Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

RecE-independent recombination of plasmids in Bacillus subtilis cells

The pUB110 and pE194 plasmid cointegrates have been isolated and examined in rec+ and recE4 strains of Bacillus subtilis. Cointegrates were shown to be formed by recombination at the specific site present on both parental plasmids as a short region of homology designated RSA. The RSA consists of 63 nucleotides in pE194 and 49 in pUB110; the length of its fully conserved core segment is 10 nucleotides. All cointegrates examined were formed by single crossover event taking place within the core segment, and as a result they have identical nucleotide sequences of recombination junctions. No conversion of mismatched base pairs to nucleotide sequences originally belonging to one of the parental plasmids was found. Though the action of RecE gene did not affect the frequency of cointegrate formation, it was reduced in rec149 host by one order of magnitude. Cointegrates retained their stability during transformation.     

Macroevolution of plasmids: a model for plasmid speciation.

A new evolutionary model for diversification in plasmid incompatibility groups (plasmid speciation) is suggested. The model is based on the formation of plasmid cointegrates from two compatible plasmids. The existence of plasmid cointegrates is well known, however, their potential key role in plasmid macroevolution has not yet been recognized. In a hypothesis presented here, one of the rep genes is supposed to be relaxed from selection in plasmid cointegrates and thus becomes free to accumulate mutations. These mutations can lead to a change in incompatibility specificity. Evidence supporting this hypothesis comes from the common occurrence of multi-replicon plasmids in nature as well as from experimental studies on plasmid cointegrate formation. A more speculative extension of this model hypothesizes an evolutionary scenario for origin of the eubacterial single-replicon genome and the eukaryotic multi-replicon genome, as well as the place of plasmids and viruses in this picture.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination—Deficient Escherichia Coli strains

The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied. The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling. Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome. The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells. The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9]. In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions. These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells. In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.     

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.     

A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK

A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.     

Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C.

In the circular plasmid pKD1, which stably replicates in Kluyveromyces lactis, the three open reading frames encode a site-specific recombinase (gene A) and two proteins involved in mitotic stability (genes B and C). A recombination analysis of plasmids in which gene B or C is inactivated reveals that unlike the 2 microns plasmid of Saccharomyces cerevisiae, these genes are also required for the site specificity of plasmid recombination.     

Cointegration and resolution mediated by IS101 present in plasmid pSC101

Summary A certain class of cointegrate plasmids was found to occur between a pSC101 derivative and a second plasmid pBV320 in E. coli F^- cells. Cleavage analysis and DNA sequencing showed that the cointegrate plasmid contained direct repeats of an insertion sequence IS101 at the recombination junctions, indicating that formation of cointegrates was mediated by IS101, which is a natural constitutent of pSC101. These cointegrates were formed only in cells which contained the transposon gamma-delta, suggesting that the gamma-delta sequence, which provides transposase, is responsible for cointegration. Whenever the cointegrate plasmids were present in cells containing gamma-delta or its related transposon Tn3, the cointegrates were dissolved to give pBV320::IS101 due to recombination at duplicated IS101 sequences in the cointegrates, suggesting that both gamma-delta and Tn3, which provide a resolvase, are responsible for the resolution of the cointegrates. Comparison between the nucleotide sequence of IS101 and those of gamma-delta and Tn3 shows a high degree of homology in the regions that have been shown to be the binding sites of resolvases, as well as in the terminal inverted repeats. However, there is no homology between IS101 and the other element, gamma-delta or Tn3, in the internal resolution site, at which the resolution event may occur.Abbreviations Tc tetracycline - Cm chloramphenicol - Ap ampicillin - bp base pairs - kb kilobase pairs     

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids

Summary A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the mutagenized Ti plasmid fragment cannot replicate in these cells, they can be rescued by recombination with the homologous region of the Ti plasmid. The cointegrates formed were resolved in a second recobination event, which was detected by loss of the drug resistance marker of the E. coli plasmid. Subcloning of the Ti plasmid fragments labeled with Tn5 showed that the frequency of rescue of the hybrid plasmid as a cointegrate and its segregation in agrobacteria depend on the degree of homology with the Ti plasmid. We also applied the strategy for site-directed Tn5 mutagenesis to insert specifically the replication origin of bacteriophage fd and the thymidine kinase gene from Herpes virus into the T-DNA of Ti plasmid-C58.     

