Compatibility and molecular properties of plasmid Rms 149 in Pseudomonas aeruginosa and Escherichia coli

Two recombinant plasmids have been constructed which code for chloramphenicol resistance but otherwise have properties similar to those of the tetracycline resistance plasmid pT181 and its temperature-sensitive replication (Tsr) mutant pSA0301, respectively, concerning their stability in the host cell and behavior in incompatibility tests. Incompatible heteroplasmid clones have been established using these four plasmids, and their segregation during growth under nonselective conditions has been followed. The results obtained proved the ability of a wild-type plasmid to complement an incompatible Tsr plasmid mutant for its autonomous maintenance at restrictive temperatures. On the other hand, at permissive temperatures, the rate of heteroplasmid segregation was higher for wild-type/wild-type plasmid pairs than for wild-type/Tsr pairs, while at restrictive temperatures the two types of heteroplasmid clones presented similar segregation rates. The possible implications of these results are discussed.     

Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.     

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.     

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.     

Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer.

The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer.     

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.     

On plasmid incompatibility

In this paper is presented a brief review of the current state of information on plasmid incompatibility followed by a detailed mathematical model dealing with incompatibility between autonomous homogenic plasmids and based on the assumption that the intracellular plasmid copy pool is randomized with respect to assortment during cell division. Two cases are considered: one in which each plasmid copy replicates once in each generation of cell growth (regular replication) and one in which plasmids are chosen at random for replication from a common pool, irrespective of their replication history (random replication). In both cases, it is assumed that the partition of plasmid copies to daughter cells at cell division is regular—existing plasmid copies are divided equally among the two daughter cells (equipartition). In the case of regular replication coupled with equipartition, it is shown that the survival of heteroplasmid cells (cells containing at least one copy of each of two incompatible plasmids) during exponential growth in a nonselective medium is given by H = H₀[1 − 1/(2N − 1)]ⁿ, where H₀ and H are the numbers of heteroplasmid cells after 0 and n generations of growth, respectively, and N is the plasmid copy number in newborn cells. In the second case, (random replication-equipartition), it is shown that the survival of the heteroplasmid population during exponential growth under nonselective conditions is given by H = H₀[(N − 1)(2N + 1)/(2N − 1)(N + 1)ⁿ. Sample calculations are presented to show that segregation is more rapid in the latter than in the former case. Finally, some of the plasmid-linked genetic determinants that might be expected to affect the expression of incompatibility between nonisogenic plasmids are briefly considered. These determinants include recognition specificity for replication origins, recognition specificity, specific activity of copy number control systems, and recognition specificity of partition systems.     

In vitro inhibitory activity of RepC/C*, the inactivated form of the pT181 plasmid initiation protein, RepC.

pT181 is a Staphylococcus aureus rolling circle plasmid that regulates its replication by controlling the synthesis of its dimeric initiator protein RepC/C and by inactivating the protein following its use in replication (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). This inactivation consists of the addition of an oligonucleotide, representing several nucleotides immediately 3' to the initiation nick site, to the active site tyrosine of one of the two subunits, generating a heterodimer, RepC/C*. Previous results suggested that the inactive form was metabolically stable and was present at a much higher level than the active form (A. Rasooly and R. P. Novick, Science 262:1048-1050, 1993). In the present study we have measured total RepC antigen as a function of plasmid copy number and have analyzed the interaction of the two forms. We find that pT181-containing staphylococci contain approximately one RepC dimer per plasmid copy over a 50-fold range of copy numbers. This is consistent with previous measurements of the rate of RepC synthesis, which suggested that one RepC dimer is synthesized per replication event (J. Bargonetti, P.-Z. Wang and R. P. Novick, EMBO J. 12:3659-3667, 1993). The RepC/C* heterodimer, which is inactive for replication, is a competitive inhibitor of the replication and the topoisomerase-like and cruciform-enhancing activities of the native protein. These results suggest that the inactive form may have a specific regulatory role in vivo. Since the known plasmid-determined controls, which maintain a constant plasmid copy number, are designed to ensure the synthesis of one RepC/C dimer per plasmid replication event, it is difficult to envision any role for yet another negative regulator of replication. Conceivably, under conditions where the initiator is overproduced, such as in the absence of the normal antisense regulation of initiator production, RepC/C* could serve as a fail-safe means of preventing autocatalytic replication.     

RepC is rate limiting for pT181 plasmid replication

The effect on pT181 plasmid replication of the concentration of the plasmid-coded initiator protein, RepC, has been analyzed. In one type of experiment, plasmid replication was found to stop immediately after the addition of an inhibitory concentration of chloramphenicol (Cm) to growing cultures. Chromosomal replication showed the slow turnoff that is usual for Cm inhibition. Because plasmid replication rate is determined autogenously, no host factor can be rate limiting, suggesting that the specific factor affected is Rep C. In another type of experiment, we constructed a translational fusion between the repC coding sequence and a translationally inducible Cm-acetylase gene, cat-86, using pUB110 as the carrier replicon. The fusion plasmid showed an eightfold amplification of its own copy number and a similar amplification of a co-resident pT181 plasmid upon Cm induction. The amplified plasmids did not show autocatalytic runaway replication but rather established stable elevated copy numbers, indicating the existence of a secondary level of regulation. These results suggest that RepC is rate limiting for pT181 replication and support the hypothesis that pT181 replication is regulated at the level of RepC synthesis. The nature of the secondary regulation is unknown.     

Effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pT181 on the competition between incompatible plasmids

The deletion of the 560-bp HindIII C fragment from pT181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell. The disadvantage of the deleted plasmids seems to be manifested at the level of replication. It results that for plasmid pT181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, incompatible plasmids.     

cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein.

pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the homoplasmid state the deletions affected neither copy number nor plasmid stability. The Cmp phenotype is orientation independent, and cmp defects could not be complemented in trans.     

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.     

Sociobiological Control of Plasmid Copy Number in Bacteria

All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up “cheater” mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS) like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.     

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.