Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants

Summary Symbiotic mutants of Rhizobium meliloti were isolated following Tn5 mutagenesis. Besides four nodulation mutants (Nod^-) unable to induce nodule formation on alfalfa, five infection mutants (Inf^-), which induce the formation of root nodules without detectable infection threads or bacteroids, were obtained. The Inf^- mutants were subdivided into two classes. One class contains mutants which fail to synthesize acidic exopolysaccharide (EPS^-). The other class is comprised of mutants which produce excess amounts of acidic exopolysaccharide (EPS^*). ¹³C nuclear magnetic resonance spectroscopy of the exopolysaccharide isolated from one of the latter type of Inf^- mutant, 101.45, revealed that the side chain of the repeating octosaccharide unit lacks the terminal pyruvate residue. Complementing cosmids were isolated for all Inf^- mutants. In the case of the Inf^- EPS^- mutants the complementing cosmids contain DNA segments which overlap and are part of megaplasmid 2. For two mutants the mutations were found to map on a 7.8 kb EcoRI fragment. In the case of the Inf^- EPS^* mutants the complementing cosmids carry chromosomal DNA. The mutations of two Inf^- EPS^* mutants were localized on a 6.4 kb EcoRI fragment. Coinoculation of alfalfa plants with Nod^- and Inf^- EPS^- mutants resulted in effective symbiosis. The nodules appeared wild type and fixed nitrogen. In constrast, coinoculations with Nod^- mutants and the Inf^- EPS^* mutant 101.45 did not result in the formation of effective nodules.     

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.     

An RP4-prime containing a 285 kb fragment of rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym

Summary We integrated the RP4 plasmid into a selected region of the pSym megaplasmid of Rhizobium meliloti 2011 by homologous recombination between pSym and a cloned fragment of pSym present in the RP4. This cointegrate was used to mobilize into Escherichia coli a Tn5 transposon located on pSym in the vicinity of the site of integration of the RP4. By this technique we obtained a series of RP4-primes that contained large fragments of the pSym megaplasmid and that were most probably generated by IS8 promoted deletions in the RP4-pSym cointegrate. One of them, pGMI42, which carries nitrogenase genes nifD and H as well as nodulation genes, was used for mutagenesis of the corresponding region of pSym after insertion of the Mu prophage into the tet gene. When various (pGMI-42:: Mu)::Tn7 were introduced into R. meliloti 2011 by conjugation, homologous recombination allowed insertion of Tn7 into pSym whereas the pGMI42::Mu was lost due to the suicide effect of Mu. In this way we obtained several symbiotic mutants deficient in either nodulation (Nod^-) or nitrogen fixation (Fix^-) in association with the host plant Medicago sativa.This paper is affectionately dedicated to the memory of Jean-Simon Julliot who initiated and inspired this work and who was killed by an avalanche on February 21, 1982     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa

It is known that the Rhizobium galegae genomes contain megaplasmids. The suicide vector pSUP2111 with nifH gene of R. meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R. galegae inducing effective nodules on Galega orientalis plants. The formation of self-transmissible megaplasmids was observed. The megaplasmid transfer into non-nodulating R. meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G. orientalis plants. Among the Agrobacterium strains harbouring R. galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M. sativa (five strains); (4) Nod- with both plant hosts (11 strains).     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Conjugal Transfer of Megaplasmid 2 between Rhizobium meliloti Strains in Alfalfa Nodules

A DNA fragment containing the RP4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of Rhizobium meliloti 0540 (Inf⁻ EPS⁻), resulting in PG101 (Inf⁻ EPS⁻). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and R. meliloti 2526 (Nod⁻). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 × 10⁻⁷. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 × 10⁻⁵. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.     

Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti

Summary Large plasmids of molecular weight varying from 90 to around 200×10⁶ have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation-phenol extraction procedure. With a less destructive method (Eckhardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300×10⁶ (=megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids.All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nif DH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nif DH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.     

Site specific transposition of Tn7 into a Rhizobium meliloti megaplasmid

Summary Transposon Tn7 was shown to insert specifically into the megaplasmid of different Rhizobium meliloti strains. Tn7 transposition could not be detected in other Rhizobium strains such as R. trifolii, R. leguminosarum, R. phaseoli and R. japonicum. In R. meliloti strains, two unique sites in the megaplasmid were observed into which Tn7 can transpose at different frequencies. Only one copy of Tn7 could be detected in the megaplasmid and the insertion sites for Tn7 are outside the nif and nod region. Tn7 transposition in R. meliloti showed a marked preference for sites on plasmid RP4 compared to the megaplasmid sites. Attempts to cure Tn7 from the megaplasmid were unsuccessful. This site specific transposition of Tn7 in R. meliloti provides an additional genetic tool to further manipulate this important plasmid in symbiotic nitrogen fixation.     

