Genetic Analysis of Plasmid-specific Pheromone Signaling Encoded by pPD1 in Enterococcus faecalis

Certain plasmids in Enterococcus faecalis encode a mating response to recipient-produced peptide sex pheromones. Targeted disruption of tra genes on pPD1 suggested that TraA plays a central role in the plasmid-specific pheromone signaling pathway. TraA functioned as a negative regulator for the pheromone-inducible conjugal transfer. Complementation analysis of pPD1 tra gene mutants by pAD1 suggested that the pheromone binding function of TraC was non-specific between these plasmids, but the function of TraA and the pheromone shutdown function of TraB are plasmid-specific.     

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10⁻⁵ to 10⁻⁸ per recipient. A total of 12 distinct plasmids, designated pB1–pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10⁻¹ to 10⁻² per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPβ plasmids at the DNA sequence level. In order to characterize the gene “load” of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified. Received: 11 October 1999 / Accepted: 11 January 2000     

DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type="Italic">Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

Identification and Characterization of a Shuttle Plasmid with Antibiotic Resistance Gene from <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

While studying antibiotic-resistant plasmids from multi-drug-resistant nosocomial Staphylococcus aureus strains, we isolated a small (2.889 kb) chloramphenicol-resistant (Cm^r) plasmid, which was designated as pMC524/MBM. The molecular size of pMC524/MBM was close to that of pC194 (2.910 kb), a well-known Cm^r staphylococcal plasmid. Unlike pC194, this plasmid can replicate and express itself efficiently and stably in Escherichia coli. However, Cm is needed for stable maintenance of pMC524/MBM in different hosts. In this study, the nucleotide sequences of these two plasmids were compared after sequencing of pMC524/MBM [EMBL Accession No. AJ312056 SAU312056]. Although these two plasmids have striking nucleotide sequence homology, the Plus Origin, Minus Origin, the replication protein (Rep), and the chloramphenicol acetyl transferase (Cat) have considerable variations. Possibly, these changes have modulated pMC524/MBM into an efficient shuttle-plasmid. Received: 8 April 2002 / Accepted: 27 July 2002     

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.     

Cloning, E. coli overexpression, purification and binding properties of TraA and TraC, two proteins involved in the pheromone-dependent conjugation process in enterococci

The bacteriocin encoding plasmid pPD1 from Enterococcus faecalis is involved in a mating response to the sex pheromone cPD1 produced by recipient bacterial cells devoid of pPD1. Previous studies showed that cPD1 is internalized into donor cells in a process in which TraC plays the role of cell surface pheromone receptor. Inside the recipient cells, the pheromone binds to the plasmid-encoded cytoplasmic protein TraA, able to recognize specific DNA sequences and to modulate the conjugation process. To avoid self-induction of the conjugation process, donor cells produce the inhibitor iPD1, which competes with cPD1. This study was designed to produce recombinant TraA and TraC in a functionally active state and to evaluate their main functional properties. We have isolated the sequences encoding TraA and TraC from the plasmid pPD1 and cloned them in suitable expression vectors. The two recombinant proteins were successfully obtained in a soluble form using Escherichia coli as expression host and a T7 inducible expression system. TraC and TraA were purified to homogeneity by three or two chromatographic steps, respectively, leading to a final yield up to 4 mg/l of cell culture for TraC and up to 10 mg/l of cell culture for TraA. The ability of TraA and TraC to bind the specific pheromone and inhibitor peptides has been assessed by means of ESI-mass spectrometry. Moreover, the ability of recombinant TraA to bind DNA has been demonstrated by means of electrophoretic mobility shift assay. Overall these results are consistent with the heterologously expressed TraC and TraA being functionally active.     

Syntheses and Flavors of Four Possible Optical Isomers of Marmelo Lactones

We have isolated a tetracycline-resistant (Tc^r) Bacillus species (named HE-1) which carries multiple plasmids. HE-1 was identified as Bacillus cereus and found to bear four plasmids. Tetracycline resistance could be attributed to one of four plasmids (designated as pTIT β2 (4.7 kb)) indistinguishable from pBC16, a Tc^r plasmid formerly found in B. cereus [K. Bernhard, H. Schrempf, and W. Goebel, J. Bacteriol., 133, 897 (1978)]. All the other three plasmids (named pTITα (4.0 kb), pTIT β1 (4.7 kb) and pTIT γ (12.4 kb)) were cryptic and did not correlate with bacterial phenotypic traits such as antibiotic resistance or antibiotic and bacteriocin production. B. cereus HE-1 also showed resistance to penicillin, but this seemed very likely to be chromosomally determined in B. cereus. Of interest was the fact that pTITα, pTIT β1, and pTITγ had a noticeable DNA homology among them in blot hybridization. pTIT β2 alone did not shared sequence homology with the other three plasmids.     

Sequence arrangements of the Escherichia coli chromosome and of putative insertion sequences, as revealed by electron microscopic heteroduplex studies.

