Rhizobium plasmids in bacteria-legume interactions

World Journal o( Microbiology & Blotechnology 12, 119-125

Rhizobium plasmids in bacteria-legume interactions

A. García-de los Santos, S. Brom and D. Romero".

The functional analysis of plasmids in Rhizobium strains has concentrated mainly on the symbiotic plasmid (pSym). However, genetic information relevant to both symbiotic and saprophytic Rhizobium life cycles, localized on other 'cryptic' replicons, has also been reported. Information is reviewed which concerns functionaI features encoded in plasmids other than the pSym: biosynthesis of ceH surface polysaccharides, metabolic processes, the utilization of plant exudates, aromatic compounds and diverse sugars, and features involved symbiotic performance. In addition, factors which affect plasmid evolution through their influence on structural features of the plasmids, such as conjugative transfer and genomic rearrangements, is discussed. Based on the overall data, we propose that together the plasmids and the chromosome constitute a fully integrated genomic complex, entailing structural features as weIl as saprophytic and cellular functions.

Keywords: free-living metabolism, genomic organization, plasmid, Rhizobium, symbiosis,

An Overview of the Components in the Rltizobíum Genome

Rhizobium spp. are well known among Gram-negative bacte­ria because of the huge amount of genebc information carried in an extrachromosomal pool. Large circular plas­mids ranging in size from 150 to 1500 kb, are common components of the Rhizobium genome. In fad, the megaplas­mids oE R. melilofi (1200 to 1500 kb) are among the !argest known bacterial plasmids (Campbell 1993). The number of different plasmids present in Rhizobium is very variable both intra- and inter-species, ranging from two to ten. Calculations based on assumed sizes for the chromosome, indicate that these plasmids may represent between 25 and 50% of the total genome (Martínez el al. 1990). In most Rhizobium species many genes involved in nodulation and nitrogen fixation are encoded on a single large plasmid, the symbiotic plasmid or pSym.

Plasmids have long been considered (by definition) as unessential components of bacterial geno mes, not required for the survival and replication of individual bacteria. Their recognized function is to confer additional, albeit dispensa-

The authors are wilh the Depto. de Genética Molecular. Centro de Investigación sobre Fijación de Nitrógeno. UNAM. A. Poslal 565-A, Cuernavaca, Morelos, México, fax: (73) 175581. 'Corresponding aulhor.

ble traits that enhance the ability of bacterial populations to colonize and compete in natural communities. The pSyms conform to this definition by conferring the ability to colonize a particular niche, the legume root nodule. The basic and applied implications of nodulabon and N 2 fixabon have motivated intense research into the gene tic basis of these phenomena which has been concentrated almost invariably on the pSyms (see Fischer 1994 and Schultze et al. 1994 for recent reviews).

pSyms however, represent a comparatively small part of the extrachromosomal pool. The remaining plasmids have been considered until recently, as 'cryptic' pJasmids. Are they truly crypbc? Is it possible for a bacterial cell to harbor such a sizabJe amount of DNA without any detrimen­tal effed? Are these plasmids dispensable components or do they carry essentiaJ functions? What role do these plasmids play in bacteria-legume interactions? In this article, we review the relevant informabon pertaining to these issues, which has appeared during the past few years.

Genetic Strategies Employed in Plasmid Analysis

In what way do these plasmid structures affect the cellular functions? Different experimental approaches have sought to answer this question. A widely used technique has been

A. Carcía-de los Sanfos, S. Brom and D. Romero

employed for the dired visualizabon of large plasmids (Eckhardt 1978). A gross analysis of plasmid-encoded func­bons involves the isolation of derivatives which are cured of the plasmids. These have been obtained either through heat-curing procedures (see Martínez ef al. 1990 for a review) or by employing new procedures that allow the positive se/ection for loss of fundion (Hynes el al. 1989; Flores ef al. 1993). These methods have permitted the systematic analysis of whole plasmid sets in R. efli, and in R. legumirlOsarum bvs. viceae and irifolii (Hynes & McGre­gor 1990; Baldani ef al. 1992; Brom el al. 1992; Stepkowski ef al. 1993). Although the megaplasmids of R. rneliloií have been recalcitrant to cure, their analysis has been carried out by elegant genetic techniques that have lead to the direded generabon of large deletions (Charles & Finan 1991).

