Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid

Summary Selection was made for the transposition of the kanamycin resistance transposon Tn5 from a location on the chromosome of R. leguminosarum into a transmissible, bacteriocinogenic plasmid that also carries genes required for the induction of nitrogen-fixing nodules on peas.One hundred and sixty independent insertions into transmissible plasmids were isolated. When these plasmids were transferred by conjugation into a non-nodulating strain, which carries a deletion in one of its resident plasmids, of the 160 isolates tested 14 yielded transconjugants that formed nodules that did not fix nitrogen (Fix^-) and in a further 15 cases the transconjugants were unable to form nodules (were Nod^-). When transferred to a symbiotically proficient strain (i.e. Nod⁺ Fix⁺) none of the transconjugants was symbiotically defective; thus the mutations were not dominant.When kan was transduced from the clones that generated Fix^- transconjugants into a Fix⁺ recipient the majority of transductants inherited Fix^- indicating that the insertion of Tn5 had induced the symbiotic mutations. Transduction of kan, from the clones that failed to donate Nod⁺ by conjugation to strain 6015, occurred at barely detectable frequencies and it was not possible to demonstrate transduction of Nod^-. kan was co-transduced with Nod⁺ from some of the clones and some of these transductants also inherited the ability to produce medium bacteriocin and to transfer at high frequency by conjugation. Thus the genes for all these characters are closely linked.     

Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R. leguminosarum nod DNA

In Rhizobium phaseoli strain 8002, a large indigenous plasmid, pRP2JI, had previously been shown to carry many of the genes necessary for the induction of nitrogen-fixing nodules on Phaseolus beans. A cosmid clone library was constructed using DNA from strain 8002. From this library, two overlapping recombinant plasmids (pIJ1097 and pIJ1098) were isolated which spanned about 43 kb of pRP2JI DNA. These plasmids could restore nodulation to some, but not all nodulation-deficient strains of R. phaseoli, indicating that the nodulation genes were not clustered within one small region of pRP2JI. The cloned R. phaseoli nodulation region shared extensive DNA homology with the nodulation genes of R. leguminosarum, and on the basis of DNA hybridization, the nitrogenase genes were found to be within 10 kb of the R. phaseoli nodulation genes. Close to the nodulation genes of R. phaseoli was located a sequence that was repeated on pRP2JI but which was not present elsewhere in the genome of strain 8002.     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

Effect of Inoculation with Various Rhizobia Strains on the Fatty Acid Composition of Peribacteroid and Bacteroid Membranes of the Nodule Symbiosomes

The fatty acid (FA) composition of bacteroid and peribacteroid membranes was studied in the symbiotic pairs differing in their nitrogen-fixing efficiency; the results are compared with the FA composition of plasmalemma and free-living rhizobia. The experiments involved lupine plants inoculated with strains of Bradyrhizobium lupini359a (Nod⁺Fix⁺) and 400 (Nod⁺Fix L) manifesting high and low nitrogen-fixing efficiency, respectively, and broad bean plants inoculated with strains of Rhizobium leguminosarum97 (Nod⁺Fix⁺) and 87 (Nod⁺Fix L) of high and low nitrogen-fixing efficiency, respectively. We showed that the rhizobia of the strains 359a and 97 were able to form nodules with peribacteroid membranes containing FA mainly or exclusively of plant origin. These strains were able to develop effective symbiotic pairs with legume plants. The use of strains 400 and 87 resulted in the formation of nodules with peribacteroid membranes containing typical bacterial (branched-chain) FAs; these strains were characterized by an ineffective symbiosis.     

