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.     

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     

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.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.     

Diversity of Plasmid Profiles and Conservation of Symbiotic Nitrogen Fixation Genes in Newly Isolated Rhizobium Strains Nodulating Sulla (Hedysarum coronarium L.)

Forty-five Rhizobium strains nodulating sulla (Hedysarum coronarium L.), isolated from plants grown in different sites in Menorca Island and southern Spain, were examined for plasmid content and the location and organization of nif (nitrogen fixation) and nod (nodulation) sequences. A great diversity in both number and size of the plasmids was observed in this native population of strains, which could be distributed among 19 different groups according to their plasmid profiles. No correlation was found between plasmid profile and geographical origin of the strains. In each strain a single plasmid ranging from 187 to 349 megadaltons hybridized to Rhizobium meliloti nifHD and nodD DNA, and in three strains the spontaneous loss of this plasmid resulted in the loss of the nodulation capacity. In addition to the symbiotic plasmid, 18 different cryptic plasmids were identified. A characteristic cryptic plasmid of >1,000 megadaltons was present in all strains. Total DNA hybridization experiments, with nifHD and portions of nodC and nodD genes (coding for common nodulation functions) from R. meliloti as probes, demonstrated that both the sequence and organization of nif and common nod genes were highly conserved within rhizobia nodulating sulla. Evidence for reiteration of nodD sequences and for linkage of nodC to at least one copy of nodD was obtained for all the strains examined. From these results we conclude that Rhizobium strains nodulating sulla are a homogeneous group of symbiotic bacteria that are closely related to the classical fast-growing group of rhizobia.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum bv. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

Construction and Properties of Cloning Vectors Based on a 7.2-kb Rhizobium meliloti Cryptic Plasmid

Cloning vectors (pFD1001, pFD1192, pFD1194, and pFD1212) were constructed by extension of the host range of a 7.2-kb Rhizobium meliloti cryptic plasmid (pRm1132f) with the ColE1-based plasmids, pBR322, pACYC177, pACYC 184, pSUP301, or pHC179; mobilization was facilitated by introduction of the ori T region from pRK2, a broad-host-range plasmid. The vector plasmids transferred readily into a wide range of gram-negative bacteria and had relatively low copy number in R. meliloti; two constructs, pFD1001 and pFD1212, were completely stable in R. meliloti isolated from nodules of alfalfa (Medicago sativa). A representative of the vector constructs (pFD1001) could be maintained in R. meliloti in the presence of the broad-host-range shuttle plasmid pRK290. These two vector plasmids could be introduced into R. meliloti, either simultaneously or singly when pRK290 was the resident plasmid; however, entry of pRK290 was blocked when pFD1001 was the resident plasmid. The cloning vectors constructed in this study should prove to be useful for the genetic manipulation of Rhizobium.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

The two megaplasmids of Rhizobium meliloti are involved in the effective nodulation of alfalfa

Summary We have shown by physical and genetic means that there are two megaplasmids in all strains of Rhizobium meliloti that we have studied. Megaplasmids from several strains of R. meliloti were mobilized to Agrobacterium tumefaciens and to other Rhizobium strains using the Tn5-Mob system. We were also able to resolve these two megaplasmids in agarose gels for most strains, and to show that only one of them hybridized to nif and nod genes. Transfer of this plasmid, the pSym, to Agrobacterium, R. leguminosarum, and R. trifolii strains conferred on these recipients the ability to nodulate alfalfa ineffectively. The second megaplasmid did not appear to have a direct role in nodule initiation. However, we were able to complement extracellular polysaccharide (EPS^-) mutants of R. meliloti by transferring this second megaplasmid into them. Furthermore, Tn5-induced EPS^- mutants of R. meliloti 2011, which produced ineffective (Fix^-) nodules of abnormal morphology, were shown by hybridization and complementation to carry mutations in this second megaplasmid. This demonstrates that both megaplasmids of R. meliloti are necessary for the effective nodulation of alfalfa.     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

Plasmids from bacterial endosymbionts of hump-killer paramecia

Six isolates ofCaedibacter taeniospiralis, collected from four continents, were screened for plasmid DNA. Plasmid DNA species containing between 41.5 and 49.5 kilobase pairs (kb) were observed in all strains. Physical maps of plasmids were constructed by determining relative positions of the restriction endonuclease (BamHI,SalI,XhoI,SacI,PstI,AvaI, andEcoRI) recognition sequences in each plasmid. The physical map of the smallest plasmid (41.5 kb), pKAP30, is reflected in each of the plasmids isolated from the other strains ofC. taeniospiralis. Plasmid DNA from three of the isolates (strains 51 and 116 both from Indiana and strain 169 from Japan) each contain 43 kb, where 41.5 kb appear to be identical to pKAP30 (obtained from the Australian strain, A30). The extra 1.5 kb present in pKAP51, pKAP116, and pKAP169 is included as a single polynucleotide sequence. The 1.5-kb inclusion is located at apparently identical positions in pKAP116 and pKAP169 and at a totally different position in pKAP51. The two remaining plasmids, pKAP47 (from California strain 47) and pKAP298 (from Panama strain 298), both contain 49 kb to include a continuous 41.5-kb sequence that is apparently identical to pKAP30. The results indicate that the polynucleotide sequences of these plasmids are highly conserved and that the observed variations among them may be accounted for by transposable elements.     

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     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

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.