Two gene clusters of Rhizobium meliloti code for early essential nodulation functions and a third influences nodulation efficiency.

A pLAFR1 cosmid clone (pPP346) carrying the nodulation region of the symbiotic plasmid pRme41b was isolated from a gene library of Rhizobium meliloti 41 by direct complementation of a Nod- deletion mutant of R. meliloti. Agrobacterium tumefaciens and Rhizobium species containing pPP346 were able to form ineffective nodules on alfalfa. The 24-kilobase insert in pPP346 carries both the common nodulation genes and genes involved in host specificity of nodulation. It was shown that these two regions are essential and sufficient to determine the early events in nodulation. A new DNA region influencing the kinetics and efficiency of nodulation was also localized on the symbiotic megaplasmid at the right side of the nif genes.     

Frankia基因文库的构建及与根瘤菌结瘤基因区同源克隆的筛选

应用无色肽酶(Achromopeptidase)加溶菌酶系统破壁,提取分别来自色赤杨、细枝木麻黄和沙棘的3株代表性Frankia菌株的总DNA。以可在很多革兰氏阴性细菌中稳定复制和诱动转移的广谱寄主性质粒pLAFR1为载体,构建了其基因组文库。基于经EcoRI酶切后的Frankia总DNA中有与根瘤菌结瘤基因同源性的片段,以豌豆根瘤菌结瘤基因为探针,通过菌落原位杂交对文库进行了筛选,较强杂交克隆经斑点杂交复筛,初步得到了几个阳性克隆,为进一步研究Frankia的结瘤基因及有关共生固氮的其它基因奠定了基础。     

Reiteration of genes involved in symbiotic nitrogen fixation by fast-growing Rhizobium japonicum.

By using cloned Rhizobium meliloti nodulation (nod) genes and nitrogen fixation (nif) genes, we found that the genes for both nodulation and nitrogen fixation were on a plasmid present in fast-growing Rhizobium japonicum strains. Two EcoRI restriction fragments from a plasmid of fast-growing R. japonicum hybridized with nif structural genes of R. meliloti, and three EcoRI restriction fragments hybridized with the nod clone of R. meliloti. Cross-hybridization between the hybridizing fragments revealed a reiteration of nod and nif DNA sequences in fast-growing R. japonicum. Both nif structural genes D and H were present on 4.2- and 4.9-kilobase EcoRI fragments, whereas nifK was present only on the 4.2-kilobase EcoR2 fragment. These results suggest that the nif gene organizations in fast-growing and in slow-growing R. japonicum strains are different.     

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).     

Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains.

We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains. While one of the reiterated DNA regions, pFR2503, is closely linked to the B. japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B. japonicum USDA 438. The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B. japonicum serocluster 123 strains. Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B. japonicum and Bradyrhizobium spp. strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians. The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B. japonicum serocluster 123 strains.     

Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains.

We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains. While one of the reiterated DNA regions, pFR2503, is closely linked to the B. japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B. japonicum USDA 438. The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B. japonicum serocluster 123 strains. Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B. japonicum and Bradyrhizobium spp. strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians. The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B. japonicum serocluster 123 strains.     

Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants.

Total cellular DNA from Rhizobium trifolii, R. melitoti, and R. japonicum strains 110 and 117 were prepared. DNA fragments generated with restriction endonuclease EcoRI from these DNA samples were compared in agarose gels after electrophoresis. DNA cleavage patterns generated from R. japonicum strain 110, R. trifolii, and R. meliloti were clearly distinguishable from each other. Restriction endonuclease cleavage patterns of DNA from R. japonicum strain 110 and presumptive R. trifolii mutant strains that nodulate soybean were found to be similar. Rhizobium trifolii mutant strains were also lysed by a phage specific for R. japonicum strain 110. These results show that "R. trifolii mutant strains" are indeed derivatives of R. japonicum strain 110 and not R. trifolii.     

Isolation and characterization of an ndvB locus from Rhizobium fredii

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).     

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.     

At least two nodD genes are necessary for efficient nodulation of alfalfa by Rhizobium meliloti

A Rhizobium meliloti DNA region (nodD1) involved in the regulation of other early nodulation genes has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicate a large open reading frame with opposite polarity to nodA, -B and -C, coding for a protein of 308 (or 311) amino acid residues. Tn5 insertion within the gene caused a delay in nodulation of Medicago sativa from four to seven days. Hybridization of nodD1 to total DNA of Rhizobium meliloti revealed two additional nodD sequences (nodD2 and nodD3) and both were localized on the megaplasmid pRme41b in the vicinity of the other nod genes. Genetic and DNA hybridization data, combined with nucleotide sequencing showed that nodD2 is a functional gene, while requirement of nodD3 for efficient nodulation of M. sativa could not be detected under our experimental conditions. The nodD2 gene product consists of 310 amino acid residues and shares 86.4% homology with the nodD1 protein. Single nodD2 mutants had the same nodulation phenotype as the nodD1 mutants, while a double nodD1-nodD2 mutant exhibited a more severe delay in nodulation. These results indicate that at least two functional copies of the regulatory gene nodD are necessary for the optimal expression of nodulation genes in R. meliloti.     

Identification of Bradyrhizobium nod genes involved in host-specific nodulation.

Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes. Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species. lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product. A mutation in one of the loci was found to result in an alteration of the host range of B. japonicum. This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.     

Nucleotide sequence of Rhizobium meliloti nodulation genes

A Rhizobium meliloti DNA region, determining nodulation functions common in different Rhizobium species, has been delimited by directed Tn5 mutagenesis and its nucleotide sequence has been determined. The sequence data indicates three large open reading frames with the same polarity coding for three proteins of 196, 217 and 402 (or 426) amino acid residues, respectively. We suggest the existence of three nod genes on this region, which were designated as nodA, B and C, respectively. Comparison of the R. meliloti nodA, B, C nucleotide and amino acid sequences with those from R. leguminosarum, as reported in the accompanying paper, shows 69-72% homology, clearly demonstrating the high degree of conservation of common nod genes in these Rhizobium species.     

Heterologous hybridization of Frankia DNA to Rhizobium meliloti and Klebsiella pneumoniae nif genes

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nif D and nif H genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nif K, we have localized a DNA region hybridizing to a R. meliloti probe containing nif A and nif B genes. The extent of the homology was greater for nif B than for nif A.     

Physical and genetic map of a Rhizobium meliloti nodulation gene region and nucleotide sequence of nodC.

Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.     

Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency.

We examined the contribution of a cryptic plasmid, pRmeGR4b, to the nodulation of Medicago sativa by strain GR4 of Rhizobium meliloti. A 905-base-pair PstI DNA fragment in pRmeGR4b was found to hybridize DNA of the R. meliloti fixA promoter region as a probe. Sequence analysis of the PstI fragment showed a 206-base-pair region displaying high homology with the DNA upstream of the RNA start points of the P1 and P2 symbiotic promoters. Putative nif promoter consensus sequences were conserved in this DNA segment. Expression of DNA downstream of the nif promoterlike sequence, monitored by beta-galactosidase activity of different lacZ fusions, was demonstrated to depend on a functional nifA gene, both in microaerobically free-living cells and in nodules. Individual transposon Tn3-HoHo1 insertions in this DNA region caused a reduced nodulation competitiveness. This new symbiotic region, occupying approximately 5 kilobases of pRmeGR4b DNA, was called nfe (nodule formation efficiency).