[Frontiers in Bioscience 3, d43-62, January 1, 1998]

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Received: 8/10/98
Accepted:11/16/98

Send correspondence to:

Dr Luis A. Actis,
Department of Microbiology,
Miami University,
Oxford, Ohio 45056-8900,

Tel:(513)-529-5424,
Fax:(513) 529-2431,
E-mail: Actisla@muohio.edu

KEY WORDS

Plasmids, DNA Replication, Copy number, Incompatibility, Antisense RNA, Iterons, Antibiotic resistance

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Copyright © Frontiers in Bioscience, 1995

BACTERIAL PLASMIDS: REPLICATION OF EXTRACHROMOSOMAL GENETIC ELEMENTS ENCODING RESISTANCE TO ANTIMICROBIAL COMPOUNDS

Luis A. Actis,1 Marcelo E. Tolmasky,2 and Jorge H. Crosa31

Department of Microbiology, Miami University, Oxford, Ohio 45056-8900, 2Department of Biological Science, School of Natural Science and Mathematics, California State University Fullerton, Fullerton, California 92821-6850, 3Department of Molecular Microbiology and Immunology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Replication
3.1. Iteron-regulated plasmids: the R6K plasmid
3.2. RNA-regulated plasmids: the ColE1-type plasmids
3.3. Rolling-circle replicating plasmids: the pT181-type plasmids
3.4. Replication of linear plasmids: the Streptomyces and Borrelia plasmids
4. Plasmid copy number and incompatibility
4.1. Antisense RNAs
4.2. DNA iterons
5. Acknowledgments
6. References
1. ABSTRACT

Plasmids are self-replicating extrachromosomal DNA molecules found in Gram-negative and Gram-positive bacteria as well as in some yeast and other fungi. Although most of them are covalently closed circular double-stranded DNA molecules, recently linear plasmids have been isolated from different bacteria. In general, plasmids are not essential for the survival of bacteria, but they may nevertheless encode a wide variety of genetic determinants, which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. The medical importance of plasmids that encode for antibiotic resistance, as well as specific virulence traits has been well documented and demonstrated the important role these bacterial genetic elements play in nature. Although they encode specific molecules required for initiation of their replication, plasmids rely on host-encoded factors for their replication. Plasmid replication initiates in a predetermined cis-site called ori and can proceed either by a rolling circle or a theta replication mechanism. Some of the plasmid-encoded elements required for their replication, such antisense RNA molecules and DNA repeated sequences located close to ori, determine plasmid attributes like copy number and incompatibility.

2. INTRODUCTION

Extrachromosomal replicons known as plasmids are found in Gram-negative and -positive bacteria as well as in some lower eukaryotes. Their existence was initially revealed as the "F factor" in Escherichia coli even before the double-helix structure of DNA was elucidated by Watson and Crick (1-3). Since then, molecular and genetic work on plasmids resulted in extraordinary contributions to the modern fields of molecular genetics and molecular biology (4). Molecular and genetic analysis of bacterial plasmids led to basic concepts such as "the operon" and "the replicon", and has provided essential information on DNA conjugation and fertility, control of gene expression, gene transfer and genetic recombination, and transposable elements. Studies of essential plasmid functions have resulted in important findings about basic aspects of initiation of DNA replication and its regulation, DNA partitioning, and plasmid copy number and incompatibility (5). In pathogenic microorganisms, plasmids that contribute directly to microbial pathogenicity in plants and animals, such as for instance iron transport in several pathogens or the presence of adhesins, invasins or antiphagocytic proteins, is well documented (6, 7). In addition, numerous studies have shown the role played by plasmids in bacteria of importance in other areas such as agriculture and plant molecular biology (8, 9). In a more applied vein, plasmids played a central role in the initial development of recombinant DNA technology, gene cloning, and the constant evolution of molecular biology (4).

One of the features that keep plasmids at the forefront of Microbiology is their ability to carry and transmit genes encoding resistance to antimicrobial compounds. This type of plasmids is widespread in bacteria and can be transferred between different microorganisms, a genetic property that represents a very serious medical problem in human and animal medicine. These plasmids, called R plasmids, harbor a variety of genes encoding resistance to a wide spectrum of antimicrobial compounds, which include antibiotics, heavy metals, resistance to mutagenic agents like ethidium bromide, and even disinfectant agents such as formaldehyde (10, 11). Furthermore, genetic and molecular analysis of plasmids proved to be essential in understanding the structure of transposons and integrons and the role these genetic elements play in the transmission of resistance to antimicrobial agents (12-14). The presence of these mobile genetic elements in transmissible plasmids, some of them capable of replicating in bacterial strains belonging to different species, makes matters quite serious since they contribute to the transmissibility of resistance genes from strains to strains as well as between different replicons within any given strain.