Another approach widely used for the comprehensive analysis of plasmid fundions is the consrrudion of deriva­tives which acquire whole plasmids through gene tic transfer­ence (see Martínez el al. 1990 for a review). The generabon of complete physical maps of rhizobial plasmids (Prakash el

al. 1981; Girard el al. 1991; Perret ef al. 1991; Honeycutt ef al. 1993), are very useful assers to the study of plasmids, including the determination of transcriptional patterns of plasmid regions (David el al. 1987; Fellay el al. 1995; L.

Girard, unpublished work). The study of plasmid stability (Mercado-Blanco & Olivares 1993; Mercado-Blanco &

Olivares 1994), dynamics (Brom el al. 1991; Romero ei al. 1991; Flores el al. 1993; Romero el al. 1995b), and transmissi­bility (see below), are other useful aids for their functional and evolutionary scrutiny.

Besides the research direded to the analysis of whole plasmids, other studies have been aimed at specific genes, employing the whole array of genetic methodologies devel­oped for Rhizobium (reviewed in Martínez et al. 1990). New strategies for the isolabon of specific DNA sequences (Bjourson el al. 1992) and for the integrabon of genetic information (Uchimi el al. 1993; Uchimi el al. 1995) are continually devised.

The charaeterization of plasmids themselves, by their presence, size and restrietion fragment length polymor­phisms has been used for taxonomic purposes (Schofield ef al. 1987; Young & Wexler 1988; Demezas et al. 1991; Laguerre el al. 1992).

Although research in Rhizobium plasmids has been fo­cused mainly on the symbiobc plasmids, the parbcipation of other replicons in symbiotic and other celJular functions has been evidenced.

Deciphering the Genetic Functions Encoded in Rhízobium 'Cryptic' Plasmids

Biosyrllhesis of Cell Surface Polysaccharides The functions of exopolysaccharides (EPS) in the free­living state have been associated with nutrient gathering,

12O World Jou",al 01 MlaoblOlo8.Y & 8IOfec!m%g'y, Vol 12, 1996

attachment and stress-protedion, while in symbiosis they are required (in hosts which form indeterminate nodules) for successful nodule invasion and development (Leigh &

Walker 1994), Strains of Rhizobiurn rneliloli carry a 24 kb cluster of EPS biosynthetic genes on a megaplasmid distinct from the pSym (Leigh & Walker 1994). Regulation of EPS biosynthesis is apparently controlled by plasmids in other Rhizobiurn species. For instance, the psi gene in the pSym of R.Iegurninosarum by. phaseoli inhibits EPS synthesis (Borthakur & Johnston 1987); a sequence with a similar fundion in S, fredii appears to be located in a plasmid other than the pSI/m (Barbour & Elkan 1990).

Lipopolysaccharide (LPS) molecules are essential compo­nents of the baderial outer membrane. In R. leguminosarum by. viciae (Hynes & McGregor 1990), R. legurninosarum by. trifolii (Baldani et al. 1992), and R. efli (Brom el al. 1992), genes involved in LPS biosynthesis have been localized on non-symbiotic plasmids. Mutations in plasmid-borne LPS genes cause strong alterations on the cell surface, affeding not only free-living features such as morphology, moblity and aggregation, but also effedive nodulation in hosts which form determinate noduJes. Functional complementa­bon (Hynes & McGregor 1990; Noel 1992; A Garcia de los Santos, S, Brom & D, Romero unpublished work) and structural homology studies (unpublished work) strongly suggest that plasmid-borne LPS genes may be highJy con­served between R, legumirlOsarum and R. efli strains. Nota­bly, R. me1iloli Rm41 also carries a gene involved in LPS biosynthesis, IpsZ on the eps megaplasmid; this gene is absent in other R. melilofi strains, such as RmSU47 (Williams el al. 1990).

