Integration of R plasmid Rts1 to the gal region of the Escherichia coli chromosome.

An R plasmid Rts1 was integrated into the gal region of the chromosome of Escherichia coli XA-7012 (galE) strain by the directed transposition technique. The integration of the Rts1 genome was confirmed mainly by conjugation studies and also by transduction experiments using phage P1. As a result, it was found that the integrated genome contained genes responsible for kanamycin resistance, conjugal transferability, and for autonomous replication. As reported previously, Rts1 is temperature sensitive in replication and inhibits the growth of the host at nonpermissive temperature. However, although a plasmid derived from the integrated Rts1 genome still demonstrates temperature sensitivity upon transfer and high level of kanamycin resistance, this plasmid no longer displays temperature sensitivity in replication and the inhibitory effect on the host. These results indicate that the temperature sensitivity of replication of Rts1 and its inhibitory effect on the host cell are due to the presence of a gene or gene cluster on the Rts1 genome and that the gene(s) is clearly discriminated from the one responsible for the temperature sensitivity of transfer.     

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.     

Instability of Rts1 (Drug-Resistant Factor) Replicon: Stabilization by DNA Fragments Derived from Rts1

Rts1 is a large naturally occurring plasmid which has a kanamycin resistance gene and exhibits various temperature-sensitive phenotypes. A smaller derivative of plasmid, pOK, contains the Rts1 replicon and the kanamycin resistance gene of Rts1. This plasmid, pOK, is much more unstable than Rts1 at 42.5°C. A DNA fragment, G₃, 1590 nucleotides long from Rts1 DNA, stabilized pOK completely at 42.5°C but only in thecisconfiguration. G₃did not change the copy number of pOK. The pOK derivative containing G₃was destabilized by the presence of a compatible plasmid containing G₃. G₃has four inverted repeats, two 14-base direct repeats, and three ORFs. Smaller fragment of G₃also had a stabilization effect and these studies showed that the ORF does not play any role in stabilization.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria.

Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.     

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.     

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.     

Control of replication and segregation of R plasmid Rts1.

A mutant plasmid, pTW2, which was derived from the integrated Rst1 genome in the Escherichia coli chromosome, was studied as to its mode of replication at 30 degrees C. When Proteus mirabilis Pm17 harboring pTW2 was grown in broth at 30 degrees C, a considerable number of R- segregants (approximately 40%) were consistently observed. This indicates that pTW2 is unstable even at the permissive temperature for the replication of Rts1. The pTW2+ cells in a culture were heterogeneous with respect to the level of kanamycin resistance, ranging from 500 to 4,000 mug of the drug per ml. The amount of pTW2 deoxyribonucleic acid (DNA) relative to the Pm17 chromosomal DNA was about fivefold as large as that of Rts1 DNA in an exponentially growing culture. In addition, pTW2 in P. mirabilis continued to replicate after the chromosome had ceased to replicate, which was shown in the study of the inhibition of protein synthesis. Contrary to pTW2, the parent plasmid Rts1 is highly stable, and the relative percent Rts1 DNA is maintained at approximately 7% in any cultural conditions at a permissive temperature. These results suggest that copies of pTW2 may not segregate evenly into the host progeny upon cell division and that the replication of pTW2 does not coordinate with that of the chromosome. A remarkable instability of pTW2 as well as an increase in the relative percent pTW2 DNA was also shown when E. coli were used as the host cells. These results suggest the possibility that there is a gene or a gene cluster on the Rst1 genome responsible for the control of both replication and segregation of Rts1.     

Acceptance and transfer of plasmid Rts1 by bacteria of the genus Erwinia]

The ability of 13 Erwinia strains to accept, to inherit and to transmit the Rts1 factor by conjugation was studied. 11 strains accepted the Rts1 factor from Escherichia coli K-12 CSH-2 with the frequency of about 10(-7)--10(-3). The Rts1 factor was genetically stable in the Erwinia cells and was not eliminated by acriflavine and under the temperature of 37 and 42 degrees C. All the R+ exconjugants were characterized with more high degree of the resistance of kanamycin than E. coli cells harbouring the same R factor. Erwinia strains harbouring the Rts1 plasmid transferred it by conjugation into homologic (Erwinia) and heterologic (E. coli) bacteria. The study of kinetics of the transfer of the Rts1 factor in different mating systems showed that the transfer of this plasmid from R+ Erwinia into R- Erwinia and R- E. coli--in the liquid medium. It is concluded that Erwinia can be the host and the donor of the Rts1 factor.     

Replication of the R Factor Rts1 in Proteus mirabilis

The replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density-gradient centrifugation. The proportion of Rts1 deoxyribonucleic acid (DNA) relative to the host chromosomal DNA (% R-DNA) was 7% in both exponential and stationary growth phases in Penassay Broth and supplemented M9 minimal medium at 30 C. The chromosomal DNA content per cell varied over a threefold range in the different growth media. In agreement with previous genetic observations, the replication of Rts1 was found to be temperature-sensitive and Rts1 DNA was diluted from the cells during exponential growth at 42 C. (14)N-(15)N medium transfer experiments have shown that individual copies of Rts1 are selected at random for replication during the duplication of the multicopy episome pool.     

Further characterization of the R plasmid Rts1 and its mutant pTW2: replication and incompatibility of the plasmid.