Physical characterization of Rhizobium meliloti megaplasmids

Intact megaplasmids of Rhizobium meliloti 2011 have been isolated and visualized by electron microscopy. The contour lengths of 64 megaplasmid molecules were determined. One definite class of molecules of 400 micron length and a range of larger molecules with lengths of up to 560 micron was observed. The contour lengths of the megaplasmids pRme2011a and pRme2011b were measured after isolation from plasmid-free Agrobacterium strains into which they had been individually transferred. Plasmid pRme2011a corresponds to the 400-micron class of megaplasmids while plasmid pRme2011b belongs to the 560-micron class. Preparatively isolated megaplasmids pRme2011a and b showed completely different restriction patterns. The pattern of total megaplasmid DNA from R. meliloti 2011 is composed of those from pRme2011a and b, suggesting that no more than two different megaplasmids exist. Because the length distributions of measured molecules were broad, R. meliloti 2011 megaplasmids seem to vary in length in vivo. Because only pRme2011a hybridized with a nifHD probe, this is the Sym plasmid. For R. meliloti strain MVII-1, which carries the megaplasmids pRmeMVII-1f and pRmeMVII-1g, pRmeMVII-1f was shown to be the Sym plasmid. Buoyant density determinations of R. meliloti 2011 and MVII-1 megaplasmids gave a value of 1.717 g/cm3 for pSym, which is that of Agrobacterium DNA. The buoyant density of the second megaplasmid was 1.721 g/cm3, corresponding to the density of the R. meliloti chromosome. As determined by reassociation kinetics, pRme2011a and b are unrelated. The degree of relatedness between strains MVII-1 and 2011 was 82%.     

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).     

Identification and cloning of nodulation genes from the wide host range Rhizobium strain MPIK3030

Summary A 70 kbp segment of the megaplasmid from a broad host range Rhizobium strain (MPIK3030) was mapped with the aid of cosmid clones made in the vector pJB8. A 7.9 kbp EcoRI fragment from this region, 55 kbp away from the nif gene cluster, was shown to hybridize to the common nod genes from R. meliloti. Using several R. meliloti nod probes it was possible to delimit an 830 bp region as being the center of greatest homology. Sequence data from two sections of this region gave a nucleotide homology of 73.7% to the nodC gene of R. meliloti. Using Tn5 mutagenesis a clone was isolated carrying Tn5 in the highly homologous region. When tested on Macroptilium atropurpureum, this MPIK3030 derivative was shown to have a Nod^– phenotype. When the wild-type allele was reintroduced into the Tn5 mutant, nodulation was restored. Interspecies complementation also showed that both R. meliloti and Rhizobium sp. MPIK3030 nod regions were able to restore nodulation to Tn5-induced nodC mutants from either strain.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

General Method for the Identification of Plasmid Species in Fast-Growing Soil Microorganisms

Using a horizontal gel electrophoresis method, we demonstrated reproducibly the presence of indigenous plasmids in different Rhizobium, Agrobacterium, and Pseudomonas strains. The method yields a large amount of plasmid DNA and is sensitive in detecting megaplasmids with molecular weights higher than 5 × 10⁸. In two Rhizobium meliloti strains, a megaplasmid other than the low-mobility plasmid already known was detected.     

Competitive abilities of Tn5 Tox- mutants of a rhizobacterium inhibitory to wheat growth

Antibiosis has been thought to impart a competitive advantage to soil microorganisms. A rhizobacterium of the genus Pseudomonas produces a toxin that inhibits the growth of other microorganisms and winter wheat (Triticum aestivum L.). The bacterium was mutagenized with the Tn5 transposon to obtain toxin-negative (Tox^-) mutants or was selected for its spontaneous resistance to rifampicin. Tox^- mutants were used to determine the role of the toxin in wheat root inhibition, root colonization, and rhizosphere competitiveness. Four Tox^- (loss of inhibition of both E. coli and wheat root growth) and four partial Tox⁺ (partial loss of inhibition of E. coli and wheat root growth) Tn5 mutants were isolated. Seven of the mutants had different Tn5 chromosomal insertions, which suggests that toxin production is the result of several gene loci. Competitive root-colonization abilities of the Tox^- isolates were studied in winter wheat rhizospheres using varied population levels in autoclaved and nonautoclaved soil. Toxin production did not affect the competitive abilities of these organisms with native soil microflora. Results here indicate that toxin production by these organisms is not the primary mechanism of their competitive advantage in root colonization. Thus, opportunities exist for biological control of plant-suppressive bacteria using these Tox^- strains.     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Transposon mutagenesis in Azospirillum brasilense: isolation of auxotrophic and Nif- mutants and molecular cloning of the mutagenized nifDNA

Summary We report the successful mutagenesis of Azospirillum brasilense 29710 Rif Sm with transposon Tn5. The narrow host-range plasmid pGS9 (p15A replicon), which possesses broad host-range N-type transfer genes, was used as the suicide vehicle to deliver Tn5 in Azospirillum. Out of 900 colonies tested, 0.8% proved to be auxotrophic. One mutant altered in indoleacetic acid (auxin) biosynthesis was isolated and, in addition, three mutants completely defective in nitrogen fixation (nif) were obtained. All the mutants tested contained a single copy of Tn5 integrated randomly in the genome. The Tn5-mutagenized EcoRI fragments were cloned from the three Nif^- mutants. Physical analysis of cloned DNA showed that Tn5 was present on a different EcoRI fragment in each case, ranging in size from 15–17 kb. The nitrogenase structural genes (nifHDK) in A. brasilense 29710 Rif Sm were localized on a 6.7 kb EcoRI fragment. We found that Tn5 is not inserted in the nifHDK genes in the Nif^- mutants reported here. Site-directed mutagenesis using the cloned, Tn5-containing DNA from mutant Nif27(pMS188), produced a large number of Nif^- transconjugants of the A. brasilense 29710 Rif wild-type strain, showing the linkage between Tn5 insertion and the Nif^- phenotype. This is the first time that transposon-mutagenized auxotrophic, Nif^- and other mutants have been available for genetic analysis in Azospirillum. This should greatly facilitate the cloning and mapping of genes involved in nitrogen fixation as well as in many other phenotypic characteristics of Azospirillum.