Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

In vitro studies of the initiation of staphylococcal plasmid replication. Specificity of RepD for its origin (oriD) and characterization of the Rep-ori tyrosyl ester intermediate

Several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (Rep) which is homologous with that of the inc3 tetracycline resistance plasmid pT181. The rep gene sequences of six pT181-like plasmids are known, each encoding proteins of molecular mass 38 kDa with 62% overall amino acid sequence identity. The initiation of replication in vivo by each of the Rep proteins is plasmid specific, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.     

Replication termination for staphylococcal plasmids: plasmids pT181 and pC221 cross-react in the termination process.

We present data which indicate that (i) the origin of replication of plasmids pT181 and pC221 can also function as termination signals; (ii) termination of replication occurs when a round of replication initiated either by RepC at the pT181 origin or by RepD at the pC221 origin reaches either of these origins, proving that the two plasmids cross-react for termination of replication; and (iii) the replication initiated at the origin of another staphylococcal plasmid, pE194, does not terminate at the origin of pT181 or pC221, indicating the existence of a specific relationship between the initiation and termination of a replication event.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.     

Comparative analysis of five related staphylococcal plasmids

The genomic organization of five small multicopy staphylococcal plasmids comprising the pT181 family has been analyzed. In addition to pT181, the family presently includes the streptomycin resistance plasmid pS194 and the chloramphenicol resistance plasmids pC221, pC223, and pUB112. Although they belong to five different incompatibility groups, the five plasmids have similar basic replicons, use the same basic copy control mechanism, and have a common structural organization. It has been demonstrated previously that pT181 and pC221 encode trans-active replication proteins (RepC and RepD, respectively) which specifically recognize the respective plasmid's origin of replication in both cases is initiated by site-specific nicking and 3' extension. The other three plasmids in this family encode similar replication proteins; 63% of the predicted amino acid residues are identical for all five and the least similar pair shows 75% identity at the amino acid level. However, despite this homology, the replication proteins and origins of replication of different members in this family did not show cross complementation in vivo. Outside of the basic replicon, which comprises about one-third of each plasmid's genome, functional organization is also conserved. The resistance determinants are all located in the same position, immediately downstream of the replication protein coding sequence, and all are transcribed in the same direction. The three chloramphenicol resistance determinants encode highly homologous chloramphenicol transacetylases which are unrelated to the tet and str gene products. Three of the five plasmids form relaxation complexes and the involved genome segments are closely related. The other two are not homologous to these three in the corresponding region, but are homologous to each other and encode a site-specific recombinase, Pre. It is suggested that the replication, resistance, and relaxation complex regions of these plasmids can be regarded as conserved segments ("cassettes") assembled in various combinations, but always with the same spatial arrangement.     

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.     

Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element.

The leading strand replication origin of pT181 plasmids consists of two adjacent inverted repeat elements (IR-II and IR-III), which are involved in origin recognition by the initiator (Rep) protein. The conserved core element, IR-II, which contains the initiation nick site, is induced by Rep to form a cruciform structure, probably the primary substrate for the initiation of rolling circle replication. The divergent repeat, IR-III, constitutes the determinant of origin recognition specificity. We show here that the distal arm of IR-III is not required for sequence-specific recognition, whereas the proximal arm and central region are required. Since the initiator is dimeric, we presume that it binds symmetrically to IR-III. A unique type of DNA-protein interaction is proposed, in which the lack of sequence requirement for the distal arm is a consequence of binding to the adjacent IR-II, which thereby polarizes the stringency of binding to the two arms of IR-III. In addition, genetic evidence indicates that both the spacing and the phasing of IR-II to IR-III are crucial for function and that the central segment of IR-III may serve to position the two flanking half-sites for optimal interaction of Rep with IR-III.     

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.     

Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase

The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro. This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3. Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate. The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.     

Sequence requirements for the termination of rolling-circle replication of plasmid pT181

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.     

An enhancer of DNA replication.

cmp, a nucleotide sequence element in the plasmid pT181 of Staphylococcus aureus, acts as an enhancer of DNA replication. When cmp is present on an unrelated vector along with the pT181 origin of replication, it increases the ability of the linked pT181 origin to compete with a coresident pT181 plasmid for the initiator protein RepC. cmp is contained within a 156-base-pair segment, and its deletion from pT181 reduces by twofold the frequency of plasmid replication under derepressed conditions. The enhancer sequence contains a locus of DNA bending, and enhancer activity decreases with distance from the replication origin.     

Lactobacillus hilgardii plasmid pLAB1000 consists of two functional cassettes commonly found in other gram-positive organisms.

A Lactobacillus hilgardii plasmid, pLAB1000, was studied to understand the organization of autonomous replicons from lactobacilli. Two cassettes could be identified. First, the replication region consisted of a sequence coding for a replication protein (Rep) and its corresponding target site, similar to those from plasmids pUB110, pC194 (Staphylococcus aureus), pFTB14, pBAA1 (Bacillus sp.), and pLP1 (Lactobacillus sp.). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate Rep production. The results also suggested that pLAB1000 replicates via a single-stranded DNA intermediate, and a putative lagging-strand initiation site was found that had similarities to those of alpha 3, St-1, and G4 isometric bacteriophages. The second cassette of pLAB1000 consisted of a sequence coding for a putative mobilization protein (Mob) and its corresponding RSA site. This cassette was similar to those found in pT181, pUB110, pE194 (S. aureus), and pG12 (Bacillus sp.), and it was found to be conserved among different Lactobacillus plasmid replicons. The origin and evolution of these functional cassettes are also discussed.     

Bacillus anthracis and Bacillus cereus PcrA helicases can support DNA unwinding and in vitro rolling-circle replication of plasmid pT181 of Staphylococcus aureus

Replication of rolling-circle replicating (RCR) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid DNA by the PcrA helicase. In this report, we demonstrate that heterologous PcrA helicases from Bacillus anthracis and Bacillus cereus are capable of unwinding Staphylococcus aureus plasmid pT181 from the initiator-generated nick and promoting in vitro replication of the plasmid. These helicases also physically interact with the RepC initiator protein of pT181. The ability of PcrA helicases to unwind noncognate RCR plasmids may contribute to the broad-host-range replication and dissemination of RCR plasmids in gram-positive bacteria.     

Complete nucleotide sequence of pTZ12, a chloramphenicol-resistance plasmid of Bacillus subtilis

The complete nucleotide sequence of pTZ12, a chloramphenicol-resistance (CmR) plasmid (2517 bp) derived from Corynebacterium xerosis plasmid pTZ10, has been determined after propagation in Bacillus subtilis. The nucleotide sequence of pTZ12 suggests that a recombination event may have occurred naturally within the open reading frames for the Rep protein of pT181 (or a pT181-like plasmid) and pC221 (or a pC221-like plasmid).     

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.     

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.     

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.     

Kinetic mechanism of initiation by RepD as a part of asymmetric, rolling circle plasmid unwinding

Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s(-1) at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.