These findings cast some doubt about the commonly­heId view that plasmids encode determinants of episodic, dispensable nature. The profound effects of EPS or LPS loss on cell physiology clearly illustrate their necessity for bacterial survival.

Sequer¡ces Re1aled fo Metabo/ic Processes Rhizobium plasmids may carry genomic sequences required for central metabolic pathways. R. tropici harbours duplica te genes for citrate synthase, one on the chromosome and the other on the pSym. Mutations in the plasmid-borne copy provoke a decreased enzyme aetivity, at least in sorne growth conditions and also lead to diminished nodulation (Pardo el al. 1994), The R. melilofi eps megaplasmid al50 contains C.-dicarboxylate transport genes, which have a pleiotropic effect on the utilization of many TeA cycle intermediates as growth substrates (Watson el al. 1988). Two loei involved in thiamine biosynthesis have also been ascribed to the same megaplasmid (Finan el al, 1986). Superoxide dismutase enzyme adivity is plasmid-condi­tioned in R. leguminosarum by. frífolii, and in this organism there are two eledromorphs of both hexokinase and car­bamate kinase; one eledromorph of each enzyme is lost

upon plasmid curing. Enzymes required for the use of nitrate as nitrogen source also depend on the presence of plasmids (Baldani el al. 1992). Comparison of growth rates among parental and plasmid-cured derivatives indicates the existence of other undefined metabolic traits ín R. legurni­rlOsarlArn by. viciae (Hynes & McGregor 1990) and R. etli (Brom et al. 1992). Derivatives cured of sorne plasmids of R. leglAminosarum by. Irifolí; (Baldani el al. 1992) and R. etli (Brom et al. 1992) could not be obtained. In R.meliloli, approximately 200 kb of the eps megaplasmid were recalci­trant to delebon (Charles & Finan 1991). These data suggest that these plasmids may carry genes whose fundions are essenbal for cellular viability.

Wili:z.afion of Plarl{ Mefabolites Plant root exuda tes provide a rich source of nutrients for mícroorganisms. Genes required for the catabolism of sorne of these compounds have been localized on Rhi:z.o­bilAnl plasmids. The mos and moc genes, involved in the synthesis and catabolism of rhizopíne, an opine-like com­pound ín alfalfa, have been shown to be closely hnked on the pSym of a R. rneliloti strain. Moreover, the expres­sion of the mos genes is regulated through the NifAI NtrA system, whích controls the expression of N, fixabon genes (Saint et al. 199.3). In another R. rnelilofi strain, genes involved in the catabolism of trigonelline, stachydrine and carnibne, a group of betaines produced by Medicago sativa , are located on the pSym (Goldmann el al. 1991). Trigonell­íne utilization has been shown to be associated with a non-Sym plasmid in R. efli (unpublished work). Calystegin catabolic genes were found in one R.meliloti strain out of 42 tested rhizosphere bacteria. The genes were located on a non-Sym self-hansmissible plasmid (Tepfer ef al. 1988).

H is possible that Rhi:z.obium strains containing this kind of gene may have a se!ective growth advantage in the rhizosphere microenvironment. Thus, these genes corre­spond to the class more commonly found on plasmids: genes for episodic use, in this case, for baderia-plant interactions.

Ufili:z.afion of Aromafic Compounds and Diverse Sugars The soil environment contains many aromabc compounds originating from root exudates and plant degradabon. These compounds play multiple roles in Rhi:z.obiurn-legume interactions as inducers of nodule development (Schultze et al. 1994), as chemoattractants (Parke et al. 1985), and as growth substrates for R?¡j:z.obium (Parke & Ornston 1984; Hartwig et al. 1991). In R. melilofi, delebons of the exo megaplasmíd impair the abílity to use the aromabcs proto­catechuate and quinate as sole carbon sources (Charles &

Finan 1991). The ability to utilize the aromabc compound catechol is encoded in the pSym of a Rhi:z.obium legurnino­sarurn by. {rifolíi strain (Baldani et al. 1992).

