Isolation and characteristics of a temperature-sensitive plasmid pRP19.6--an RP1 derivative containing the duplicated IS21 sequence

When analysing the antibiotic resistant, temperature-independent derivatives of Proteus mirabilis cells, carrying the plasmid RP1ts12, a derivative of the latter (pRP19.6) with an elevated frequency of integration into E. coli K12 chromosome, has been isolated. The structure and properties of pRP19.6 was studied. As revealed from the data of structural and genetic analyses pRP19.6 is identical to the factor R68.45 described earlier by Haas and Holloway. Similarly to R68.45, the plasmid under study contains two copies of IS21 sequence and mobilises nonconjugative plasmid pBR325 with high efficiency. Using the temperature sensitive replication of pRP19.6, frequency of it's integration into the chromosomes of E. coli rec+ and recA- stains is determined. It is demonstrated that the clones carrying the plasmid in integrated state are Hfr-strains. The possibilities to use the temperature sensitive R68.45 like plasmid for isolation of Hfr-strains in the broad range of gram-negative bacteria and for insertional inactivation of chromosomal genes are discussed.     

Integration of plasmid RP1 with the chromosome of Escherichia coli K-12 recA. 2 classes of Hfr strains

Integration of broad host range RP1 plasmid into the chromosome of Escherichia coli K-12 recA- cells has been studied. Using temperature-sensitive for replication plasmids pVD1 and pVD3, the derivatives of RP1, it has been shown that integration of RP1 into the bacterial chromosome results in formation of two classes of Hfr strains. Properties of these Hfrs have been examined. From the data obtained, it has been concluded that the plasmid integration and formation of one of the Hfrs classes appear to be mediated by transposon Tn1 residing on RP1. The other class of Hfr strains is formed due to a stable integration of RP1. In the course of analysis of R+ transconjugants arising at low frequency in crosses between stable Hfrs and E. coli rec+ recipients, it has been found that the significant part of them contain plasmid-chromosome hybrids (R-prim plasmids). On the basis of the latter results, a new simple method for R' plasmids selection has been proposed. Using restriction endonuclease analysis, the structure of plasmids that were excised from chromosomes of the stable Hfr strains and were comparable in their size to RP1, has been investigated. Probable mechanisms of the stable Hfr strains formation are discussed.     

Genetic control of the formation of plasmid F'. I. Effect of recA- and seg-2 mutations in an Hfr donor strain on the character of plasmid F' formation]

Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

Formation of stable Hfr strains in R factor RP1 integration with the chromosome of E. coli K12 recA and their use for R-plasmid selection

Analysis of thermoindependent derivatives of E. coli K12 JC1553 recA (p VD1) carrying a replication thermostable mutant pVD1 of R factor RP1 IncP Ap Km Tc showed that formation of about 5 per cent of them was associated with stable integration of the plasmid with the bacterial chromosome. The respective bacteria had the following features: (1) preserved all the markers of plasmid pVD1, (2) according to the data of the electrophoretic analysis had no extrachromosomal DNA on prolonged cultivation under nonselective conditions, (3) were effective donors of the chromosomal genes, (4) had a low rate of the plasmid marker transfer on crossing with R- recipient. The latter feature was suggested to be used as a test for identification of stable Hfr strains. Investigation of the properties of the transconjugants obtained on crossing of stable Hfr strains with R-recipients rec+ showed that same of them had plasmid DNA with a higher molecular mass as compared to that of plasmid pVD1 DNA. The presence of this DNA was connected with formation of R' plasmid as a result of an irregular exclusion of plasmid pVD1 from the chromosome of stable Hfr bacteria. On the basis of the results a simple method was proposed for selection of R' plasmids having a number of advantages over the classical ones. The perspectives of using thermostable derivatives of RP1 for cloning the chromosome genes are discussed.     

Immunity to repeated transposition of the insertion sequence IS21

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.     

Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome

R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.     

A method of plasmid classification by integrative incompatibility

A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the rec A function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these rec A Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 10^?2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility was coded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. This complicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of rec-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

Characteristic features of the structure of co-integrating plasmids and simple insertions formed during transposition of the IS1 element in transposon Tn9'

Earlier we have studied unstable dissociating IS1/Tn9'-mediated cointegrates between the plasmids pDK57 (pBR322::Tn9') and pRP3.1, a deletion derivative of RP1, and two types of such cointegrates containing three and four copies of IS1 were revealed. In the present paper we studied the structure of stable IS1/Tn9'-mediates cointegrates and simple insertions formed by interaction between the plasmids pDK57 and pRP3.1 in the E. coli recA- cells. It was shown, that the stable cointegrates were formed by insertion of pDK57 in different loci of pRP3.1 and these cointegrates contain three copies of IS1, i.e. one copy of IS1 and a copy of Tn9' at the junction of the two replicons. The cointegrates are formed predominantly due to the activity of the left copy of Tn9', which occupies a proximal position in regard to the promoter of the cat gene. It was found that the integration of pDK57 into the kan gene region of pRP3.1 leading to the formation of the KmS cointegrates occurs only in one of the two possible orientations. Meanwhile the insertions of the transposon Tn9' into the kan region of pRP3.1 leading to simple insertions occurs in the orientation opposite to the orientation of the transposon in the KmS cointegrates. It is proposed that simple insertions are not the products of direct transposition of Tn9', but they are formed from unstable cointegrates under the action of IS1-specific resolvase.     

Spontaneous deletions of the chromosome-mobilizing plasmid R68.45 in Pseudomonas aeruginosa PAO

In Pseudomonas aeruginosa strain PAO the chromosome-mobilizing IncP-1 plasmid R68.45 was unstable whereas the parent plasmid R68 was stable. The instability of R68.45 was observed in rec+ and rec strains within about 100 generations after conjugal transfer of the plasmid and, to a lesser extent, in established R68.45 donor strains. Two phenotypically distinct classes of R68.45 derivatives were obtained: (i) CbR (carbenicillin-resistant), TcR (tetracycline-resistant), KmR (kanamycin-resistant), Tra+ (transfer proficient), Cma- (chromosome-mobilizing ability), lacking the duplicated IS21 copy typical of R68.45 and indistinguishable from R68 by restriction enzyme analysis; (ii) CbR TcR KmS Tra- Cma-, due to deletion of one IS21 copy, the adjacent KmR gene, and a variable part of the Tra-1 region including, in most cases, the origin of transfer (oriT). Both types of deletion derivatives were stable. R68.45 derivatives lacking the Tra-2 region were not recovered spontaneously, but could be constructed in vitro and were stable in strain PAO. Deletion formation of type ii as well as Cma did not depend on homologous recombination and can be ascribed to functions of the duplicated IS21. Chromosome mobilization does not appear to require obligatory transfer of the entire R68.45 plasmid. Four ClaI restriction sites were mapped on R68 extracted from P. aeruginosa. One of these sites was cryptic, presumably because of methylation, when the plasmid was prepared from Escherichia coli (dam+).     

Replication of Fpoh+ plasmid in mafA mutants of Escherichia coli defective in plasmid maintenance.

A class of F' plasmids, designated Fpoh+, was previously shown to be able to replicate extra-chromosomally on Hfr strains by virtue of carrying the specific site or region poh+ (permissive on Hfr) of the E. coli chromosome (Hiraga, 1975, 1976a). These plasmids were now found to replicate on E. coli mafA mutants (mafA1 and mafA23) that cannot support vegetative replication of F and some other F-like plasmids. The derivatives of Fpoh+ that have lost the poh+ site, on the other hand, failed to replicate on mafA mutants. These mutants harboring Fpoh+ (but not Poh- derivatives thereof) exhibit abnormal cell division and form elongated cells, presumably due to competition between Fpoh+ and the host chromosome for some factor(s) essential for the initiation of DNA replication of the both replicons. It is tentatively concluded that the poh+ site is required for F' plasmids to replicate on mafA mutants as well as on Hfr strains. In view of the fact that the mechanism of inhibition of autonomous F DNA replication in mafA mutants and in Hfr strains are clearly different, the present data seem to provide strong support to the notion that the poh+ region contains the replication origin of the E. coli chromosome.     

Replication control of IncP plasmids

Clones of Escherichia coli with a chromosomally integrated RP4-prime plasmid were isolated and characterized. Chromosome transfer was increased about 50-fold and the Hfr still carried an autonomous plasmid indistinguishable from the original RP4-prime. This could be eliminated by pRP64 or R751, two distinguishably marked incompatible plasmids, giving rise to strains which stably retained the resistance patterns of both plasmids and which continued to transfer the chromosome at enhanced levels. In both cases, however, the copy number of the autonomous plasmid was reduced by the presence of a chromosomal RP4 such that the total number of P plasmid genomes (integrated and autonomous) remained constant. The results are consistent with the idea that copy number is controlled by diffusible inhibitors or initiators of replication.     

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.     

Dissociation of unstable cointegrate plasmids formed during transposition of IS1 element: the role of IS1 encoded recombinase

The properties of IS1/Tn9'-mediated cointegrates between plasmids pDK57 (pBR322:: :: Tn9') and pRP3.1--the deletion derivative of RP1 were investigated. It was found that IS1/Tn9'-mediated integration of pDK57 into the active transcribed regions of pRP3.1 (in particular kan and tet genes) leads to formation of unstable cointegrates capable of resolving in E. coli K-12 rec+ and recA cells. The structure of dissociation products of unstable cointegrates was studied. According to the data received in rec+ cells, the unstable cointegrates mainly produced plasmids pDK57 and pBR322::IS1--Cms-derivative of pDK57 as resolution products. In recA cells the cointegrates dissociate in different ways, and this process leads to the formation of not only pDK57 and pBR322::IS1, but also to the production of the deletion derivatives of these plasmids as well as to the derivatives of pDK57 and pBR322::IS1, containing duplications of IS1 or separate parts of Tn9'. It was concluded that the IS1-specific recombinase is involved in the dissociation (resolution) of unstable IS1/Tn9'-mediated cointegrates. This recombinase recognizes the sites localized in both inverted termini of IS1 as well as in the adjacent DNA segments. Hence, it is possible, that the IS1 recombinase is involved also in the generation of IS1-adjacent delations.     

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.     

The sym plasmid pRP2JI and at least two other loci of Rhizobium leguminosarum biovar phaseoli can confer resistance to infection by the virulent bacteriophage RL38

Abstract The virulent Rhizobium bacteriophage RL38 did not form plaques on R.leguminosarum by phaseoli but did so at high efficiency on a derivative of that strain lacking its symbiotic plasmid pRP2JI. Other strains with large deletions in pRP2JI which removed many nod and nif genes retained resistance to RL38, showing that the gene which confers phage resistance lies elsewhere on the plasmid. Although the wild-type strain of R. leguminosarum bv. phaseoli failed to plate RL38, it was possible to transduce chromosomal markers into this strain, indicating that the 'block' was not at an early stage in the infection process. Two different recombinant plasmids obtained from a clone bank of genomic DNA of R. leguminosarum bv. phaseoli , which appeared to have no DNA in common, both conferred resistance to RL38. Surprisingly, the DNA cloned in each of these plasmids did not originate from pRP2JI. Therefore, several different loci both on the Sym plasmid and elsewhere on the bacterial genome can be involved in conferring resistance to this bacteriophage.