Hooykaas et al., 1981; Johnston et al., 1978). Moreover, Rhizobium plasmids have been transferred to Agrobacterium tumefaciens and the recipients have gained the ability to initiate and in some cases establish a symbiotic process with the appropriate host legumes (Hooykaas et al., 1982; Kondorosi et al., 1982; van Brussel et al., 1982; Martinez et al., 1987; Brom et al., 1988). A fundamental question is whether these transfers occur naturally in soil populations (see below).

Besides those carried by the sym plasmid, genes relevant to nodulation and nitrogen fixation have been found in the chromosome and in other plasmids. It is not known where genes necessary for other important traits such as saprophytic or host competitive ability reside.

8.6 EXCHANGE OF GENETIC INFORMATION AMONG SOIL BACTERIA

The current Rhizobium systematics considers it to be a member of the bacterial family Rhizobiaceae. This family is composed of four different genera: Rhizobium, Bradyrhizobium, Agrobacterium and Phylobacterium (Jordan, 1984). These gram-negative bacteria are able to interact with plants eliciting cortical hypertrophies. Rhizobium and Bradyrhizobium species form nitrogen-fixing nodules on the roots of many leguminous species, and in some cases on the stems of certain leguminous trees. Phylobacterium species produce nodules on the leaves of certain plants. Agrobacterium species (with the exception of Agrobacterium radiobacter) produce gall hypertrophies on stems and roots of various dicotyledonous plants. Bacteria of the genera Rhizobium and Bradyrhizobium were originally classified on the basis of host nodulation ability. Criticism to this host-specificity-based taxonomy arose early (Wilson, 1944). Its major limitations have been pinpointed frequently. In particular, these are the existence of cross infection, the low representativity of host plants involved in the classification (around 1% of the described nodulated leguminous species), and, more recently, the discovery in Rhizobium species of plasmids carrying symbiotic determinants.

Results from numerical taxonomy (Graham, 1964; Moffett and Colwell, 1968), DNA hybridization (Gibbins and Gregory, 1972; Jarvis et al., 1980; Hollis et al., 1981), and protein patterns (Roberts et al., 1980) of bacteria of the genus Rhizobium indicate that different clusters may be defined on the basis of chromosomal characters. The approach of bacterial population genetics has allowed the definition of bacterial clusters based on chromosomal characters and to investigate the level of chromosomal recombination occurring in natural populations (Selander, 1985; Piñero et al., 1988). This approach could be successfully used to understand the fate of strains introduced to the soil.

A critical problem is to define the boundaries of specific plasmids, in particular the symbiotic plasmids, in regard to chromosomal clusters. This problem may be approached by comparing dendrograms based on chromosomal characters with those based on plasmidic characters.

The mismatch of the two types of dendrograms would be an indication of the exchange of plasmids among certain chromosomal clusters, while the dendrograms superposition will indicate the confinement of plasmids. In our current studies dendrograms are being established for both the chromosomes and symbiotic plasmids. These dendrograms have been made by using electrophoretic mobility of enzymes for the chromosome and restriction fragment length polymorphism for plasmids. Preliminary experiments indicate that these methodologies are sensitive enough to detect differences in very closely related bacteria. For a particular type of Rhizobium phaseoli we have found evidence for plasmid exchange. These studies will be extended to cover different soil bacteria, both symbiotic and pathogenic, with the aim of defining the genospecies clusters.

8.7 CONCLUDING REMARKS

At the present time, there are studies indicating that plasmid transfer among strains may occur in nature (Schofield et al., 1987; Broughton et al., 1987; Richaume et al., 1989). However, a linkage between plasmids and chromosomes has been observed (Young and Wexler, 1988), indicating that there may be an adaptation of plasmids and chromosomes and that the boundaries for plasmid spread may be somehow restricted. More experiments will be needed to define new guidelines for the safe and successful introduction of strains in agricultural fields. These guidelines should be established based on the knowledge of the ecology of bacterial populations in soil and of the rules operating in ecological time in regard to the stability and interchange of genetic information.

ACKNOWLEDGEMENTS

We acknowledge Lorenzo Segovia for reviewing the manuscript. This work was supported in part by the US National Academy of Sciences/National Research Council by means of a grant from the US Agency for International Development, by a grant C II*-0104-MEX(A) from La Communauté Économique Européenne and by grants from the Consejo Nacional de Ciencia y Tecnologia (México).

REFERENCES

Better, M., Lewis, B., Corbin, D.,Ditta, G. and Helinski, D. (1983) Structural relationships among Rhizobium meliloti symbiotic promoters. Cell, 35, 479-85.

Brom, S., Martinez, E., Dávila, G. and Palacios, R. (1988) Narrow and broad host range symbiotic plasmids of Rhizobium spp. strains that nodulate Phaseolus vulgaris. Apl. Environ. Microbil. 54, 1280-3.

Christensen, A.H. and Schubert, K.R. (1983) Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a rhizobium leguminosarum plasmid. J. Bacteriol. 156, 592-9.

Djordjevic, M.A., Zurkowski, W. and Rolfe, B.G. (1982) Plasmids and stability of symbiotic properties of Rhizobium trifolii. J. Bacteriol. 151, 560-8.

Downie, J.A., Knight, C.D., Johnston, A.W.B. and Rossen, L. (1985) Identification of genes and gene products involved in the nodulation of peas by Rhizobium leguminosarum. Mol. Gen. Genet. 198, 255-62.

Flores, M., González, V., Pardo, M.A. Leija, A., Martinez, E., Romero, D., Piñero, D., Dávila, G. and Palacios, R. (1988) Genomic instability in Rhizobium phaseoli. J. Bacteriol. 170, 1191-6.

