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.     

NifA-NtrA regulatory system activates transcription of nfe,a gene locus involved in nodulation competitiveness of Rhizobium meliloti

We have previously demonstrated that the Rhizobium meliloti large plasmid pRmeGR4b carries the gene locus nodule formation efficiency (nfe) which is responsible for nodulation efficiency and competitive ability of strain GR4 on alfalfa roots. In this study we report that expression of nfe-lacZ fusions in Escherichia coli is activated in the presence of the cloned nifA gene of R. meliloti. This activation was found to be oxygen sensitive and to require the E. coli ntrA gene product. In contrast to the R. meliloti nifA, the cloned nifA gene of Klebsiella pneumoniae was able to activate expression of nfe in aerobically grown cells of both E. coli and R. meliloti. Hybridization experiments did not show homology to nfe in four R. meliloti wild-type strains tested. These strains were uncompetitive when coinoculated with a GR4 derivative carrying plasmid pRmeGR4b, but were competitive when coinoculated with a GR4 derivative carrying a single transposon mutation into the nfe region. When nfe DNA was introduced into the four wild-type strains, a significant increase in the competitive ability of two of them was observed, as deduced from their respective percentages of alfalfa root nodule occupancy in two-strains coinoculation experiments.     

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation.

A 6 kb DNA segment of the R. meliloti 2011 pSym megaplasmid, which contains genes controlling host specificity of root hair infection and of nodulation, was cloned and sequenced. The DNA sequence analysis, in conjunction with previous genetic data, allowed identification of four nod genes designated as E, F, G and H. nodH is divergently transcribed with respect to nodFE and nodG. A conserved nucleotide sequence was found around 200 bp upstream of the translation start of nodF, nodH and nodA. This sequence is also present upstream of common nodA and species specific nodF genes of other Rhizobium species. The predicted protein products of nodF and nodG show homology with acyl carrier protein and ribitol dehydrogenase, respectively. The nodH product contains a rare sequence of four contiguous proline residues. Comparison with the nod gene products of R. leguminosarum shows that species specific nodFE products are as well conserved as those of common nodABC and nodD genes.     

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.     

Sequence of ISRm4 from Rhizobium meliloti strain GR4.

ISRm4, an IS-like sequence structurally similar to Pseudomonas cepacia insertion element IS402, was identified by sequence analysis. This 933-bp element carries 17-bp putative terminal inverted repeats with five mismatches and a putative direct target duplication of 3 bp.     

Role of galactosyltransferase activity in phage sensitivity and nodulation competitiveness of Rhizobium meliloti.

A stock culture of Rhizobium meliloti 102F51 contains colonies of two distinct phenotypes (Handelsman et al., J. Bacteriol. 157:703-707, 1984); one colony type is agglutinated by high dilutions of the alfalfa agglutinin, is sensitive to phage F20, and is resistant to phage 16B, and the other is agglutinated only by low dilutions of the alfalfa agglutinin, is resistant to phage F20, and is sensitive to phage 16B. Cells of the latter phenotype have an inner-membrane-bound galactosyltransferase activity that transfers galactose from UDP-galactose to a water-insoluble anionic polymer. This enzymatic activity is absent in cells of the first phenotype. All of the phage 16B-resistant mutants selected from a sensitive strain were agglutinated by high dilutions of the alfalfa agglutinin, were sensitive to phage F20, and lacked galactosyltransferase activity. The galactose-containing polymer prepared in vitro was immunologically cross-reactive with the cell surface.     

Analysis of Rhizobium meliloti nodulation mutant WL131: novel insertion sequence ISRm3 in nodG and altered nodH protein product.

