Effects of Inhibition and Restoration of Protein Synthesis on the Replication of the R Factor Rts1 in Proteus mirabilis

The effect of inhibition of protein synthesis on the replication of the R factor Rts1 in Proteus mirabilis was examined by using the technique of CsCl density gradient centrifugation. Only 12% of the copies of Rts1 were found to replicate during amino acid starvation, whereas there was a 30% increase in the amount of P. mirabilis chromosomal deoxyribonucleic acid (DNA) during the same period. Essentially the same amount of Rts1 and host chromosome replication was observed when chloramphenicol was used to inhibit protein synthesis. The replication of Rts1 DNA was also examined in experiments in which cultures were starved for amino acids in (14)N-labeled medium and then transferred to (15)N-labeled medium containing the required amino acids. These experiments showed that Rts1 replication took place throughout the first generation in (15)N-labeled medium and that each copy of Rts1 was replicated one time during the first generation of chromosomal DNA synthesis in (15)N-medium.     

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 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.     

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.     

Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae.

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.     

Conjugation in Rhodopseudomonas sphaeroides mediated by R plasmids

Plasmids R68.45, RP4, RP4::Mu cts62, RP1ts::Tn10, RP1ts::Tn9, Rts1 and RP41 were transferred into cells of photosynthetic nitrogen-fixation bacterium Rhodopseudomonas sphaeroides from Escherichia coli and Pseudomonas aeruginosa. The transfer of plasmids occurred with high frequency of 10(-1) to 10(-2) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell in all cases. Mobilization of R. sphaeroides 2R chromosome was obtained by RP4 and Rts1 plasmids at a frequency of 10(-7) to 10(-8) per donor cell. Bacteriophage Mu cts62 could be induced from the plasmid DNA in R. sphaeroides 2R cells and was capable of the lytic growth and producing phage progeny. It was demonstrated that an increase in the efficiency of donor chromosomal genes transfer into recipient cells could be achieved in crosses with the donor carrying RP4::Mcts62 plasmid.     

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.     

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.     

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.     

The role of cyclic AMP in the thermosensitive lesion of the formation of closed covalent circular Rts 1 DNA.

The formation of closed covalent circular DNA of R factor, Rts 1, does not take place at non-permissive temperature, 42°C, in $\text{E. coli}$ 20SO. However, when Rts 1 was placed in mutants having a low level of cyclic AMP or lacking cyclic AMP receptor protein, the thermosensitive lesion is overcome. Addition of cyclic AMP caused inhibition of the formation of ccc DNA in mutants with low cyclic AMP level, but not in mutants lacking cyclic AMP receptor protein.     

Molecular Nature of the Deoxyribonucleic Acid of a Thermosensitive R Factor, Rts1

The deoxyribonucleic acid of the thermosensitive R factor, Rts1, has been examined by the technique of sedimentation in alkaline sucrose, electron microscopy, and radiation target size. All these methods yielded a molecular weight of approximately 120 million for Rts1 deoxyribonucleic acid in Escherichia coli. Sedimentation analysis revealed that Rts1 deoxyribonucleic acid in Proteus mirabilis was also 120 million daltons. Rts1 did not segregate into E. coli minicells under the conditions where another smaller non-thermosensitive R factor could.     

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.     

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.     

Bacteriophage T4-related macromolecular synthesis under restriction of plasmid Rts1.

Rts1 is a plasmid which confers upon the host bacteria the capacity to restrict T4 bacteriophage growth at 32 degrees C but not at 42 degrees C. Pulse-labeling of phage-infected cells showed that Rts1 restricts the synthesis of T1 DNA. Despite efficient restriction of T4 phage growth and DNA synthesis, infected Escherichia coli 20SO harboring Rts1 synthesized both early and late T4 phage RNA. Synthesis of early T4 phage RNA under restrictive conditions (32 degrees C) was almost equal to that found under nonrestrictive conditions, and a lesser, but significant, amount of late T4 phage RNA was made in almost complete absence of T4 DNA synthesis. Moreover, very little, if any, T4 phage-coded lysozyme was detected in the infected E. coli 20SO/Rts1 at 32 degrees C, whereas normal amounts of lysozyme were present 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.     

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.     

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.     

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.     

The kanamycin resistance transposon Tn2680 derived from the R plasmid Rts1 and carried by phage P1Km has flanking 0.8-kb-long direct repeats

Phage P1Km carries within the invertible DNA segment a 5-kb insertion with 0.8-kb terminal direct repeats flanking the kanamycin resistance determinant. The same structure was also found on the R plasmid Rts1, from which the Km resistance segment of P1Km was derived. Obviously, this Km resistance segment translocated as a unit to the P1 genome and it is therefore called Tn2680. Loss of the Km resistance determinant due to recombination between the flanking direct repeats occurs during vegetative growth of P1Km. Amplification of Tn2680 to tandem oligomers is documented and is thought to result from recombination between the flanking direct repeats. The flanking 0.8-kb repeats are different from known IS elements.     

The region controlling the thermosensitive effect of plasmid Rts1 on host growth is separate from the Rts1 replication region.

Rts1 is a high-molecular-weight (126 x 10(6)) plasmid encoding resistance to kanamycin. It expresses unusual temperature-sensitive phenotypes, which affect plasmid maintenance and replication, as well as host cell growth. We have cloned the essential replication region of Rts1 from pAK8, a smaller derivative which is phenotypically similar to Rts1. Restriction endonuclease digests of isolated pAK8 deoxyribonucleic acid were allowed to "self-ligate" (ligation without an additional cloning vector) and subsequently were used to transform Escherichia coli strain 20SO to kanamycin resistance. Screening of these strains for the phenotypes of thermosensitive host growth and temperature-dependent plasmid elimination demonstrated that these two properties were expressed independently. Furthermore, it was shown that the Rts1 replication locus per se is not necessarily responsible for altered host growth at the nonpermissive temperature. The kanamycin resistance fragment of pAK8 was also cloned into pBR322. Electrophoretic analysis of BamHI restriction enzyme digests of this plasmid and similar digests of an Rts1 miniplasmid has allowed the identification of an 18.6-megadalton fragment carrying the replication locus and a 14.1-megadalton fragment carrying the kanamycin resistance gene.