Total Escherichia coli DNA from strain DK445 (which is CSH50 F^?, R^?, deletion lac and pro, lysogenized with lambda cIts857 Sam7 lac5: :Mu cI⁺) was denatured, reannealed, and observed by electron microscopy. The single-strand DNA lengths ranged from about 50 to 150 kilobases (kb). In some molecules a short duplex region with a single-stranded fork at each end was observed. The duplex lengths were 0.75 kb, 1.30 kb, 5.22 kb, 5.62 kb, which correspond to those of IS1; of IS2, IS3, or IS4; of the ribosomal RNA genes; and of the γδ sequence, respectively. Duplexes of 1.0 kb and 0.5 kb were also found. Most of the duplexes of 0.5, 0.75, 1.0 and 1.3 kb were observed as intramolecular stem-loop structures and were therefore interpreted to be sequence duplications in inverted order on the same DNA strand. The most frequent separations of the putative inverted insertion sequences were around 22 and 27.5 ± 1.5 kb. About 14% of the E. coli chromosome is estimated to be involved in the sequence arrangements that give rise to stem-loop structures upon denaturation and reannealing. The copy numbers of the putative insertion sequences and other elements that form the “stems” of the stem-loop structures are also estimated.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv, tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Large Linear Plasmids of Borrelia Species That Cause Relapsing Fever

Borrelia species of relapsing fever (RF) and Lyme disease (LD) lineages have linear chromosomes and both linear and circular plasmids. Unique to RF species, and little characterized to date, are large linear plasmids of ∼160 kb, or ∼10% of the genome. By a combination of Sanger and next-generation methods, we determined the sequences of large linear plasmids of two New World species: Borrelia hermsii, to completion of its 174-kb length, and B. turicatae, partially to 114 kb of its 150 kb. These sequences were then compared to corresponding sequences of the Old World species B. duttonii and B. recurrentis and to plasmid sequences of LD Borrelia species. The large plasmids were largely colinear, except for their left ends, about 27 kb of which was inverted in New World species. Approximately 60% of the B. hermsii lp174 plasmid sequence was repetitive for 6 types of sequence, and half of its open reading frames encoded hypothetical proteins not discernibly similar to proteins in the database. The central ∼25 kb of all 4 linear plasmids was syntenic for orthologous genes for plasmid maintenance or partitioning in Borrelia species. Of all the sequenced linear and circular plasmids in Borrelia species, the large plasmid''s putative partition/replication genes were most similar to those of the 54-kb linear plasmids of LD species. Further evidence for shared ancestry was the observation that two of the hypothetical proteins were predicted to be structurally similar to the LD species'' CspA proteins, which are encoded on the 54-kb plasmids.     

Plasmids of the pRM/pRF Family Occur in Diverse Rickettsia Species

The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.     

Genome Organization and Localization of the pufLM Genes of the Photosynthesis Reaction Center in Phylogenetically Diverse Marine Alphaproteobacteria

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996^T to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.     

The tap‐tpg gene pair on the linear plasmid functions to maintain a linear topology of the chromosome in Streptomyces rochei

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2‐L (211 kb), pSLA2‐M (113 kb) and pSLA2‐S (18 kb), their complete nucleotide sequences having been determined. Restriction and sequencing analysis revealed that the telomere sequences at both ends of the linear chromosome are identical to each other, are 98.5% identical to the right end sequences of pSLA2‐L and pSLA2‐M up to 3.1 kb from the ends and have homology to those of typical Streptomyces species. Mutant 2‐39, which lost all the three linear plasmids, was found to carry a circularized chromosome. Sequence comparison of the fusion junction and both deletion ends revealed that chromosomal circularization occurred by terminal deletions followed by nonhomologous recombination. Curing of pSLA2‐L from strain 51252, which carries only pSLA2‐L, also resulted in terminal deletions in newly obtained mutants. The tap‐tpg gene pair, which encodes a telomere‐associated protein and a terminal protein for end patching, is located on pSLA2‐L and pSLA2–M but has not hitherto been found on the chromosome. These results led us to the idea that the tap‐tpg of pSLA2‐L or pSLA2‐M functions to maintain a linear chromosome in strain 7434AN4. This hypothesis was finally confirmed by complementation and curing experiments of the tap‐tpg of pSLA2‐M.     

Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants

Summary The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Km^r) and pUB110dB (1.5 MDa, Km^r), were obtained from pTB913 (2.9 MDa, Km^r, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Km^r, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids.     

