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DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.

Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place.

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3'-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can "sense" and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.

Replication of the streptococcal plasmid pLS1 is controlled by two plasmid-encoded gene products: the repressor protein CopG and the antisense RNA, RNA II. Two different mutants in rnaII have been isolated. The 5'-end and the levels of RNA II synthesized by pneumococcal cells harbouring the wild-type pLS1 or mutant plasmids (affected in either genes copG or rnaII) were analysed. One of the rnaII mutants exhibited a high-copy-number phenotype, whereas an in vitro-constructed mutation, which affects the -10 region of the rnaII promoter, resulted in plasmids lacking copy-number phenotype. The latter mutation had a pleiotropic effect: It abolished RNA II synthesis, but it also affected the initiation of translation signals of the gene encoding the RepB initiator protein. Transcriptional and translational fusions, together with in vitro inhibition of RepB synthesis by specific oligonucleotides, showed translational inhibition of RepB synthesis by RNA II, perhaps by directly blocking the accessibility of the ribosomes to the repB initiation of translation signals.

Many factors can influence the ability of plasmids to colonize different hosts, efficient replication probably being the most critical one. Two major strategies seem to facilitate promiscuous plasmid replication: (i) initiation independent of host initiation factors; and (ii) versatile communication between plasmid and host initiation factors. Appropriate communication between a replicon and the different hosts, which becomes crucial at the initation of plasmid replication, plays a major role in plasmid promiscuity. Fused replicons or mechanisms that rescue collapsed replication forks may increase the efficiency of plasmid propagation. However, their contribution to plasmid promiscuous replication remains to be fully evaluated. Several examples of host-specific adaptation of promiscuous plasmids point to an enormous flexibility of these replicons.

Two elements, the products of genes copG and rnaII, are involved in the copy-number control of plasmid pLS1. RNA II is synthesized in a dosage-dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG, rnaII or both elements at various gene dosages into pLS1-compatible plasmids. Assays of incompatibility towards wild-type or mutant pLS1 plasmids showed that: (i) the rnaII gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG-rnaII circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy-number fluctuations, indicating that, in the homoplasmid steady-state situation, the control of pLS1 replication is exerted by the co-ordinate action of CopG and RNA II.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.

This review summarises current information on rolling circle replicating plasmids originally isolated from Gram-positive bacteria with a low guanine and cytosine content in their DNA. It focuses on the peculiar biological features of these small, high copy number plasmids that replicate via an asymmetric RC mechanism. The regulation of plasmid copy number is also discussed.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RSB.

Rolling circle-replicating plasmids constitute a group of small, promiscuous multicopy replicons spread among eubacteria. Until recently, rolling circle replication seemed to be limited to small plasmids from Gram-positive hosts and to single-stranded bacteriophages from Gram-negative bacteria. However, characterization of two small plasmids from Gram-negative hosts has shown that this replication mechanism is general among eubacteria. This review focuses on a family of highly related promiscuous plasmids that replicate by the rolling circle mechanism, and that have been isolated from various Gram-positive bacteria and from the Gram-negative bacterium Helicobacter. They all share homologies at the leading-strand origins and at the initiator of replication proteins. The plasmids of this family have directly repeated sequences at their plus origin of replication, which is located 5' from the start point of the mRNA for the initiation of replication protein. Replication is controlled by an antisense RNA and by a transcriptional repressor protein. The features and regulatory circuits of replication of this plasmid family seem to be unique among rolling circle-replicating plasmids. Members of this family replicate autonomously in Gram-positive and -negative hosts.

The broad host range streptococcal plasmid pLS1 encodes the 24.2 kDa protein RepB, which is involved in the initiation of plasmid replication by an asymmetric rolling circle. RepB was overproduced in an Escherichia coli expression system and the protein was purified and characterized. Determination of the amino-terminal sequence of RepB protein showed that translation starts from the first AUG codon, which is preceded by an atypical ribosome-binding site sequence. RepB protein has in vitro-specific endonuclease and topoisomerase-like activities on the plasmid ori(+). Footprinting experiments showed that RepB protein binds to a DNA region that includes three direct repeats of 11 base-pairs. Initiation of replication of pLS1 could start by a RepB-generated specific nick introduced on the plasmid coding strand. However, as a striking difference with other Gram-positive replicons, the nick generated by RepB lies 86 base-pairs upstream from its binding region. To explain the action of RepB at a distance, complex structures of the pLS1 ori(+) are proposed.

pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably maintained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30 degrees C. The optimal concentration of template DNA was 40 micrograms/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its derivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.

The replication origin of the fully sequenced broad-host-range streptococcal plasmid pLS1 has been determined by the use of an in vitro replication system prepared from Escherichia coli, a host in which the plasmid can be established. Replicative intermediates were isolated from reaction mixtures that contained dideoxythymidine triphosphate, thus limiting the average extent of in vitro synthesis. Analysis of HinfI-cleaved intermediates demonstrated that the origin of replication is included within a 443-bp fragment. Replication proceeds unidirectionally in the same direction as transcription of plasmid mRNAs. Isolation of deletion derivatives allowed us to define the replication origin of pLS1 within a region of 284 bp. Replication of pLS1 occurs through single-stranded intermediates by a rolling circle mechanism. Cleavage of supercoiled plasmid DNAs with endonuclease S1 followed by restriction mapping, allowed the positioning of three major specific S1 sites in regions of high potential to form secondary structures. One of these inverted repeats is located in the region where the origin of replication of pLS1 has been defined.

The promiscuous streptococcal plasmid pLS1 encodes for the 5.1 kDa RepA protein, involved in the regulation of the plasmid copy number. Synthesis of RepA was observed both in Bacillus subtilis minicells and in an Escherichia coli expression system. From this system, the protein has been purified and it appears to be a dimer of identical subunits. The amino acid sequence of RepA has been determined. RepA shows the alpha helix-turn-alpha helix motif typical of many DNA-binding proteins and it shares homology with a number of repressors, specially with the TrfB repressor encoded by the broad-host-range plasmid RK2. DNase I footprinting revealed that the RepA target is located in the region of the promoter for the repA and repB genes. Trans-complementation analysis showed that in vivo, RepA behaves as a repressor by regulating the plasmid copy number. We propose that the regulatory role of RepA is by limitation of the synthesis of the initiator protein RepB.