Rhizobium plasmids irl bacferia-legume irlteracfions

Utilizabon of diverse sugars appears to depend on plas­mids other than the pSym. The R. meliloti exo megaplasmid has been related to the utilizabon of melibiose, raffinose, acetoacetate and p-hydroxybutyrate (Charles & Finan 1991). Two p-galadosidase activities have been deteded in R.melilofi; one of them is ladose-inducible and has been ascribed to the exo megaplasmid (Charles ef al. 1990). The study of the plasmid-cured derivatives R. legumirlosarum by. trifolú has shown that sequences required for the utilizabon of rhamnose, sorbitol, adonitol, arabinose, glycerol, lactose and malate are distríbuted among díverse plasmids (Baldani el al. 1992).

The avaílability of these additional fundions, contributes to the metabolic diversity of these microorganisms, and probably confers on them an adaptative advantage over other soil bacteria. Their assígnment to plasmíds facihtates their distribution in the populabon and is in agreement with the classical view of plasmids.

Nodulation Competítiverless and Symbiotic Nilrogen Fixation In agricultural fields, native Rhizobium strains usually dominate nodulation over introduced strains. This poses a problem for the increase in legume productivity by inocula­bon with selected highly effective Nz-fixing bacteria. Analy­ses of plasmid-cured derivabves of R. legurninosarurn by. viciae (Hynes 1990), R. etli (Brom ef al. 1992), and R. [redii (Barbour & Elkan 1990), have shown that sequences required to achieve wild type competibve levels are distrib­uted among various non-Sym plasmids. R. elli transconju­gants improved their competibveness for nodule occupancy upon the introduction of aa R. tropici plasmid (Martínez­Romero & Rosenblueth 1990). A DNA region related to nodulation efficiency and compebbvity, tenned n/e (nodule formatíon efficiency), has been assigned to a plasmid of R. meliloEi (Soto et al. 1994).

The presence of detenninants involved in N l fixation, has also been shown in R. leguminosarum by. viciae (Hynes &

McGregor 1990), R. efli (unpublished work) and R. melilofi (Rastogí et al. 1992) non-Sym plasmids.

Whereas the aboye mentioned studies indicate a positive involvement of plasmid sequences in symbiosis, other reports show that sorne plasmids may have a negative effed on symbiol:ic effedivity. Loss of a 'crypbc' plasmid in strains of R. loti enhances theír N, fixation and nodulation compebtiveness (Pankhurst et al. 1986). Deletions in a 'cryptic' R. legumirlOsarum by. viciae plasmid, result in effec­tive nodulation on a previously ineffedive host (Selbitschka & Lotz 1991). Also in a strain of R. meiilofi, an increase in effedive N, fixation was correlated to the loss of a 'cryptic' plasmid (Velázquez et al. 1995). The molecular basis of many of these effeds is still unknown.

The symbiotic capacity of Rhizobium strains depends on the interadíon of a great number of fadors sorne of them being diredly involved in the process while others ad

WDrld ]O"",QI DI MicrDbiolo8Y & lJiotechnolo8J/. Vol l2, 1996 121

A. García-de los Santos, S. Brom and D. Romero

indiredly. Genetic sequences reJated to this array of faetors are distributed among the different plasmids.

Olher Functiorls Chaperanin-encoding genes, such as groEL. have been located both on the chramosome and on megaplasmids of R. melilofi (Rusanganwa & Gupta 1993). Studies ín both Bradyrhizobium japonicum (Fischer, 1994) and R. melilofi (Long et al. 1991) suggest their involvement in symbiosis.

Melanin produetion is a widespread charaderistic among Rhizobi,.rm species (Cubo ef al. 1988). Although ies fundion is unknown, it has been praposed as a defence against toxic plant phenoJs. Melanín biosynehesis is also plasmid-encoded either by the pSym (Cubo et al. 1988) or by 'cryptic' plasmíds (Hynes & McGregor 1990; Mercado-Blanco et al. 1993).

An acid-tolerant phenotype in a strain of R. leguminosa­mm by. Irifohí has also been associated with a plasmid (Chen el al. 1993).