A host-vector system for gene cloning in the cyanobacterium Synechocystis PCC 6803

Summary Synechocystis 6803 contains at least four cryptic plasmids of 2.27 kb (pUS1, pUS2 and pUS3) and 5.20 kb (pUS4). The 1.70 kb HpaI fragments of the related plasmids pUS2 and pUS3 were cloned into the Ap^r gene of the E. coli plasmid pACYC177, yielding the Km^r hybrid plasmids pUF12 and pUF3 respectively. pUF3 recombines in Synechocystis 6803 with a 2.27 kb plasmid giving the Km^r shuttle vector pUF311. The 1.35 kb HaeII fragment containing the Cm² gene of the E. coli plasmid pACYC184 was cloned in pUF311 generating the Cm^r Km^r shuttle vector pFCLV7. Wild-type cells of Synechocystis 6803 are transformed, albeit poorly, by the plasmids pUF3, pUF12 and pFCLV7. pFCLV7 very efficiently transforms the SUF311 strain of Synechocystis 6803 containing pUF311 as a resident plasmid. This is due to recombination between the homologous parts of pFCLV7 and pUF311. For the same reason the strain SUF311 is also efficiently transformable by E. coli plasmids, as shown for pLF8, provided that they have some homology with the E. coli part of pUF311.The combined use of Synechocystis 6803 strain SUF311 and of plasmids pFCLV7 and pLF8 generates an efficient host-vector system for gene cloning in this facultatively heterotrophic cyanobacterium.     

Cloning and expression in Escherichia coli of the homoserine kinase (thrB) gene from Brevibacterium lactofermentum

Summary Five DNA fragments carrying the thrB gene (homoserine kinase E.C. 2.7.1.39) of Brevibacterium lactofermentum were cloned by complementation of Escherichia coli thrB mutants using pBR322 as vector. All the cloned fragments contained a common 3.1 kb DNA sequence. The cloned fragments hybridized among themselves and with a 9 kb BamHI fragment of the chromosomal DNA of B. lactofermentum but not with the DNA of E. coli. None of the cloned fragments were able to complement thrA and thrC mutations of E. coli. Plasmids pULTH2, pULTH8 and pULTH11 had the cloned DNA fragments in the same orientation and were very stable. On the contrary, plasmid pULTH18 was very unstable and showed the DNA inserted in the opposite direction. E. coli minicells transformed with plasmids pULTH8 or pULTH11 (both carrying the common 3.1 kb fragment) synthesize a protein with an M _r of 30,000 that is similar in size to the homoserine kinase of E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - SDS sodium dodecyl sulphate - TSB tripticase soy broth - m-DAP meso-diaminopimelic acid - Sm^r, Cp^r, Km^r, Am^r, Ap^r, Tc^r, MA15^r resistance to streptomycin, cephalotin, kanamycin, amykacin, ampicillin, tetracycline and microcin A 15, respectively     

High-resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres

A technique to detect DNA sequences on extended DNA fibres (EDF) prepared from interphase nuclei from tomato (Lycopersicon esculentum) and Arabidopsis thaliana leaves by fluorescence in situ hybridization (FISH) is described. Three nuclear lysis procedures have been tested for their ability to decondense chromatin and to generate highly extended intact DNA fibres on microscopic slides. DNA probes of various sizes have been used in FISH experiments to EDFs to establish the resolution and sensitivity of the technique. The fluorescent signals of a 5S rDNA probe hybridized to tomato EDFs revealed continuous strings of about 200 µm, that corresponded to a molecular size of about 660 kb. In A. thaliana, a contig of three cosmids spanning a genomic region with a total length of about 89 kb was analysed. By means of multi-colour hybridization the physical positions of the cosmids were visualized as red and green fluorescence strings with overlapping regions in yellow. Comparison of the length of the fluorescent signals with the molecular data revealed a stretching degree of the DNA fibres at 3.27 kb µm^?1, which is close to the Watson-Crick DNA length estimate of 2.9 kb µm^?1. Other experiments on small size molecular probes with both lambda clones (13.5–17 kb insert sizes) and plasmids (4.2 and 5 kb) in a contig of A. thaliana, and the 5S rDNA region in tomato showed close agreement with molecular data. The lower limit of the detection, which was established in a hybridization experiment with two DNA probes from the 45S ribosomal gene on extended fibres of tomato, was about 0.7 kb. Consistent patterns of alternating fluorescent red and green spots were obtained reflecting the tandemly repeated arrangement of the 18S and 25S ribosomal sequences. On the basis of the microscopic distance between these hybridization spots the size of the ribosomal unit was estimated at 8.2 kb. This implies a drastic improvement of high-resolution physical mapping of DNA sequences by FISH on plant DNA.     

Characterization of the transfer-related <Emphasis Type="Italic">tra</Emphasis> region of the conjugative plasmid p19 from a <Emphasis Type="Italic">Bacillus subtilis</Emphasis> soil strain

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.     

Excision of the 40kb segment of the TOL plasmid from Pseudomonas putida mt-2 involves direct repeats

Summary The TOL plasmid pWWO gives rise to the Tol^– plasmid pWWO-8 due to a specific excision event involving 40 kb of TOL DNA. This event occurs by means of reciprocal recombination involving a pair of directly repeated 1.4 kb sequences. This 40 kb segment is not involved as such in the formation in vivo of hybrid plasmids between TOL and resistance plasmids, notably RP4.     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.