Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region.

Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.     

Decreased symbiotic effectiveness of Rhizobium leguminosarum strains carrying plasmid RP4

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncPα classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.     

Chromosomal transfer promoted by the promiscuous plasmid RP4.

We have studied the properties of the recombinant plasmid RP4λatt. This plasmid possesses the EcoRI-generated fragment of phage λ containing the genes att-int-xis (srIλ2–3) inserted into the single EcoRI site of the promiscuous plasmid RP4. The insertion of this λ fragment has no detectable effect on normal plasmid functions. However, it confers the ability to promote low-frequency polarized chromosomal transfer by int-promoted integration into the host λ attachment site attλ. We have succeeded in isolating an Hfr derivative which has the plasmid stably integrated at attλ. The Hfr derivative is unusual in having both an integrated and an autonomous RP4λatt stably coexisting in the same cell.     

Detection of plasmid RP4 transfer in soil and rhizosphere,and the occurrence of homology to RP4 in soil bacteria

Plasmid RP4 transfer between introduced pseudomonads was studied in non-rhizosphere and rhizosphere soil. The addition of nutrients to the non-rhizosphere soil stimulated plasmid transfers between introduced donor and recipient cells, and no transfer was detected in nonamended soil. Transfer was also detected in soil in a model rhizosphere, but not in corresponding non-rhizosphere soil. Colony hybridization with whole plasmid RP4 DNA as a probe was employed to detect transfers to indigenous organisms in soil. Although transfers to introduced recipient cells were easily detected in parallel controls, no indigenous organisms were identified that had received RP4. Background levels of soil organisms with the RP4 resistance pattern were considerable, and about 10% of these populations contained DNA sequences with homology to RP4. However, no plasmids could be detected in any of 20 isolates, nor was resistance transfer to aPseudomonas fluorescens recipient detected in filter matings.     

Isolation of transposon TnA from plasmid RP4 carrying two copies of this element

Employing heteroduplex and restriction analyses, two inverted copies of a 3.2 · 10⁶ dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative of the plasmid RP4. Integration of the second copy of TnA causes loss of the conjugative properties of RP4. Both TnA sequences in RP4:: TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4::TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coli QD5003, HB101[pCRI] and JC7623 with the isolated transposon were unsuccessful.     

Conjugal transfer of enterococcal transposons in Bacillus megaterium

The conjugative enterococcal transposons Tn916 and Tn919 were introduced into Bacillus megaterium by a filtermating technique. The transfer frequencies obtained ranged from 1.3×10^-6 to 6.6×10^-7. The transposons integrated stably into the B. megaterium chromosome. Tn916 could generate auxotrophs and was transferred from B. megaterium Tn916 transconjugants to other species.     

Effect of plasmid F'Col+trp+ on the transfer of plasmid RP4]

Interaction of conjugative plasmids F'colV colB trp and PR4 in Escherichia coli host was studied during the transfer of the plasmids from cell to cell. The plasmid F'colV colB trp is found to stimulate the transfer of RP4 from the diplasmid strain. This seems to be due to stabilization of the conjugating pairs which require normal pili coded by the plasmid F'colV colB trp.     

High-frequency chromosome transfer in Rhodopseudomonas sphaeroides promoted by broad-host-range plasmid RP1 carrying mercury transposon Tn501.

Insertion of the mercury resistance transposon Tn501 into broad-host-range plasmid RP1 greatly enhanced the ability of this plasmid to promote chromosome transfer in the photosynthetic bacterium Rhodopseudomonas sphaeroides. Compared with the wild-type RP1, which produced less than 10(-8) recombinants per donor cell, RP1::Tn501 produced between 10(-3) and 10(-7) recombinants per donor cell depending upon the marker selected. Plasmid RP1::Tn501 promoted polarized transfer of the chromosome from one or perhaps two origins on the chromosome, giving rise to two linkage groups. All of the biosynthetic and antibiotic resistance genes that have been mapped, including those involved in photosynthesis, occur on one or another of these linkage groups.     

Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid

The high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia. Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB-presenting (asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH(-) strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.     

