A new insertion sequence from Sinorhizobium meliloti with homology to IS1357 from Methylobacterium sp. and IS1452 from Acetobacter pasteurianus

The insertion sequence ISRm8 was identified by sequence analysis of the cryptic plasmid pRmeGR4b of Sinorhizobium meliloti GR4. ISRm8 is 1451 bp in length and carries 22/24-bp terminal imperfect inverted repeats with seven mismatches and a direct target site duplication of 3 bp. ISRm8 carries a unique open reading frame whose putative protein showed significant similarity to the insertion sequences IS1357 and IS1452, isolated from Methylobacterium sp. and Acetobacter pasteurianus, respectively. Two copies of this IS element were found in strain GR4; one of them is linked to plasmid pRmeGR4b, whereas the other is localized out of the non-pSym plasmids. In S. meliloti field populations ISRm8 shows a limited distribution (50% of the strains tested carry the IS element), with a copy number ranging from 1 to 6.     

Ammonia Inhibition of Plasmid pRmeGR4a Conjugal Transfer between Rhizobium meliloti Strains

We have examined nutritional factors influencing conjugal transfer of the two nonsymbiotic large plasmids, pRmeGR4a and pRmeGR4b, of Rhizobium meliloti GR4. To monitor transfer, each plasmid was tagged with a different antibiotic resistance marker. Transfer of plasmid pRmeGR4b was dependent upon the presence of plasmid pRmeGR4a on the same donor cell. Transconjugants for pRmeGR4b were obtained at frequencies 5-to 10-fold higher than transconjugants carrying both plasmids, indicating that mobilization of pRmeGR4b by pRmeGR4a probably occurred in trans. Conjugal transfer of the tagged plasmids between R. meliloti strains was tested on minimal medium supplemented with single amino acids, nitrate, or ammonium as the single nitrogen source. A higher number of transconjugants was obtained when glutamate was the only nitrogen source, whereas conjugation was virtually undetectable on ammonium. No relationship was found between donor or recipient growth rate and plasmid transfer rate on a given nitrogen source. Furthermore, in media containing both glutamate and ammonium as nitrogen sources, transfer was reduced almost 100-fold compared with that in media containing glutamate alone. Inhibition was readily detected at 2.5 mM or higher concentrations of either ammonium chloride or ammonium sulfate and appeared to be specific for exogenously supplied ammonium. Inhibition of conjugal transfer between R. meliloti strains by ammonium was only observed for rhizobial plasmids, not for a heterologous plasmid such as RP4. Apparently, ammonium did not affect the plasmid-encoded transfer machinery, as it had no influence on rhizobial plasmid transfer from R. meliloti to Agrobacterium tumefaciens. The effect of ammonium seemed to take place on R. meliloti recipient cells, thereby reducing the efficiency of plasmid conjugation, probably by affecting mating pair formation or stabilization.     

Comparative analysis of the genetic structure of a Rhizobium meliloti field population before and after environmental release of the highly competitive R. meliloti strain GR4

Abstract: Rhizobium meliloti strain GR4, which exhibits a highly competitive ability for alfalfa root nodule occupancy, was used in a field release experiment in Granada, Spain. In order to analyze the ecological impact of the GR4 release, we characterized the R. meliloti indigenous population of the field site by ERIC-(enterobacterial repetitive intergenic consensus) PCR and IS (insertion sequence) fingerprinting. Both fingerprinting methods resulted in the same grouping of the isolates. Data obtained were compared with a previous analysis by plasmid based sequence-specific PCR. Isolates belonging to the major infective group, as defined by dominant plasmid types, were shown to have identical or nearly identical ERIC and IS fingerprint patterns. Hence, we conclude that all three typing methods are suited to characterize the genetic structure of the field population. The possible impact of the introduction of strain GR4 was examined two years after its release in its original environment. No effect on the genetic structure of the indigenous R. meliloti field population was observed.     

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.     

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).     

Ectopic transposition of a group II intron in natural bacterial populations

Self-splicing group II introns are thought to be the evolutionary progenitors of eukaryotic spliceosomal introns. The invasion of novel (ectopic) sites by group II introns is considered to be a key mechanism by which spliceosomal introns may have become widely dispersed. However, the dynamics of these events in populations are unknown. In bacteria, only two group II introns have been shown to splice and to be mobile in vivo. One of these introns, RmInt1 from Sinorhizobium meliloti, which encodes a protein with no endonuclease domain, has been shown to invade the ectopic oxi1 site independently of recombinase. In this study, we analysed ectopic transposition of the RmInt1 intron in a natural population of S. meliloti. We characterized S. meliloti isolates by polymerase chain reaction amplification of a gene, dapB, which is found only on the pRmeGR4b plasmid diagnostic of GR4-type strains. The diversity within this specific field population of bacteria was analysed by restriction fragment length polymorphism using ISRm2011-2 (homing site of RmInt1) and RmInt1 as probes. We found that ectopic transposition of RmInt1 to the oxi1 site occurred in this natural bacterial population. This ectopic transposition was also the most frequent genetic event observed. This work provides further evidence that the ectopic transposition of group II introns is an important mechanism for their spread in natural bacterial populations.     