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod⁺) and is able to invade these nodules (Inv⁺), but fails to fix nitrogen (Fix^–). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod⁺Inv⁺Fix⁺) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod⁺Inv^–Fix^–) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP. Received: 8 October 1996 / Accepted: 11 December 1996     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Selection and symbiotic properties ofRhizobium leguminosarum biovarphaseoli strains harboring pRtr5a

TheRhizobium leguminosarum biovartrifolii symbiotic plasmid pRtr5a has been transferred toR. leguminosarum biovarphaseoli RCR 3644-S1. The transconjugant selection had been done byTrifolium pratense plants. All transconjugants lacked the resident pSym, but had complete pRtr5a, and were Fix⁺ onT. repens andT. alexandrinum, Fix^– onT. subterraneum, and formed a few small white and Fix^– nodules onPhaseolus vulgaris. It is shown that this nodulation onP. vulgaris is due to pRtr5a. The presence of pRtr5a and/or the passage throughTrifolium pratense nodules provoke(s) the recipient strain symbiotic plasmid loss.     

Genetic Analysis of Rhizobium leguminosarum bv. Phaseoli Mutants Defective in Nodulation and Nodulation Suppression

Nodulation-defective rhizobia and their nodule-forming derivatives containing cloned DNA from the wild type were used to study nodulation suppression in Phaseolus vulgaris L. Non-nitrogen-fixing derivatives which formed rhizobia-containing white nodules induced partial suppression. Comparison of this with the complete suppression by Fix⁺ derivatives and a Fix^- mutant which formed rhizobia-containing pink nodules suggests that the extent of suppression may be related to successive stages of nodule development.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

Summary R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod^-) or nitrogen fixation (Fix^-) have been readily obtained. In some Nod^- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. ³²P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod^- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod^- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod^- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.     

Phosphorus uptake by bean nodules

As part of a breeding program to improve the nitrogen-fixing symbiosis between common bean (Phaseolus vulgaris) and Rhizobium etli, we developed a rapid screen for common bean accessions that preferentially nodulate with KIM5s, a high nitrogen fixing strain of R. etli. We constructed a mutant of KIM5s that did not fix nitrogen (Fix^-) but was otherwise indistinguishable from KIM5s. We screened plants for symptoms of nitrogen deficiency when grown in a Honduran soil containing indigenous common bean-nodulating rhizobia (10⁴ per gram) and KM6001, the Fix^- mutant of KIM5s (10⁴/seedling added 7 days after planting). Leaf color was scored on a scale of 1 to 5, in which 1 was dark green and 5 was bright yellow. Of 820 genetically diverse accessions of P. vulgaris screened, 51 were scored 1, 626 were scored 2 or 3, and 143 were scored 4 or 5. Selfed seed was produced from common bean plants of the accessions scored 1, 4 or 5. Twenty-four accessions that scored 1, and 58 that scored 4 or 5 were screened in soil containing indigenous rhizobia and the wild type KIM5s (Fix₊), and nodule occupancy was determined by antibiotic resistance. On the 24 common bean accessions that were scored 1, KIM5s occupied 0-6% of the nodules, on 26 of the accessions that were scored 4 or 5, KIM5s occupied 90%-100% of the nodules, and on the remaining 34 that scored 4 or 5, there was a distribution of nodule occupancy. Foliar color was highly correlated with nodule occupancy (r = 0.786,p = 0.01). The results indicate that the rapid visual screen using the Fix- mutant accurately identified common bean accessions that preferentially nodulate with the wild-type KIM5s (Fix⁺) strain in soil containing indigenous rhizobia. This screen will facilitate introduction of the preferential nodulation trait into superior cultivars and provides the foundation for studies of the genetic basis of preferential nodulation.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Insertion and deletion mutations within the nif region of Rhizobium japonicum