Nodulation (nod) genes are required for invasion of legumes by Rhizobium bacteria. Mutant WL131 is a derivative of 102F51 that has a severe Nod- phenotype on alfalfa. Upon examination of the extended DNA region containing host-specific nodulation genes nodFEG and nodH, we found that the nodG gene of WL131 bears a novel insertion sequence, ISRm3. Complementation studies implied, however, that the phenotype on alfalfa correlated with the nodH locus. We found that nodH in WL131 encodes an altered gene product. Correlation of the WL131 defect with nodH was also supported by phenotypic behavior. Each mutation affected nodulation more severely on alfalfa (Medicago sativa) than on sweet clover (Melilotus albus). However, we found that the degree of requirement for nodH in nodulation varied with the conditions under which the plant was grown.     

Stability and transmissibility of the cryptic plasmids of Rhizobium meliloti GR4

Rhizobium meliloti strain GR4 harbours two cryptic plasmids sharing extensive regions of homology between them and with other non-symbiotic plasmids of different strains of R. meliloti. They both are very stable showing a segregation rate of less than 0.1% loss per generation. pRmeGR4a (115 MDa) is a self-transmissible plasmid at a variable frequency to other species, and it is also responsible for promoting, at low frequency, the contransfer of pRmeGR4b (140 MDa), the other cryptic plasmid of the strain. A 4.8 kb PstI fragment of pRmeGR4a, responsible for the high stability in cis of this plasmid, has been isolated and several recombinant plasmids have been constructed showing different segregation rates in the strains used in this study. Their stabilities can be considerably improved by insertion of the stabilization mrs/par region of RK2.     

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100

Abstract The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty verions were identified by electron microscoy at densities of 1.29–1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.     

Localization of nodulation gene on the chromosome of Rhizobium meliloti

Using N-methyl-N'-nitro-N-nitrosoguanidine mutant RM54 of Rhizobium meliloti L5-30 defective in the nodulation process (Nod-) and in the biosynthesis of adenine was obtained. Nod- phenotype of this mutant was not caused by the auxotrophic mutation. The nod gene is located on the chromosome. The wild type strain of R. meliloti and Nod- mutant RM54 harbour two indigenous plasmids having a molecular weight of 90 Mdal and about 300 Mdal.     

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.     

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.     

Insertion sequence (IS) hybridization supports classification of Rhizobium meliloti by phage typing

Sixty-one isolates of Rhizobium meliloti from two field sites which had been previously classified into 15 phage types on the basis of sensitivity to 16 typing phages, were subjected to insertion sequence (IS) hybridization using DNA probes for ISR m 3 and ISR m 5. Isolates from all but one phage type contained ISR m 3 (apparent copy no. 1–11) and all isolates contained ISR m 5 (apparent copy no. 3–11). The isolates were placed into 24 IS classes based on differences in their respective ISR m 3 and ISR m 5 hybridization profiles. At either field site, isolates representing different phage types possessed IS hybridization profiles that differed from each other, while those comprising a specific type had identical or closely related profiles. Isolates from one phage type were unusual since they did not react with any of the typing phages and were shown by IS hybridization to constitute a heterogeneous group. Evidence for spatial effects were provided by isolates from two of six types present at both sites which fell into separate IS classes on the basis of their site of origin. These data have ecological implications and suggest that for a particular site, phage typing may be employed for the rapid assessment of the genetic diversity among field isolates.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn5-mob with a helper plasmid RP4-4 was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased nodulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.     

Enhanced competitiveness for nodulation of Medicago sativa by Rhizobium meliloti transconjugants harbouring the root-inducing plasmids of Agrobacterium rhizogenes strain 15834

Abstract The root-inducing plasmid of Agrobacterium rhizogenes strain 15834 marked with transposon Tn 5 -mob RP4-4 with a helper plasmid was mobilized into nitrogen-fixing Rhizobium meliloti strain CIAM 1759. The resulting transconjugants did not induce ‘hairy’ root syndrome but developed nitrogen-fixing nodules on alfalfa. Among the 9 transconjugants tested, 6 strains had increased modulation rates. The competitiveness of 2 of these R. meliloti (pRi) strains was significantly enhanced as compared with the parent strain CIAM 1759; this was confirmed both in tube and in pot tests.