Gottfert, M., Horvath, B., Kondorosi, E., Putnoky, P., Rodriguez-Ouifiones, F. and Kondorosi, A. (1986) At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti. J. Mol. Biol. 191, 411-20.

Hennecke, H. (1981) Recombinant plasmids carrying nitrogen fixation genes from Rhizobium japonicum. Nature (Lond.) 291, 354-5.

Hombrecher, G., Brewin, N.J. and Johnston, A.W.B. (1981) Linkage of genes for nitrogenase and nodulation ability on plasmids in R. leguminosarum and R. phaseoli. Mol. Gen. Genet. 182, 133-6.

Jarvis, B.D.W., Dick, A.G. and Greenwood, R.M. (1980) Deoxyribonucleic acid homology among strains of Rhizobium trifolii and related species. Int. J. Syst. Bacteriol. 30, 42-52.

Johnston, A.W.B., Beynon, J.L., Buchanan-Wollaston, A.V., Setchell, S.M., Hirsch, P.R. and Beringer, J.E. (1978) High frequency transfer of nodulating ability between strains and species of Rhizobium. Nature (Lond.) 276, 634-6.

Kaluza, K., Hahn, M. and Hennecke, H. (1985) Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome. J. Bacteriol. 162, 535-42.

Kondorosi, A., Kondorosi, E., Pankhurst, C.E., Broughton, W.J. and Banfalvi, Z. (1982) Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium. Mot. Gen. Genet. 188, 433-9.

Lamb, J.W. and Hennecke, H. (1986) In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nif A and Fix A. Mol. Gen. Genet. 202, 512-17.

Ma, Q.S., Johnston, A.W.B., Hombreeher, G. and Downie, J.A. (1982) Molecular genetics of mutants of Rhizobium leguminosarum which fail to fix nitrogen. Mot. Gen. Genet. 187, 166-71.

Martinez, E., Palacios, R. and Sánchez, F. (1987) Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids. J. Bacteriol. 169, 2828-34.

Morrison, N.A., Hau, C.Y., Trinick, J.M., Shine, J. and Rolfe, B.G. (1983) Heat curing of a sym plasmid in a fast-growing Rhizobium sp. that is able to nodulate legumes and the non-legume Parasponia sp. J. Bacteriol. 153, 527-31.

Piñero, D., Martinez, E. and Selander, R.K. (1988) Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli. Appl. Environ. Microbiol., 54, 2825-32.

Prakash, R.K., Schilperoort, R.A. and Nuti, M.P. (1981) Large plasmids of fastgrowing Rhizobia:homology studies and location of structural nitrogen fixation (nif) genes. J. Bacteriol. 145, 1129-36.

Quinto, C., de la Vega, H., Flores, M., Leemans, J., Cevallos, M.A., Pardo, M.A., Azpiroz, R., Girard, M.L., Calva, E. and Palacios, R. (1985) Nitrogenase reductase: a functional multigene family in Rhizobium phaseoli. Proc. Natl Acad. Sci. USA, 82, 1170-4.

Reanney, D.C. (1978) Coupled evolution: adaptive interactions among the genomes of plasmids, viruses and cells. Int. Rev. Cytol. Suppl. 8, 1-68.

Roberts, G., Leps, W.T., Silver, L.E. and Brill, W.J. (1980) Use of two dimensional polyacrylamide gel electrophoresis to identify and classify Rhizobium strains. Appl. Environ. Microbiol. 39, 414-22.

Rostas, K., Kondorosi, E., Horvath, B., Simoncsits, A. and Kondorosi, A. (1986) Conservation of extended promoter regions of nodulation genes in Rhizobium. Proc. Natl Acad. Sci. USA 83, 1757-61.

Ruvkun, G.B., Long, S.R., Meade, H.M., van den Bos, R.C. and Ausubel, F.M. (1982) ISRm1: a Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes. J. Mol. Appl. Genet. 1, 405-18.

Sapienza, C. and Doolittle, W.F. (1982) Unusual physical organization of the Halobacterium genome. Nature (Lond.) 295, 384-9.

Schofield, P.R., Djordjevic, M.A., Rolfe, B.G., Shine, J. and Watson, J.M. (1983) Molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii. Mol. Gen. Genet. 192, 459-65.

Scott, K.F., Rolfe, B.G. and Shine, J. (1983) Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene. DNA 2, 149-55.

Soberón-Chávez, G., Nájera, R., Olivera, H. and Segovia, L. (1986) Genetic rearrangements of a Rhizobium phaseoli symbiotic plasmid. J. Bacteriol. 167, 487-91.

van Brussel, A.A.N., Tak, T., Wetselaar, A., Pees, E. and Wijffelman, C.A. (1982) Small leguminosae as test plants for nodulation of Rhizobium leguminosarum and other rhizobia and Agrobacteria harboring a leguminosarum sym plasmid. Plant Sci. Lett. 27, 317-25.

Wang, C.L., Beringer, J.E. and Hirsch, P.R. (1986) Host plant effect of hybrids of Rhizobium leguminosarum biovars viceae and trifolii. J. Gen. Microbiol. 13, 2063-70.

Young, J.P.W. and Wexler, M. (1988) Sym plasmid and chromosomal genotypes are correlated in field populations of Rhizobium leguminosarum. J. Gen. Microbiol. 134, 2731-9.

Zurkowski, W. (1982) Molecular mechanism for loss of nodulation properties of Rhizobium trifolii. J. Bacteriol. 150, 999-1007.