In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons

Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii.

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen-fixing nodules by Rhizobium leguminosarum

A system which allows direct selection for curing of plasmids in Gram-negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRleVF39a, carries genes for the production of a melanin-like pigment as has been previously reported. Plasmid pRleVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix- nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRleVF39c contained few, if any, bacteria. Analysis of washed cells by SDS-PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum by. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

紫云英根瘤菌质粒功能研究

紫云英根瘤菌CH203含有3条质粒(pRHa,97MI);pRHb,168MD;pRHc,251MD为共生质粒),用带蔗糖敏感基因Tn5-sacB进行菌株质粒消除和质粒缺失突变株筛选,获得一系列突变株。与野生型菌相比,质粒pRHa的丢失导致菌株结无效根瘤,质粒pRHb的丢失使菌株失去共生能力,在TY培养基平板上菌落变得粗糙,失去了脂多糖(LPSI)。质粒pRHc(共生质粒)的丢失显然失去其菌株的共生能力,同时使菌株抗酸性明显减弱。质粒回复能恢复突变株的表现特征和共生能力。此外,紫云英根瘤菌CH205含有5条大小不同的质粒(分子量42MD～230MD),该菌株某些质粒的消除能显著增强菌株的结瘤固氮能力。研究结果也表明除共生质粒外,紫云英根瘤菌其它质粒明显影响菌株的共生效应。     

Characteristics of Plasmids in Rhizobium huakuii

Rhizobium huakuii nodulates Astragalus sinicus, an important green manuring crop in Southern China, which can be used as forage. The plasmid profiles of 154 R. huakuii strains were examined with the Eckhardt procedure. The plasmid number of the strains varied from one to five, and their molecular weights were estimated from 42 to 600 mDa or more. The plasmids were hybridized with probes nodABC and nifHDK. The results showed that there was one plasmid carrying the nod and nif genes in the strains that harbor two or more plasmids, and the molecular weights of the symbiotic plasmids varied from 117 to 251 mDa. Homology was not observed on plasmids in the strains having only one plasmid; presumably the symbiotic genes are on the chromosome. Plasmid curing was carried out with the Bacillus subtilus sacB to generate derivatives of Rhizobium huakuii strain CH203, which harbors three plasmids, pRHa(97MD), pRHb(168MD), and pRHc(251MD). The largest plasmid (pRHc) carried both nodulation and nitrogen fixation genes. When pRHc was cured, the strain lost its symbiotic ability. The other two plasmids were also related to symbiosis. The derivative cured of pRHb did not nodulate on the host plant, had an altered lipopolysaccharide, and grew much more slowly than the parent strain. Curing of the smallest plasmid (pRHa) resulted in delaying the strain nodulation and made it lose nitrogen fixation ability. Curing of each plasmid in strain CH203 reduced its acid tolerance. Complementation of plasmid-cured strains with appropriate plasmids restored their original phenotypes. Received: 18 December 1996 / Accepted: 28 March 1997     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

The function of three indigenous plasmids in Mtesorhizobium huakuii 2020 and its symbiotic interaction with Sym pJB5JI of Rhizobium leguminosarum

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.     

Formation of Rhizobium phaseoli symbiotic plasmids by genetic recombination

We report here the formation of symbiotic plasmids (pSyms), by genetic recombination between rearranged pSyms, which lack symbiotic information, and resistance plasmids carrying parts of different symbiotic plasmids (R's). This recombination was found to occur both between plasmids derived from different Rhizobium phaseoli isolates, and between plasmids derived from strains obtained from the same original isolate. We also present evidence on the formation of a functional symbiotic plasmid by recombination of an R', carrying nif and nod genes from strain CFN42, and an indigenous plasmid present in this strain (pCFN42e), which was thought to be unrelated to its symbiotic plasmid (pCFN42d). These data are discussed with respect to the stability and transfer of Rhizobium symbiotic information.     

Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti.

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid. The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R. trifolii mutants. In addition, lucerne nodulation was restored to a Hac- R. meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain. The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.     

Relationship of the Presence and Copy Number of Plasmids to Exopolysaccharide Production and Symbiotic Effectiveness in Rhizobium fredii USDA 206

Rhizobium fredii USDA 206 harbors four large plasmids, one of which carries nodulation and nitrogen fixation genes. Previously isolated groups of plasmid-cured derivatives of strain USDA 206 were compared with each other to determine possible plasmid functions. Mutant strain 206CANS was isolated as a nonmucoid (Muc⁻) derivative of strain 206CA, a mutant that was cured of two plasmids. The Muc⁻ phenotype of 206CANS was only expressed when the strain was grown on certain media, particularly those with polyols as carbon sources. Plasmid pRj206b of strain 206CANS was previously shown to have a higher copy number than the same plasmid in strains USDA 206 and 206CA. When this plasmid was transferred to Muc⁺ strains, it conferred a nonmucoid phenotype on recipient strains. The symbiotic effectiveness of the wild-type and cured strains was compared. Overall, few differences were shown, but strains 206CA and 206CANS were found to have higher nitrogenase activities than the other strains. Thus, there appeared to be a possible relationship among exopolysaccharide synthesis, plasmid copy number, and symbiotic effectiveness.     

