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.     

Identification and nucleotide sequence of Rhizobium meliloti insertion sequence ISRm3: similarity between the putative transposase encoded by ISRm3 and those encoded by Staphylococcus aureus IS256 and Thiobacillus ferrooxidans IST2.

The insertion sequence ISRm3 was discovered simultaneously in different Rhizobium meliloti strains by probing Southern blots of total cellular DNA with 32P-labeled pTA2. This plasmid is indigenous to strain IZ450 and fortuitously contained four copies of ISRm3. By using an internal EcoRI fragment as a specific probe (pRWRm31), homology to ISRm3 was subsequently detected in over 90% of R. meliloti strains tested from different geographical locations around the world. The frequency of stable nonlethal ISRm3 transpositions was estimated to be 4 x 10(-5) per generation per cell in strain SU47 when grown in liquid culture. The entire nucleotide sequence of ISRm3 in R. meliloti 102F70 is 1,298 bp and has 30-bp terminal inverted repeats which are perfectly matched. Analysis of six copies of ISRm3 in two strains showed that a variable number of base pairs (usually eight or nine) were duplicated and formed direct repeats adjacent to the site of insertion. On one DNA strand, ISRm3 contains an open reading frame spanning 93% of its length. Comparison of the putative protein encoded with sequences derived from the EMBL and GenBank databases showed significant similarity between the putative transposases of ISRm3 from R. meliloti, IS256 from Staphylococcus aureus, and IST2 from Thiobacillus ferroxidans. These insertion sequences appear to be distantly related members of a distinct class.     

Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene.

Two of the three plasmids of the wild-type Rhizobium meliloti 41 (pRme41a and pRme41c) carry a copy of ISRm2, a 2.7-kilobase-long transposable element. ISRm2 is terminated by 22-base-pair (bp) inverted repeat sequences, exhibiting some homology to the inverted repeats of elements generating 9-bp target sequence duplication. Transposition of ISRm2 results in a duplication of 8 bp in length, rather rare among transposable elements. DNA sequences homologous to an internal fragment of ISRm2 were found in several Rhizobium species. Transposition of ISRm2 into fixation and nodulation genes located on the symbiotic plasmid pRme41b was detected at a high frequency. Exact locations of two copies of ISRm2 which transposed into the nod-nif region on the megaplasmid were determined. In one case, integration into the protein-coding region of the hsnD gene that determines a host specificity function of nodulation occurred. In the other mutant, ISRm2 was localized upstream of nifA, where a short open reading frame coding for a new fix gene (fixX) was identified. The product of fixX is a ferredoxin carrying a characteristic cluster of cysteine residues. On the basis of the observation that the arrangement of the ISRm2 copies is identical in the free-living wild-type cells and in nitrogen-fixing nodules, we concluded that the involvement of ISRm2 transposition in the development of nitrogen-fixing symbiosis is unlikely.     

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.     

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.     

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.     

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.     

The nucleotide sequence of ribosomal 5S RNA from Rhizobium meliloti: comparison with 5S rRNA of Agrobacterium

The complete nucleotide sequence of R. meliloti 5S ribosomal RNA has been determined and compared with the already known sequence of A. tumefaciens 5S rRNA (Vandenberghe et al., 1985, Eur. J. Biochem., 149, 537-542) and of other 5S rRNAs from Rodobacteria Alpha-2 (Wolters et al., 1988, Nucleic Acids Res., 16, rl-r70). The differences found at eight positions (23, 73, 83, 72 in helical fragments; 16, 40, 88 in loops; 54 in bulge), which might affect secondary structures of 5S rRNA, are small. Moreover, the sequence analysis specifies both variable and common positions in 5S rRNA secondary structure of Rodobacteria Alpha-2.     

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.     

DNA sequence homology in Rhizobium meliloti plasmids

Summary Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle — size plasmids of R. meliloti studied in this paper.     

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.     

Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria.

We describe the construction and use of a set of plasmid vectors of the transposons Tn1, Tn5, and Tn9 that are suicidal in Rhizobium species and therefore suitable for mutagenesis with these three transposons. The vectors are composed of the p15A replicon which functions in Escherichia coli but not in Rhizobium species and a region encoding the N type of bacterial conjugation system which is very efficient in matings between E. coli and Rhizobium species. The usefulness of the vectors has been most extensively assessed in Rhizobium meliloti. It is likely that they will be useful for mutagenesis and genome manipulation in other bacteria as well.     

Galactose metabolism in Rhizobium meliloti L5-30.

Data from previous studies of Rhizobium meliloti mutants have been consistent with the catabolism of hexoses via the Entner-Doudoroff pathway. However, galactose metabolism was not impaired in those mutants. We show here by enzymatic assay and by identification of a galactose mutant lacking 2-keto-3-deoxy-6-phosphogalactonate aldolase that the De Ley-Doudoroff pathway is used for galactose metabolism. Mutants in this pathway have not been previously reported for any organism.     

Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae.

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.     

Generalized transduction in Rhizobium meliloti.

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.     

Catabolite-repression-like phenomenon in Rhizobium meliloti.

We report a phenomenon similar to catabolite repression in Rhizobium meliloti. Succinate, which allows the highest observed rate of growth of R. meliloti, caused an immediate reduction of beta-galactosidase activity when added to cells growing in lactose. A Lac- mutant was unaltered in nodulation and nitrogen fixation capacities, but a pleiotropic mutant deficient in several catabolic properties was unable to produce effective nitrogen-fixing nodules.