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.     

ISRm4-1 and ISRm9, two novel insertion sequences from Sinorhizobium meliloti

Two novel insertion sequences, ISRm4-1 and ISRm9 have been identified in Sinorhizobium meliloti. ISRm4-1 is 936-bp in length, flanked by 17-bp putative terminal inverted repeats and a putative target duplication of 3-bp. ISRm4-1 is a member of the IS5 family of insertion sequences, closely related to ISRm4. ISRm9 is 2797-bp in length and carries 25-bp inverted repeats with target duplication of 7-bp. ISRm9 belongs to the IS21 family of insertion elements. On the non-pSym plasmid pRmeGR4b from S. meliloti strain GR4, a copy of ISRm4-1 is interrupted at nucleotide 150 from its 5′-end by a copy of ISRm9. Whereas ISRm4-like elements are widespread in S. meliloti, the distribution of ISRm9 appears to be correlated to that of pRmeGR4b-type plasmids.     

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.     

Sequence analysis of the 144-kilobase accessory plasmid pSmeSM11a, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.     

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti

The stability of the thy autoselective system, based on an essential thymidylate synthase gene, for enhanced maintenance of plasmid vectors in Rhizobium meliloti was evaluated in the greenhouse and with field-grown alfalfa. The thy autoselective system consists of a free-replicating, broad-host-range plasmid vector containing a copy of the thyA gene from Lactococcus lactis subsp. lactis and a spontaneous mutant of R. meliloti deficient in thymidylate synthase (Thy(sup-)). Under greenhouse conditions, Thy(sup-) rhizobia did not persist in rooting solution alone unless supplemented with thymidine but survived in the presence of the host plant. Nodules formed on alfalfa plants grown in thymidine-free rooting solution and inoculated with Thy(sup-) rhizobia contained only Thy(sup+) revertants. In soil, Thy(sup-) rhizobia were compromised and failed to nodulate alfalfa. Thy(sup-) mutants containing a thy plasmid survived in the rhizosphere and nodulated alfalfa like the wild-type strain. The thy autoselective system was tested in the field with Thy(sup-) strain Rm24T and pPR602, a thy plasmid vector devoid of antibiotic resistance genes and marked with constitutively expressed lacZY. At 80 days after sowing, most rhizobia isolated from the nodules of field-grown alfalfa inoculated with Rm42T(pPR602) contained pPR602. The thy autoselective system proved useful to ensure maintenance of the plasmid vector under greenhouse and field conditions in R. meliloti.     

A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.     

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.     

Monitoring the diversity of Rhizobium meliloti field and microcosm isolates with a novel rapid genotyping method using insertion elements

Rhizobium meliloti strains isolated from alfalfa plants grown in a mining recultivation field, in a model ecosystem (microcosm) and in soil core containers were characterized by two new taxonomic methods, fingerprinting and handprinting, using insertion sequence elements (IS) as hybridization probes. The diversity of strains within the field population could first be detected with IS-fingerprinting, whereby nearly three times more groups of Rhizobium meliloti strains could be identified in comparison to the groups according to plasmid profiles. This complexity and diversity of the rhizobial population was also detected in microcosm studies. Strains identified among the field population were also detected in the microcosm studies. The persistence of rhizobia in soil was demonstrated in soil core samples held in a cold room for 2 years. A decrease in the genomic diversity of the R. meliloti population upon soil storage was observed. A novel monitoring method, IS-handprinting, in which the presence of certain endogenous insertion elements within a strain is registered, was successfully employed to characterize genetically the field R. meliloti strains with simplicity and speed. In contrast to IS-fingerprinting, IS-handprinting is based on a simple plus-or-minus detection, which is sufficient for a taxonomic characterization. Both methods, using a non-radioactive detection system, are sensitive enough to detect one copy of an insertion element in a strain's genome. IS-fingerprinting, with its fine resolution, would be suitable for ecological studies of individual strains in any complex ecosystem, whereas IS-handprinting would be suitable for monitoring strains and characterizing large numbers of strains.     

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.     

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.     

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.     

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%.