Plasmid-determined nodulation and nitrogen-fixation abilities inRhizobium phaseoli

Summary InRhizobium phaseoli strain 8002, the 190 Md plasmid pRP2JI which determines the ability to produce nitrogen-fixing nodules onPhaseolus beans (Nod⁺ Fix⁺) and the production of melanin on L-tyrosine-containing media (Mel⁺), was shown to be transmissible by conjugation to otherRhizobium strains. When pRP2JI was transferred to Nod^- strains ofR. leguminosarum (which normally nodulates peas) the transconjugants gained the ability to nodulatePhaseolus beans and to make melanin.Out of 187 derivatives of strain 8002 carrying pRP2JI plasmids into which the transposon Tn5 had been inserted, six were Fix^- Nod⁺ Mel⁺, one was Fix^- Nod⁺ Mel^- and four were Fix⁺ Nod⁺ Mel^-. Three other derivatives of strain 8002 were Nod^- Mel^-; these had suffered deletions of c 30 Md in pRP2JI. Thus the genes for melanin production and nodulation appear to be closely linked, but melanin production is not necessary for the induction of nitrogen-fixing nodules onPhaseolus beans.     

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.     

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     

Symbiotic mutants of USDA191, a fast-growing Rhizobium that nodulates soybeans

Summary Symbiotic and auxotrophic mutants of Rhizobium japonicum strain USDA191 were isolated using Tn5 mutagenesis and techniques that cause plasmid deletions and plasmid curing. Characterization of several mutants that are unable to nodulate (Nod^-) or unable to fix nitrogen (Fix^_) showed that nod and nif genes are located within one regions of a 200 MD plasmid (pSym191). Blot hybridization analysis of plasmids in other fast-growing R. japonicum strains showed that nod as well as nif sequences are located on plasmids in eight strains but are apparently carried in the chromosome in two strains.     

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     

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.     

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.     

Mobilization ofEscherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob intoEscherichia coli C600

Summary Escherichia coli Rl is an Ag⁺-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb). Tn5-Mob was introduced into theE. coli Rl host replicon via conjugation on membrane filters. The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated withE. coli C600 yielded Ag⁺-resistant transconjugants. This mobilization procedure allowed transfer and expression of pJT1 Ag⁺ resistance inE. coli C600. Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag⁺-resistant determinant(s) intoE. coli by conjugation or transformation including high-voltage electroporation.E. coli C600 containing PJTI and PJT2 displayed decreased accumulation of Ag⁺ similar toE. coli R1.E. coli C600 could not tolerate 0.1 and 0.5 mM Ag⁺, rapidly accumulated Ag⁺ and became non-viable. Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms.     

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.     

Conjugational transfer of the nodulation-conferring plasmid pWZ2 in Rhizobium trifolii

Summary When the nodulating Rhizobium trifolii strain 24Vio^r containing plasmid RP4 was conjugated with the non-nodulating R. trifolii mutant strain 24Str^rNod^-35, plasmid RP4 was transferred at a frequency 10^-3–10^-4. Two out of nearly three thousand tested transconjugants which contained plasmid RP4 had acquired the ability to form nodules on clovers. Molecular studies of the DNA of both these nodulating transconjugants showed the presence of plasmid RP4 and another plasmid which was not found in the original recipient strain. The size of this second plasmid corresponded to that of the plasmid pWZ2, the elimination of which was correlated with irreversible loss of the nodulating ability of R. trifolii strain 24 (Zurkowski and Lorkiewicz 1979). Plasmid RP4 was eliminated from cells by ethidium bromide, without the loss of nodulating properties. The nodulation capacity, however, was eliminated from transconjugants after incubation of bacteria at elevated temperature. Non-nodulating clones obtained after such incubation did not contain the plasmid pWZ2. The results indicate that the plasmid pWZ2 is a necessary element for induction of nodules by R. trifolii, and that it can be mobilized by plasmid RP4.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.     

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.     

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.     

Narrow- and Broad-Host-Range Symbiotic Plasmids of Rhizobium spp. Strains That Nodulate Phaseolus vulgaris

Agrobacterium transconjugants containing symbiotic plasmids from different Rhizobium spp. strains that nodulate Phaseolus vulgaris were obtained. All transconjugants conserved the parental nodulation host range. Symbiotic (Sym) plasmids of Rhizobium strains isolated originally from P. vulgaris nodules, which had a broad nodulation host range, and single-copy nitrogenase genes conferred a Fix⁺ phenotype to the Agrobacterium transconjugants. A Fix⁻ phenotype was obtained with Sym plasmids of strains isolated from P. vulgaris nodules that had a narrow host range and reiterated nif genes, as well as with Sym plasmids of strains isolated from other legumes that presented single nif genes and a broad nodulation host range. This indicates that different types of Sym plasmids can confer the ability to establish an effective symbiosis with P. vulgaris.     

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.     

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.     

Construction of a partial diploid for the degradative pathway encoded by the TOL plasmid (pWW0) from Pseudomonas putida mt-2: Evidence for the positive nature of the regulation by the xylR gene

Summary The hypothesis that the early enzymes of the degradative pathway determined by the TOL plasmid pWW0 are positively regulated by the product of the xylR gene has been tested by constructing a strain which is a partial diploid for the TOL genes. Two parental plasmids were first constructed by in vivo methods, neither of which could determine the ability to grow on m-xylene, one of the primary substrates of the plasmid degradative pathway, because of mutations. One of these, pWW0-216, was a derivative of pWW0 but carried a xylR ^- allele and a copy of the Tn401 transposon, encoding carbenicillin resistance. The other plasmid, pWW0-152, was a derivative of the promiscuous R plasmid RP4 into which had been translocated part of a pWW0 plasmid carrying a wild type xylR ⁺ allele but with a defective xylA, the structural gene for xylene oxidase. When these two plasmids were mated into the same strain, all the transconjugants examined grew on m-xylene and one representative of these, PaW 219, was shown to contain induced levels of xylene oxidase when grown under inducing conditions. The possibility that ability to utilise m-xylene was due to recombination between or reversion of the coexisting plasmids was eliminated by demonstrating that the two parental plasmids segregated on mating out from PaW 219. It is concluded therefore that xylR ⁺ is transdominant to xylR ^-, and that its gene product is a positive regulator.     

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).     

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.