Incompatibility of the R plasmid Rts1 and its replication mutant pTW2 was studied in recA host cells of Escherichia coli. When the R plasmid R401, belonging to the same incompatibility group as Rts1, was used as a test plasmid, R401 was eliminated preferentially from (Rts-R401)+ cells irrespective of the direction of transfer. In contrast, pTW2 and R401 were mutually excluded. The decreased incompatibility of pTW2 was confirmed by a direct incompatibility test in which a derivative of Rts1 expelled pTW2 exclusively. Alkaline sucrose gradients of pTW2 and Rts1 DNA indicated that approximately one-fourth of the Rts1 genome was deleted in pTW2. In addition, both the various temperature-dependent properties of Rts1 and the inhibitory effect on phage T4 development were also lost in pTW2. A possible mechanism that regulates the stringent replication of Rts1 is discussed.     

Replication of thermosensitive Rts1 plasmid deoxyribonucleic acid at the nonpermissive temperature.

Replication of the thermosensitive drug resistance factor Rts1 was studied at the nonpermissive temperature (42 degrees C). It was concluded from the following observations that replication of this plasmid takes place at 42 degrees C without involving the covalently closed circular (CCC) form of deoxyribonucleic acid (DNA). (i) DNA-DNA- reassociation kinetics studies with purified Rts1 DNA showed that Rts1 DNA increased several-fold during cell growth at 42 degrees C while very little, if any, CCC DNA was synthesized. (ii) When Escherichia coli 20S0(Rts1) was labeled with [3H]thymidine at 42 degrees C, a significant amount of radioactive DNA hybridizable to Rts1 DNA was formed. This DNA was found in a fraction where DNA other than CCC DNA was expected in alkaline sucrose density gradient centrifugation analysis. When E. coli 20S0(Rts1) was labeled at 32 degrees C, the labeled CCC DNA did not disappear during a chase period at 42 degrees C. This indicates that preformed CCC DNA does not participate in replication at the nonpermissive temperature. These results are consistent with the hypothesis that there are two modes of replication of Rts1 DNA, one involving a CCC molecule and the other not involving this form, and that only the latter mode takes place at the nonpermissive temperature.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Complete nucleotide sequence of plasmid Rts1: implications for evolution of large plasmid genomes

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

An Rts1-derivative plasmid conferring UV sensitivity on Escherichia coli host

A deletion mutant was isolated from a kanamycin resistance R plasmid Rtsl. This mutant plasmid, pTW20, was found to enhance the lethal effect of UV irradiation on $\text{Escherichia}\text{coli}$ host, especially at 42°C. A cloning experiment with pTW20 DNA demonstrated that the gene, $\text{puv}$, being responsible for the UV sensitivity was located on the kanamycin resistance gene containing $\text{Bam}$H1 fragment of pTW20. This fragment conferred a sensitivity to methyl methane sulfonate on its host along with the sensitivity to UV, suggesting that a reapir process of the host chromosome is impaired by the presence of $\text{puv}$.     

Nucleotide sequence and copy control function of the extension of the incI region (incI-b) of Rts 1

An Rts1 derivative, pTW20, contains three incompatibility (inc) regions, incI-a (incI in previous studies), incII, and newly determined incI-b loci. By restriction analysis, we have located the incI-b adjacent to the incI-a region on the pTW20 map. Nucleotide sequence analysis of the minimal incI-b region revealed the presence of four repeated sequences, each consisting of 18 bp, which is similar to the incI-a and incII repeats existing on mini-Rts1. All four repeating units were required for expression of a strong incompatibility. In addition, RepA protein, essential for the replication of Rts1, bound specifically to the repeated sequences, suggesting that the repeats would titrate out RepA protein as do incI-a and IncII. Insertion of the incI-b to a mini-Rts1 plasmid in a natural arrangement decreases the copy number of mini-Rts1 to the same level as that of mini-F. The incI-a and incI-b might be a single constituent in incompatibility and copy number control of Rts1.     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

Isolation of the kanamycin resistance region (Tn2350) of plasmid R1drd-19 as an autonomous replicon.

We have isolated a circular form of Tn2350, an IS1-flanked kanamycin resistance transposon forming part of the plasmid R1drd-19. This circle (pTn2350::9.6 kilobases) contains a single IS1 element and probably arises by recombination between the two directly repeated Is1 sequences of Tn2350. It can be used to transform Escherichia coli to kanamycin resistance. It is capable of autonomous replication but is not maintained stably in dividing cells and segregates under nonselective conditions. Cloning of a segment of pTn2350 on a conditional plasmid vector allowed us to assign the replication functions of this plasmid to a 1.6-kilobase restriction fragment. The plasmid R1drd-19 can thus be considered as a cointegrate between two replicons separated by IS1 sequences.     

Structural and functional analysis of cloned DNA segments containing the replication and incompatibility regions of a miniplasmid derived from a copy number mutant of NR1.

A 1.45-megadalton segment of DNA cloned from a miniplasmid derived in vivo from a copy number mutant of the R plasmid NR1 has been shown to contain all functions essential for incompatibility and autonomous plasmid replication in Escherichia coli. Specific endonuclease cleavage sites within this DNA segment that localize functions required for replication have been mapped. A 0.45-megadalton fragment that specifies the FII incompatibility of NR1 has been identified within the replication region, and DNA fragments containing this incompatibility region, but lacking other functions required for replication, have been cloned.