As descríbed in thís seetion many dissimilar funetions, either housekeeping or dispensable, may be plasmid-borne. The locabon of many of these traits was disco ve red during studies on the genetic basis of a particular charaderistic, rather than from more comprehensive studies of the role of plasmid per se. Research along this line will probably reveal new plasmid-linked phenotypes in years to come.

Factors Influencing Plasmid Evolution

Conjugative Transfer Conjuga ti ve transfer of indigenous plasmids has been observed among Rhízobium legumirlOsarum strains under Jabo­ratory conditions (Johnston el al. 1982; Geniaux & Amarger 1993) or under conditions resembling the soil environment (KinkJe & Schmidt 1991; Rao et al. 1994). The transmissible plasmids vary in their transference frequency, their size and in the genetic information they carry; sometimes they correspond to the pSym. In addition to being self-transmissi­ble, some plasmids may induce the mobilization of other replicons (Johnston el al. 1982; Mercado-Blanco & Olivares 1993; unpublished work).

Very few data are available regarding the c1assification of rhizobial plasmid incompatibility groups. The incompat­ibility between aa Rj plasmid of Agrobacferium and aa R. leguminosarum by. Irifolíi pSym has been reported (O'Conell ef al. 1987). We have observed the incompatibility between aa R. etlí plasmid and a Ti plasmid from Agrobacteríum lumefa­GÍens (unpublished work). Other data show incompatibility between some viciae and phaseoli pSyms (Johnston el al. 1982), or between some R.efli and R. legumirlOsaTl-lm by. trifolii plasmids (unpublished work). Not aH pSyms corre­spond to the same incompatibility group, as strains with two pSyms, having the same (Geniaux & Amarger 1993),

122 World Jo"mal oi Mlcroblology & BiOl,c1mology. Vol 12. 1996

or a different (Hooykaas el al. 1981) host-range have been construded.

The analysis of chromosomal and plasmid charaderistics in natural populahons of R. legumínosarum bíovars, such as Irifolií (Schofield ef al. 1987), phaseoli (Geniaux el al., 1993) and viciae (Young & Wexler 1988) and also in R. galegae (Kaijalainen & Lindstrom 1989) supports the occurrence of natural plasmid exchange. A theoretical analysis of some of these data suggests that 10% (R. galegae ) or 15 to 30% (R. legumínosarum bv. ¡¡íciae) of aH genetic types in these populations have been either the source or the recipient of a plasmid transfer event (Valdés & Piñera 1992). In fad, present R. legul11inosarum by. phaseoli strains apparently arase by natural transfer of the pSym of R. elli into a R. leguminosarum genomic background (Segovia el al. 1993). Although most of the evaluations of plasmid transfer refer to the pSym, they may appJy to other plasmids as well. These data c!early reveal the ímportant role of plasmid transfer in the struduring of new gene tic types in Rhizobíum populations.

For a plasmid transfer event to be successful, several barriers must be surrnounted. Leaving aside the barriers imposed by the transfer process itself, an incoming pJasmid must defeat barriers such as restridion-modification systems and incompatibility with resident pJasmids. A frequently overlooked fador is the burden that replication of a new plasmid imposes on the recipient cel!. Experiments in Escheríchia coli show that the introduetion of a new pJasmid reduces the fitness of the host cell. However, grawth of the recipient cell under seledive condítions for as few as 500 generations ameliorates the deleterious effeet of the new plasmid, even under non-selective condüions (Levin 1993). Changes involved in this restoration of fitness affect either the recipient cel! or both the ceH and the incoming plasmid, thus being truly coevolutionary. lt is reasonable to suppose that coevolutionary pracesses similar to these have been important in the struduring of plasmid-chromosome com­plexes in Rhízobium.

In addition to being a helpful tool for the explanation of evolutive changes, plasmid transmissibility for the construc­han of improved strains has been a long coveted but unfulfilJed goal in N 2 fixation research. What are the mechanisms goveming rhizobial plasmid transfer? This prob­lem has not yet been addressed but we can speculate that the mechanisms may be similar to those acting for pTi transfer in Agrobacterium (Piper ef al. 1993) as these species belong to the same family and share certain strudural and functional features.