Cointegrates carrying two copies of a Tn3 derivative in an inverted orientation

We constructed a mutant of Tn3, Tn3 #2, that contains a 55-bp direct repeat of sequences near the amino-terminal coding region of the transposase, and an 8-bp EcoRI linker. This mutant transposase is functional. The plasmid carrying Tn3 #2, pMB8::Tn3 #2, recombines with the plasmid pHS1 at a frequency of 2.8 X 10(-7) recombinants per division cycle. This is similar to the recombination frequency of pHS1 and pMB8::Tn3+ (wild-type) which is 4.5 X 10(-6) recombinants per division cycle. One-third of the recombinants between pMB8::Tn3 #2 and pHS1 were approx. 22 kb in length. Restriction analysis and nucleotide sequencing showed that these large plasmids were Tn3 #2-mediated cointegrates formed by integration of pMB8::Tn3 #2 into pHS1. However, unlike Tn3 tnpR- -mediated cointegrates that contain direct repeats of the incoming element, Tn3 #2-mediated cointegrates carry two copies of Tn3 #2 in the form of inverted repeats. Like the tnpR- repeats, the Tn3 #2 repeats occur at both junctions between the parental plasmids, and are associated with a 5-bp direct duplication of the pHS1 target site. Furthermore, these recombinants contain a small deletion starting precisely at the end of Tn3 #2 and extending into pMB8 sequences. We propose a model for the generation of Tn3 #2-mediated cointegrates.     

Plasmids controlling synthesis of hemolysin inEscherichia coli

Summary Plasmids of three different sizes, designated as plasmid A (mw: 65×10⁶), plasmid B (mw: 41×10⁶) and plasmid C (mw: 32×10⁶) respectively, have been isolated from various hemolytic wild-type strains ofE. coli. DNA-DNA hybridization was performed to determine their relationship. The wild-type strain, PM167a, harbours plasmids of all three sizes. Hybridization studies indicate that all three plasmids share extented sequence homologies but that plasmid A is not composed of plasmids B and C. Hybridization between plasmids of the donor strain and those of appropriate transconjugants demonstrates that in some cases plasmids with identical size are not longer completely homologous in their nucleotide sequences. This indicates that despite their defined sizes these plasmids are not stable genetic entities, but rather they undergo frequently recombination and dissociation during conjugation. In one particular transconjugant strain, K12-PM152/1, a plasmid D was found which is a stable recombined molecule of plasmids B and C of the original strain. Plasmids of size B found as the only extrachromosomal elements in a hemolytic wild-type strain (P224) and two transconjugant strains (e.g. K12-CM20 and K12-PM167/1) share extended nucleotide sequence homologies but are not identical. Little sequence homology was observed between two different hemolytic plasmids and the F and the Col Ib plasmids suggesting that the former do not belong to either the F-like or the I-like group of plasmids. Another hemolytic plasmid is F-like based on its sequence homologies with the F factor.     

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.     

IS1-mediated tandem duplication of plasmid pBR322. Dependence on recA and on DNA polymerase I

Transposable-element-mediated fusion of the conjugal plasmid pOX38::Tn9 with pBR322 results in the appearance of cointegrates composed of a single copy of each plasmid, and cointegrates which carry a single copy of pOX38 but multiple tandem copies of pBR322. These plasmids are separated by directly repeated copies of the transposable element. We demonstrate here that such multimers can be generated from monomeric cointegrates, probably by unequal crossing over between the flanking Tn9(IS1) elements. Their appearance is thus not necessarily associated with the original transposition (fusion) event. Our study demonstrates that the process of duplication is strongly dependent on the homologous recombination system of Escherichia coli, since it is undetectable by our methods in recA- strains. It is also strongly dependent on the presence of a functional DNA polymerase I in the cell. The major pathway(s) for this duplication thus appears to rely on both the homologous recombination system and the replication of the duplicated segment.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Intermolecular Transposition of Is10 Causes Coupled Homologous Recombination at the Transposition Site

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 P(R) promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in δrecA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.     

Formation of Rhizobium phaseoli symbiotic plasmids by genetic recombination

We report here the formation of symbiotic plasmids (pSyms), by genetic recombination between rearranged pSyms, which lack symbiotic information, and resistance plasmids carrying parts of different symbiotic plasmids (R's). This recombination was found to occur both between plasmids derived from different Rhizobium phaseoli isolates, and between plasmids derived from strains obtained from the same original isolate. We also present evidence on the formation of a functional symbiotic plasmid by recombination of an R', carrying nif and nod genes from strain CFN42, and an indigenous plasmid present in this strain (pCFN42e), which was thought to be unrelated to its symbiotic plasmid (pCFN42d). These data are discussed with respect to the stability and transfer of Rhizobium symbiotic information.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.