Insertion and deletion mutants were used to characterize a genomic region of Rhizobium japonicum where the nitrogenase structural genes are located on two separate operons nifDK and nifH. In addition to previously described nifD:: Tn5 and nifK:: Tn5 mutations we have now generated, by localized mutagenesis, further Tn5 insertion mutations in the vicinity of nifDK as well as within and adjacent to nifH. The nifD:: Tn5, nifK:: Tn5, and nifH:: Tn5 mutant strains were of the Nod⁺ Fix^- phenotype whereas all other mutants were symbiotically fully effective (Nod⁺ Fix⁺). The nifH:: Tn5 mutation was helpful in the identification of the nifH gene product (the dinitrogenase reductase) by two-dimensional gel electrophoresis: due to its polar effect this insertion specifically abolished the synthesis of that protein under microaerobic culture conditions. The ultrastructure of soybean root nodules infected with either the nif ⁺ wild-type or with the nif ^- (but otherwise isogenic) mutant strains was analyzed by electron microscopy. All contained fully developed bacteroids, but the nitrogen non-fixing mutants showed massive accumulation of PHB.Of Tn5-containing strains, kanamycin sensitive derivatives were obtained which contained deletions. Several classes of deletion mutants were found which, as judged by their physical DNA structure and their phenotypes, allowed the following most important conclusions: (i) deletions lacking both the nifDK and nifH regions indicate linkage between the two operons whereby at least 15 kb of DNA separate them; (ii) one deletion ending upstream from nifH, and lacking only nifDK, indicates that the nifDK operon is located on the 5-flanking side of the nifH operon; (iii) all deletion mutants are Nod⁺ indicating that there are no essential nodulation gnes located between and adjacent to nifDK and nifH.     

Plant symbiotic mutants as a tool to analyse nitrogen nutrition and yield relationship in field-growth peas (Pisum sativum L.)

Pisum sativum L. is known for high seed and protein yields but also for.yield instability. Because legumes utilize two sources of nitrogen (atmospheric N₂ fixed in nodules and assimilation of soil mineral N), studies on their nitrogen nutrition is more complex than in other plants. In this work, pea symbiotic mutants (with no nodules at all ([Nod^-]), with inefficient nodules ([Nod⁺Fix^-]) or showing an hypernodulating and a ‘nitrate-tolerant symbiosis’ character ([Nod⁺⁺Nts]), their semi-leafless isogenic homologues and the parental control line cv Frisson were fertilized with three levels of mineral nitrogen (0, 25 or 50 g N m^-2) to generate a range of mineral nitrogen regimes in the same genetic background. Impact of the source and level of nitrogen nutrition was measured on reproductive development, growth, nitrogen accumulation and seed yield. It was shown that a N deficiency induced flowering termination. It also led to a large decrease in the number of seeds produced and the amount of N accumulated in forage and in seeds, when little effect was observed on the progression rates of reproductive stages along the stem. The single seed weight and the amount of dry matter accumulated in forage neither responded strongly to N deficiency. The source of nitrogen was shown to be of little importance to yield but the application of about 50 g N m^-2 was necessary to reach the yield of the control cv Frisson when exclusive assimilation was ensuring the N requirements of the plant. Despite the fact that the nitrate-tolerant and hypernodulating mutant P64 used in this study did not yield as well as the parent cv Frisson, it is proposed that [Nod⁺⁺Nts] characters could act as a yield regulating factor.     

A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not of beans by R. phaseoli and is corrected by cloned DNA from Rhizobium or the phytopathogen Xanthomonas

Summary A Tn5-induced mutant strain of R. phaseoli which failed to synthesize exopolysaccharide (EPS) was isolated and was shown to induce normal nitrogen-fixing nodules on Phaseolus beans, the host of this Rhizobium species. The corresponding wild-type Rhizobium DNA was cloned in a wide host-range vector and by isolating Tn5 insertions in this cloned DNA, mutations in a gene termed pss (polysaccharide synthesis) were isolated. These were introduced by marker exchange into near-isogenic strains of R. leguminosarum and R. phaseoli which differed only in the identity of their symbiotic plasmids. Whereas the EPS-deficient mutant strain of R. phaseoli induced normal nitrogen-fixing nodules on Phaseolus beans, the same mutation prevented nodulation of peas by a strain of R. leguminosarum which normally nodulates this host. Further, it was found that DNA cloned from the plant pathogen Xanthomonas campestris pathover campestris could correct the defect in EPS synthesis in R. leguminosarum and R. phaseoli and also restored the ability to nodulate peas to the pss::Tn5 mutant strain of R. leguminosarum.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate