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.     

Restriction and electron microscopic analyses of deletion derivatives thermosensitive with respect to maintaining plasmid pEG1

Temperature-independent deletion mutants of temperature-sensitive in self-maintenance plasmid pEG-1, derived from R-factor RP4, are mapped using the restriction endonucleases PstI, SmaI, EcoRI, BamHI and the heteroduplex analysis. The pEG1 derivatives under study are found to have deletions in the area of ampicillin transposon Tn1 and nearby genes. The left and the right ends of these deletions boundaries are localized between 37.5 MD and 7.6 MD in the RP4 map. Thus far, the area of plasmid RP4 (37.5-7.6 MD) with Tn1 and, presumably, inc gene(s) in it does not have any genes needed for stable maintenance of R-factor. A conclusion is made that the gene RP4, which carries the mutation determining thermosensitive character of pEG1 maintenance and of inhibition of the cell growth, is localized between the EcoRI site and transposon Tn1 at a distance less than 1.0 MD from the latter.     

Function of the InsA gene in the IS1 element of the Tn9' transposon: influence of oligonucleotide inserts in the InsA gene on formation of simple insertions and plasmid cointegrates]

To elucidate the role of the insA reading frame in transposition of the IS1 element of the Tn9' transposon, the derivatives of plasmids pUC19::Tn9' and pUC19::IS1 have been obtained using oligonucleotide inserts of the length equal or exceeding 9 bp and equal to 10 bp. The ability of mutant variants of the Tn9' transposon and the IS1 element to form simple insertions and plasmid cointegrates was studied. To this end, experiments were performed on mobilization of the derivatives of pUC19 containing mutant variants of the IS1 element and Tn9' as well as of the plasmids pUC19::Tn9' by the conjugative plasmid pRP3.1. According to the data obtained, mutations (inserts) in the insA gene have no influence on the frequency of transposition of the IS1 element and Tn9' from the plasmid pUC19 to pRP3.1. At the same time, the frequency of transposition events of mutant variants of Tn9' from the plasmid pRP3.1 to pBR322 is more than 10 times lower in comparison with the wild type transposon. The data obtained are in accordance with the assumption that the insA gene is not essential for transposition. A hypothesis is put forward explaining the role of the insA gene product in the process of bringing together short inverted repeats of the IS1, which are the sites for the transposase to be recognized at first stages of transposition.     

Study of the role of the insA open reading frame in the IS1 insertion element structure during transposition and resolution of the IS1 mediated cointegrates

In order to elucidate the function of the IS1 insA gene derivatives of plasmid pUC19::Tn9' with insertions of synthetic oligonucleotides were obtained. The latter are equal or multiple of 9 b.p. in length and are located in the Pst1 site within each of the two IS1 copies of the Tn9' transposon. The insertions of the nine base oligonucleotides code for the neutral amino acids and do not shift the reading frame. One of the mutant transposon obtained - Tn9'/X was studied on the ability to form simple insertions and plasmid cointegrates. For this purpose the pUC19 derivatives carrying the wild type and mutant transposon were mobilized by conjugative plasmid pRP3.1. It was found that the damage of the insA gene does not influence the ability of transposon to form simple insertions and plasmid cointegrates in both recA - and rec+ cells of E. coli. However, the frequency of the cointegrate formation in the subsequent transposition of the mutant transposon from pRP3.1::Tn9'/X to pBR322 was by 10-20 times lower in comparison to the wild type transposon. Instable (dissociating) Tn9'/X-mediated plasmid cointegrates formed by interaction pUC19::Tn9'/X and pRP3.1 were obtained. It was shown that in the E. coli recA-cells such cointegrates dissociate, as a rule, "correctly", i.e. they segregate mainly plasmids of types pUC19::Tn9'/X and pUC19::IS1/X. The data obtained correspond with the notion that the gene insA product is not essential for transposition, but is, possibly, involved in the formation of the IS1-generated deletions.     

Homology of plasmids with a wide range of hosts

According to blotting hybridization and heteroduplex analysis, plasmids R751, R906 and RP4 of Inc Pi group have continuous regions of homology. These homologous regions were mapped on the R751 and RP4-derived pRP401 deletion mutant DNAs. The plasmid pRP401 (m.w. 21.9 kg) retains the broad host range property and has two regions of intensive homology with other Inc P-1 plasmid DNAs. These regions are localized at 8.2-12.0 kb and 13.9-21.9 kb of the physical map of pRP401 plasmid. Homologous regions of pRP401 DNA include at least the replication genes (oriV, trfA, trfB) as well as genes kilB, korA, korB and probably kilC. The data strongly point out that the broad host range plasmids have the same principle of structural and functional organization.     

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.     

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.     

Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application 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.     

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.     

Temperature-sensitive replication of plasmid pKM101

A mutant of Escherichia coli K-12, IB10 carrying the ts10 mutation has been isolated. The mutation affects replication and inheritance of pKM101 plasmid. Incubation of the mutant under non-selective conditions of 42 degrees C resulted in the formation of R-cell population. The frequency of temperature-independent clones was 2,1 X 10(-5). The defect of pKM101 replication was shown to result in growth inhibition of host cells at a non-permissive temperature. The host growth only started after elimination of the plasmid. The mechanisms are likely to exist governing the participation of plasmid gene products in processes related to host growth. The influence of ts10 mutation on replication of other plasmids was studied. It was established that ts10 did not affect replication of R6K, RP4 and Flac+ plasmids. However, replication of R15, R205 as well as of pKM101 plasmid stopped under conditions of non-permissive temperature in IB10 mutant. Obviously, ts10 mutation results in defective replication of plasmids only belonging to the N-incompatibility group (IncPN). It is shown that R6K, RP4, Flac+ plasmids are not able to correct pKM101 replication in the mutant at 42 degrees C.     

质粒RP4CFIts突变体筛选及Kil表型分析

用羟胺体外诱变质粒RP4后再转化受体菌,我们选择到一抹温度敏感的菌落形成抑制突变体。其表型为在非允许温度时培养,不能由单个菌体繁殖为集落,类似于Kor基因失活引起的Kil表型。生化分析表明:在非允许条件下,突变体的生长逐步受到抑制,大约繁殖两代后即全面抑制,此时宿主菌的DNA、RNA、蛋白质等大分子合成几乎全部下降,但β-半乳糖苷酶的合成却并未停止,只是由于细胞通透性改变而大部分排出胞外,另外噬菌体感染后的产率也明显降低,并且在非允许温度处理20h内,此种状态是完全可逆的,即仍可因放回允许温度培养而形成集落,据此抑制现象的逐步发生性,大分子合成的全面抑制性和完全可逆性判断,很可能和宿主细胞在紧急状态下的整体调节机制——严紧型反应有关。     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4

Summary Plasmid pRP761 is a derivative of the promiscuous plasmid RP4, which has a Tn76 insert 1.8 kb from its EcoRI site within the trfB region (Barth 1979). This mutation was pleiotropic, having three effects: the plasmid is unstably maintained in E. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. We show that pRP761 has reduced expression from both its korA and korB genes and that Tn76 has inserted between them. Fragment exchange experiments showed that this is the only mutant region in pRP761 and is therefore solely responsible for the pleiotropic effects. A spontaneous deletion derivative pRP761-6 has lost Tn76 and its adjacent kilA and korA genes: it has reacquired stability, does not inhibit host growth but is still reduced in its host-range. The provision of cloned korA ⁺ in trans complements the first two phenotypic effects in pRP761 to a large extent, but neither korA ⁺ alone nor korA ⁺ with korB ⁺ complements the host-range reduction in pRP761 or pRP761-6. A possible explanation for these results is that there is a site between korA and korB, affected by the Tn76 insert, that is essential to stable replication of these plasmids in some of their bacterial hosts.     

Mechanism of defective lysogenization by phage P1 in a lon-mutant of Escherichia coli K-12.

Phage P1 cannot lysogenize a lon- mutant of Escherichia coli K-12, which is defective in the regulation of cellular division cycle to result in snake formation (14). P1 mutants, called P1pla, can lysogenize the lon- host. These mutations have been classified into two complementation groups: one is cis-dominant; the other is trans-dominant. A temperature-sensitive lon- mutant was isolated, which exhibited the lon- phenotype at 42 C but not at 33 C. A temperature-shift experiment of the P1-lysogenic derivative of the lon- ts mutant showed lysis of the culture and induction of the phage production. It is proposed that P1 plasmid may be under a certain regulatory circuit of the division cycle of the host bacterium by indirectly regulating the production of P1 immune repressor, or alternatively by directly derepressing the functions of P1 prophage.     

Integration of the RP4 factor with the chromosome in Escherichia coli K12 and the formation of 2 types of Hfr-strains]

The mutant RP4ts12, derived from the R-factor RP4 and thermosensitive in replication, is incorporated into the chromosome A3dna(ts) of E. coli K12, thus suppressing dnaA mutation. The integration of this factor into the chromosome leads to the formation of Hfr strains of two types: the strains of the first type transfer plasmid markers to recipient cells earlier than to chromosomal ones; the strains of the second type transfer plasmid markers to recipient cells after chromosomal ones. During conjugation the R-factor integrated into the chromosome dissociates from chromosomal DNA introduced into the recipient cell and becomes autonomous.     

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.     

Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co-integrates

The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA-independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1-mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.