DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli.

The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.     

The poison–antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison–antidote plasmid stability system. The three genes were named pasA , pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison–antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison–antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.     

Termination of DNA replication in Escherichia coli requires a trans-acting factor.

The terminus region of the Escherichia coli chromosome contains two sites that inhibit the progression of DNA replication forks. These termination sites, designated T1 and T2, are separated by 7.5 min (350 kilobases [kb]) on the genetic map and are located at the extremities of the terminus region. They demonstrate polarity (they stop replication forks traveling in one direction but not the other) and inhibit replication forks that have passed through and are about to leave the terminus. We have used deletion mutations in the terminus region to map the locations of T1 and T2 more accurately and to initiate studies on the mechanism of replication fork inhibition. We have narrowed the boundaries of T1 and T2 to 20 and 4 kb, respectively. T1 maps between kb 80 and 100 on the physical map of the terminus region (J. P. Bouché, J. Mol. Biol. 154:1-20, 1982), and T2 maps between kb 438 and 442. In addition, we report here that deletion of the region containing the T2 termination site inactivated T1. Supplying the T2 region on a plasmid restored T1 function, demonstrating that inhibition of replication at T1 requires a trans-acting factor which maps in the vicinity of termination site T2. We have called this newly identified terminus function the termination utilization substance (tus).     

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.     

Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance.

The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.     

Re-examination of F plasmid replication in a dnaC mutant of Escherichia coli.

Summary The replication of an F plasmid in a dnaC mutant, thermolabile for initiation of chromosomal replication, has been re-examined using a novel DNA-DNA annealing assay. Plasmid replication ceases rapidly at non-permissive conditions, consistent with a direct role for the dnaC product in the replication of F.     

Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2.

A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated. The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively. Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found. The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E. coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine. In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA). A region containing res-like sites was located immediately upstream of the partial tnpR gene. Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans. Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.     

Host controlled plasmid replication: Escherichia coli minichromosomes

Escherichia coli minichromosomes are plasmids replicating exclusively from a cloned copy of oriC, the chromosomal origin of replication. They are therefore subject to the same types of replication control as imposed on the chromosome. Unlike natural plasmid replicons, minichromosomes do not adjust their replication rate to the cellular copy number and they do not contain information for active partitioning at cell division. Analysis of mutant strains where minichromosomes cannot be established suggest that their mere existence is dependent on the factors that ensure timely once per cell cycle initiation of replication. These observations indicate that replication initiation in E. coli is normally controlled in such a way that all copies of oriC contained within the cell, chromosomal and minichromosomal, are initiated within a fairly short time interval of the cell cycle. Furthermore, both replication and segregation of the bacterial chromosome seem to be controlled by sequences outside the origin itself.     

Asymmetric replication of an oriC plasmid in Escherichia coli

Summary Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature-sensitive host cells were synchronized by temperature shifts. Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell. Plasmid DNA with eye structures was enriched when cytosine-1--arabinofuranoside was introduced into the culture during replication. Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon. We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric.     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.     

TOL is a broad-host-range plasmid.

We readily isolated insertions of the carbenicillin resistance element Tn401 into the TOL plasmid in Pseudomonas putida. Hybrid TOL::Tn401 plasmids stably express the Cbr phenotype in Pseudomonas aeruginosa and Escherichia coli. Whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the Pseudomonas species.     

Analysis of the mobilization region of the broad-host-range IncQ-like plasmid pTC-F14 and its ability to interact with a related plasmid, pTF-FC2

Plasmid pTC-F14 is a 14.2-kb plasmid isolated from Acidithiobacillus caldus that has a replicon that is closely related to the promiscuous, broad-host-range IncQ family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins that were related to those of the DNA processing (Dtr or Tra1) region of IncP plasmids rather than to the three-Mob-protein system of the IncQ group 1 plasmids (e.g., plasmid RSF1010 or R1162). Plasmid pTC-F14 is the second example of an IncQ family plasmid that has five mob genes, the other being pTF-FC2. The minimal region that was essential for mobilization included the mobA, mobB, and mobC genes, as well as the oriT gene. The mobD and mobE genes were nonessential, but together, they enhanced the mobilization frequency by approximately 300-fold. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3,500-fold less efficient than the mobilization of pTF-FC2. When both plasmids were coresident in the same E. coli host, pTC-F14 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-F14 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. Mob protein interaction at the oriT regions was unidirectionally plasmid specific in that a plasmid with the oriT region of pTC-F14 could be mobilized by pTF-FC2 but not vice versa. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.