Rhizobium phaseoli: A molecular genetics view

We have used molecular genetics techniques to analyze the structural and functional organization of genetic information ofRhizobium phaseoli, the symbiont of the common bean plantPhaseolus vulgaris. As in otherRhizobium species, the genome consists of the chromosome and plasmids of high molecular weight. Symbiotic determinants, nitrogen fixation genes as well as nodulation genes, are localized on a single replicon, the symbiotic (sym) plasmid. Thesym plasmid of differentR. phaseoli strains was transferred to anAgrobacterium tumefaciens strain cured of its native plasmids. In all cases, Agrobacterium transconjugants able to nodulate bean plants were obtained. Some of the transconjugants had the capacity to elicit an effective symbiosis. The genome ofR. phaseoli is complex, containing a large amount of reiterated DNA sequences. In mostR. pahseoli strains one of such reiterated DNA families corresponds to the nitrogenase structural genes (nif genes). A functional analysis of these genes suggested that the presence of reiteratednif genesis is related to the capacity of fixing atmospheric nitrogen in the symbiotic state. The presence of several repeated sequences in the genome might provide sites for recombination, resulting in genomic rearrangements. By analyzing direct descendants of a single cell in the laboratory, evidence of frequent genomic rearrangements inR. phaseoli was found. We propose that genomic rearrangements constitute the molecular basis of the frequent variability and loss of symbiotic properties in different Rhizobium strains.     

Diversity of Plasmid Profiles and Conservation of Symbiotic Nitrogen Fixation Genes in Newly Isolated Rhizobium Strains Nodulating Sulla (Hedysarum coronarium L.)

Forty-five Rhizobium strains nodulating sulla (Hedysarum coronarium L.), isolated from plants grown in different sites in Menorca Island and southern Spain, were examined for plasmid content and the location and organization of nif (nitrogen fixation) and nod (nodulation) sequences. A great diversity in both number and size of the plasmids was observed in this native population of strains, which could be distributed among 19 different groups according to their plasmid profiles. No correlation was found between plasmid profile and geographical origin of the strains. In each strain a single plasmid ranging from 187 to 349 megadaltons hybridized to Rhizobium meliloti nifHD and nodD DNA, and in three strains the spontaneous loss of this plasmid resulted in the loss of the nodulation capacity. In addition to the symbiotic plasmid, 18 different cryptic plasmids were identified. A characteristic cryptic plasmid of >1,000 megadaltons was present in all strains. Total DNA hybridization experiments, with nifHD and portions of nodC and nodD genes (coding for common nodulation functions) from R. meliloti as probes, demonstrated that both the sequence and organization of nif and common nod genes were highly conserved within rhizobia nodulating sulla. Evidence for reiteration of nodD sequences and for linkage of nodC to at least one copy of nodD was obtained for all the strains examined. From these results we conclude that Rhizobium strains nodulating sulla are a homogeneous group of symbiotic bacteria that are closely related to the classical fast-growing group of rhizobia.     

Requirement of succinate dehydrogenase activity for symbiotic bacteroid differentiation of Rhizobium meliloti in alfalfa nodules.

Transmission electron microscopy was used to study the cellular morphologies of a wild-type Rhizobium meliloti strain (L5-30), a nitrogen fixation-ineffective (Fix-) succinate dehydrogenase mutant (Sdh-) strain, and a Fix+ Sdh+ revertant strain within alfalfa nodules and after free-living growth in a minimal medium containing 27 mM mannitol plus 20 mM succinate. The results showed a requirement of succinate dehydrogenase activity for symbiotic differentiation and maintenance of R. meliloti bacteroids within alfalfa nodules and for succinate-induced cellular pleomorphism in free-living cultures. Also, the Sdh- strain had a 3.5-fold lower rate of oxygen consumption in the defined medium than did the wild type.     