Rampant horizontal transfer of SPIN transposons in squamate reptiles

Transposable elements (TEs) are highly abundant in the genome and capable of mobility, two properties that make them particularly prone to transfer horizontally between organisms. Although the impact of horizontal transfer (HT) of TEs is well recognized in prokaryotes, the frequency of this phenomenon and its contribution to genome evolution in eukaryotes remain poorly appreciated. Here, we provide evidence that a DNA transposon called SPIN has colonized the genome of 17 species of reptiles representing nearly every major lineage of squamates, including 14 families of lizards, snakes, and amphisbaenians. Slot blot analyses indicate that SPIN has amplified to high copy numbers in most of these species, ranging from 2,000-28,000 copies per haploid genome. In contrast, we could not detect the presence of SPIN in any of the turtles (seven species from seven families) and crocodiles (four species) examined. Genetic distances between SPIN sequences from species belonging to different squamate families are consistently very low (average = 0.1), considering the deep evolutionary divergence of the families investigated (most are >100 My diverged). Furthermore, these distances fall below interfamilial distances calculated for two genes known to have evolved under strong functional constraint in vertebrates (RAG1, average = 0.24 and C-mos, average = 0.27). These data, combined with phylogenetic analyses, indicate that the widespread distribution of SPIN among squamates is the result of at least 13 independent events of HTs. Molecular dating and paleobiogeographical data suggest that these transfers took place during the last 50 My on at least three different continents (North America, South America and, Africa). Together, these results triple the number of known SPIN transfer events among tetrapods, provide evidence for a previously hypothesized transoceanic movement of SPIN transposons during the Cenozoic, and further underscore the role of HT in the evolution of vertebrate genomes.     

Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion.

We have begun an analysis in Escherichia coli of the conjugal transfer functions of the broad-host-range plasmid RP4. We have isolated 19 tra mutants of RP4, generated by insertion of transposon 7, and mapped their insertion sites by restriction endonuclease analysis. These sites fall into two separate regions on either side of the kanamycin resistance determinant. The transfer rates of the mutants range from 10% of that of RP4 to an undetectable level. Spot tests with the P-1 pilus-specific phages PRR1, Pf3, and PR4 and electron microscopic examination for pili have classified the mutants into several phenotypes consistent with their having normal, retracted, or no pili. Analysis of transient plasmid heterozygotes, created by P1 transduction, divided the tra mutants into a minimum of five complementation groups. Some of these groups contain more than one phenotypic class and may represent more than one gene because of the possible polar and deletion effects of Tn7 insertion.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

Protein transfer into the recipient cell during bacterial conjugation: studies with F and RP4

Transfer of donor cell proteins to the recipient bacterium was examined in F- and RP4-mediated conjugation. Transfer of a 120 kD polypeptide, identified as the larger product of the plasmid DNA primase gene, was readily detected during RP4-promoted conjugation. The protein was transmitted to the cytoplasm of the recipient, presumably complexed to the transferred ssDNA. F DNA was transferred without detectable association with any cytoplasmic tra protein or with the ssDNA-binding protein encoded by the plasmid. However, a 92 kD protein, possibly F TraD product, was transmitted to the membrane fraction of the recipient cell.     

Construction of yeast strains containing genetically marked transposons

Haploid yeast cells contain approximately 35 Ty (transposon yeast) elements. To facilitate the study of these elements, we have constructed yeast strains in which one of these transposons carries a genetic marker. The elements that we have modified are Ty912 and Ty917, elements originally detected at the HIS4 locus in spontaneously occurring his4- mutants. The strain constructions took place in three stages: 1) cloning of the mutant HIS4 genes containing the Ty elements; 2) introduction of a HindIII restriction fragment containing the yeast URA3 gene into the cloned transposons; and 3) replacement of the chromosomal HIS4 gene with the modified genes constructed in vitro. These strains will be extremely useful in the study of Ty transposition and other Ty-promoted DNA rearrangements.     

Analysis of chromosome mobilization using hybrids between plasmid RP4 and a fragment of bacteriophage lambda carrying IS1

In vitro recombination was used to generate RP4 plasmids with an inserted restriction fragment of bateriophage λ. In some cases the λ DNA also carried the insertion sequence IS1. Comparisons were made between the abilities of these plasmids to mobilize the Escherichia coli K-12 chromosome in different genetic backgrounds. RP4-borne IS1 acting alone promoted chromosome transfer but with an efficiency 1% of that resulting from more extensive plasmid-chromosome homology. A recA mutation in the donor depressed the mobilization frequency below the level of detection. Correlation of the direction of chromosome transfer and the orientation of the cloned λ DNA allowed the direction of RP4 transfer to be determined. Studies on recombinants showed that in general they also acquired an intact, autonomous plasmid, suggesting the process of mobilization by RP4 may differ in certain features from chromosome transfer by F.