Genomic Rearrangements As components of a genome that is particularly dynamic (see Romero el al. 1995a), plasmids in Rhizobium are sub­jected to frequent structural reshuffling. High-frequency rearrangements have been observed in the plasmids of

several Rhizobi¡,¡m species (Djordjevic el al. 1982; Zurkowski

1982; Brom el al. 1991; Romero el al. 1991, 1995b;

Selbitschka & Lotz 1991; Rastogi et al. 1992; Flores el

al. 1993). Many different types of genomic rearrangements,

including cointegration, translocation, deletion and amplifica­

tion of specific sequences participate in the frequent genera­

tion of structural diversity (see Romero el al. 1995a). Recombination among reiterated sequences appears to be

responsible for such variation. Although many of the studies

have focused on the plasticity exhibited by pSyms, these rearrangements also occur in non pSym plasmids (Brom el

al. 1991; Selbitschka & Lotz 1991; Flores el al. 1993).

While the biological role of many of these rearrange­ments remains to be established, sorne of them may have

beneficial effects for the host cell. For instance, the deletions

in a R. leguminosarum by. viciae 'cryptic' plasmid discussed aboye, lead to effective nodulation on a previously ineffec­

tive host (Selbitschka & Lotz 1991). [n R. etli, a tandem

amplification in the pSym endows an increase in N l fixation,

at least in sorne genomíc backgrounds (unpublished work).

Recombination events between different pSyms have been

implied in the generabon of a hybrid symbiotic plasmid (Christensen & Schubert 1983). Additional work is required

in order to gain more insight into the forces that have

contributed to the strududng of the plasmid component as we see it today.

Conclusions and Perspectives

The data presented in this review indicate that the Rhiw­

bium genome is organized in differently sized replicons (chromosome plus plasmids) which together constitute a fundional unit.

Baderial plasmids have generally been regarded as mol­

ecules conferring addibonal features to an already complete

genome. This may not apply to RhizobÍi.¡m plasmids which have been shown to carry genes involved in basic metabolic

fundions, the synthesis of cellular components and the ublization of growth nutrients. Many of these genes in­directly affed symbiobc traits but the question remains if

sorne of them (01' others still not described) have a dired

involvement in symbiosis. The presence of indispensable genebc information on plasmids has not been conclusively

demonstrated but is strongly suspeded due to the inability to cure different Rhizobi¡,¡m strains of some plasmids. H is also

notable that these fundions are not concentrated on a

single plasmid. In the cases where a comprehensive analysis

of plasmid fundions has been made (Hynes & McGregor 1990; Baldani et al. 1992; Brom el al. 1992), curing of

almost every plasmid reveals important phenotypes, either

saprophytic, symbiobc 01' both. Furthermore, interplasmid

fundional interactions are suggested by the drastic enhance­

ment of phenotypíc effects in multiple plasmid-cured deriva-

Rhizobium plasmids in bacteria-leg¡,¡me inleracfions

bves (unpublished work). Thus, the functional organizabon

of the Rhizobi¡,¡m genome is c1eady mulbparbte, constituting a fully integrated genomic complex. What features have

converged to result in this genomíc organization? Cenomic

reiterations and insertion sequences are thought to play

principal roles in this process, due to their parbcipation in the generaban of rearrangements. The interchange of

genetic informabon caused by conjugabon also should have

an impad on its final outcome. Other questions which should be addressed in the future concem the implementa­

bon of new strategies to study the biological impad of

such genomic organizabon, the c1arification of the presence of vital genebc information, the definition of genetic trans­

fer mechanisms and the comparison with other baderia in

order to determine if this type of genomic organizabon is exclusive to the rhizobiaceae.

Acknowledgements

We thank Esperanza MarHnez-Romero and Rafael Palacios

for critical review of the manuscripL Work from the author's

laboratory was partially supported by grant IN200693

(DCAPA-UNAM).

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