Functional relationships between plasmids and their significance for metabolism and symbiotic performance of Rhizobium leguminosarum bv. trifolii

Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) is a soil bacterium establishing a highly specific symbiotic relationship with clover, which is based on the exchange of molecular signals between the host plant and the microsymbiont. The RtTA1 genome is large and multipartite, composed of a chromosome and four plasmids, which comprise approximately 65 % and 35 % of the total genome, respectively. Extrachromosomal replicons were previously shown to confer significant metabolic versatility to bacteria, which is important for their adaptation in the soil and nodulation competitiveness. To investigate the contribution of individual RtTA1 plasmids to the overall cell phenotype, metabolic properties and symbiotic performance, a transposon-based elimination strategy was employed. RtTA1 derivatives cured of pRleTA1b or pRleTA1d and deleted in pRleTA1a were obtained. In contrast to the in silico predictions of pRleTA1b and pRleTA1d, which were described as chromid-like replicons, both appeared to be completely curable. On the other hand, for pRleTA1a (symbiotic plasmid) and pRleTA1c, which were proposed to be unessential for RtTA1 viability, it was not possible to eliminate them at all (pRleTA1c) or entirely (pRleTA1a). Analyses of the phenotypic traits of the RtTA1 derivatives obtained revealed the functional significance of individual plasmids and their indispensability for growth, certain metabolic pathways, production of surface polysaccharides, autoaggregation, biofilm formation, motility and symbiotic performance. Moreover, the results allow us to suggest broad functional cooperation among the plasmids in shaping the phenotypic properties and symbiotic capabilities of rhizobia.     

Mini transposon vector mediated foreign gene expression in Mesorhizobium huakuii subsp. rengei

Among the transposable elements, mini-Tn5 transposon vector has proven to be of greater utility for insertion mutagenesis of variety of Gram negative bacteria. The mini-Tn5 vector containing promoter less egfp gene and gentamycin resistant gene was used for the present study. The transposon vector was introduced to M. huakuii from E. coli S17 by conjugation. The conjugants were screened for stable expression of egfp both in free-living and in nodules of Astragalus sinicus. The result showed that the conjugant #3 showed stable expression of green fluorescent both in free-living and bacteroid stage. The visualization of sym plasmid of wild strain and conjugants showed that conjugant #3 had a fragmentation of large sized plasmid into two but without affecting the nodulating ability. These results clearly indicated that mini-Tn5 vectors (Transposon vectors) the best alternate tools for plasmid vectors for integration of foreign genes in chromosomal DNA or symbiotic plasmid and expression, both in free-living and bacteroid stage of Rhizobium.     

豌豆根瘤菌共生基因pJB5JI与华癸中生根瘤菌7653R共生质粒的相互关系

华癸中生根瘤菌(Mesorhizobium huakuii)7653R是分离自我国南方水稻田的一株根瘤菌,含有2个内源质粒:p7653Ra和p7653Rb,其中7653Rb是共生质粒.通过Tn5-sacB的插入方法来消除质粒,获得7653Rb消除的突变株7653RD.将豌豆根瘤菌T83K3的共生质粒pJB5JI导入7653R和7653RD中,盆栽结果表明含有pJB5JI的转移接合子7653R-197的竞争结瘤能力和共生固氮能力均高于7653R.pJB5JI不能恢复7653RD在紫云英上的结瘤能力.含有pJB5JI的7653RD可以在豌豆上结无效瘤,表明pJB5JI可以在7653R的染色体背景下表达其功能.对转移接合子中的质粒稳定性进行检测,结果表明pJB5JI在人工传代的情况下可以稳定存在,但经过共生之后发生了遗传分离,对转移接合子和出发菌株及分离菌株进行kan基因的PCR扩增,除了受体菌外其他菌株都可得到PCR产物,由此推测,pJB5JI可能部分或全部整合到了受体菌的染色体基因组中.     

Attenuation of Symbiotic Effectiveness by Rhizobium meliloti SAF22 Related to the Presence of a Cryptic Plasmid

Several wild-type strains of Rhizobium meliloti isolated from alfalfa nodules exhibited different plasmid profiles, yet did not differ in growth rate in yeast-mannitol medium, utilization of 43 different carbon sources, intrinsic resistance to 14 antibiotics, or detection of 16 enzyme activities. In contrast, three measures of effectiveness in symbiotic nitrogen fixation with alfalfa (shoot length, dry weight, and nitrogen content) indicated that R. meliloti SAF22, whose plasmid profile differs from those of the other strains tested, is significantly less effective than other wild-type strains in symbiotic nitrogen fixation. Light microscopy of nodules infected with strain SAF22 showed an abnormal center of nitrogen fixation zone III, with bacteria occupying a smaller portion of the infected host cells and vacuoles occupying a significantly larger portion of adjacent uninfected host cells. In contrast, the effective nodules infected with other wild types or plasmid pRmSAF22c-cured segregants of SAF22 did not display this cytological abnormality.     

Utilization of Carbon Substrates, Electrophoretic Enzyme Patterns, and Symbiotic Performance of Plasmid-Cured Clover Rhizobia

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.