The term plasmid was originally used by Lederberg to describe all extrachromosomal hereditary determinants, and it is currently used to describe autonomously replicating extrachromosomal DNA of bacteria (2, 3). While not essential for the survival of bacteria, plasmids may encode the wide variety of genetic determinants mentioned above which permit their bacterial hosts to survive better in an adverse environment or to compete better with other microorganisms occupying the same ecological niche. Plasmid size varies from a few to several hundred kilobases (kb) and bacterial cells can harbor more than one plasmid species (15). Plasmids are found in a wide variety of microorganisms, and it is as difficult to generalize about plasmids as it is to generalize about the microorganisms that harbor them. Plasmids also include the replicative forms of filamentous coliphages and the prophage state of phages such as P1 (16).

Although bacterial genomes have long been considered to contain only covalently closed circular (ccc) double-stranded DNA molecules, the presence of linear chromosome and plasmid molecules has been described in bacteria. The first linear plasmid was found in Streptomyces rochei in 1979 (17) and now they have been detected in several bacterial genera such as Agrobacterium, Borrelia, Nocardia, Rhodococcus, Thiobacillus, and even Escherichia (18). The Streptomyces linear plasmids range in size from nine to several hundred kb and all of them contain terminal inverted repeats of different length. The Borrelia plasmids are unique among extrachromosomal elements since some of them carry critical genes such as the guaA and guaB biosynthetic genes (19) as well as genes encoding the essential major outer surface Osp or Vmp lipoproteins (20-23). In addition, some of these linear plasmids are present stably in low copy number, about one per chromosome equivalent, and can be cured by exposing Borrelia cultures to the DNA gyrase inhibitor novobiocin (20-23). These genetic properties suggest that the replication and partition processes of linear plasmids are well controlled during cell division. All these facts together with the observation that the Borrelia chromosome has attributes of a linear DNA molecule has led to the idea that these linear plasmids might be regarded more properly as minichromosomes (24).

The linear structure of DNA molecules brings an interesting biological problem associated with the protection of the DNA ends from exonuclease degradation and the complete replication of these plasmids. The ends of linear Streptomyces plasmids have telomeric structures that are also found in adenoviruses and some prokaryotic phages as well as in almost all eukaryotic plasmids. An inverted terminal repeat was found in pSL2, a linear plasmid present in S. rochei (25, 26) (figure 1A). These terminal repeats, together with specific DNA binding proteins, are thought to be involved in the juxtaposition of the two plasmid termini containing identical or very similar nucleotide sequences. This structure, known as the racket frame-like DNA model includes a terminal protein covalently attached to the 5 DNA termini which is required to protect the DNA from degradation and complete the replication of the 3 overhanging ends. The structure of the 16- and 49-kb linear plasmids of B. burgdorferi consist of a double-stranded DNA chain connected at each end by a perfect palindromic AT-rich hairpin loop (figure 1B) (27, 28). In addition, each end contains a conserved 19-base pair (bp) inverted repeat sequence, a telomeric structural feature found in all linear plasmids analyzed in Borrelia to date (18, 20, 22). These features have similarities to the telomeres of other linear double-stranded replicons, including among them viral genomes (29, 30) and the mitochondrial DNA of the yeast Pichia (31).

Figure 1. (A) Racket frame structure proposed for linear Streptomyces plasmids. The black circles represent the terminal protein attached to the 5 ends, which is required to protect the ends and complete the replication of both plasmid termini. The ovals represent juxtaposition proteins that bring together the plasmid termini by binding to specific regions of palindromic symmetry. The ori located near the center of the plasmid is depicted by the box and the arrows indicate the bidirectional DNA replication from this ori. (B) Typical structure of linear plasmids isolated from Borrelia containing terminal hairpin telomeric structures composed of inverted repeats (thick arrows) harboring the nick sites (vertical arrows) involved in DNA replication.

In general, bacterial plasmids replicate independently of the host chromosome, although usually they rely on some host-encoded factors for their replication. They are present in bacterial cells replicating at a specific number of copies per cell, which can range from one or two to several hundreds. However, replication is not enough for plasmids to be stably inherited in a cell line. Plasmid maintenance functions, particularly in low-copy number plasmids, such as F, fulfill an essential role in the equipartition of the molecules to the daughter cells (5). Plasmid partition systems, one of the maintenance mechanisms, are normally plasmid encoded, although host cellular functions may be required (32-34). The mechanism of partition apparently involves the recognition of pairs of plasmid molecules and membrane proteins followed by an active process of separation of the members of the pair to the opposite halves of the dividing bacterial cell (35). More recently, the partition mechanism of F and the role of plasmid-encoded proteins and plasmid DNA sequences in partition were examined by using the green fluorescent protein (36). This new approach allowed the analysis of plasmid partition while the cells were growing and dividing. Besides partition, plasmids have evolved other maintenance mechanisms such as site-specific recombination systems that resolve plasmid multimers. There are also anticlumping systems that destroy aggregates of plasmid molecules to allow them to diffuse freely in the cytoplasm. In addition, there are systems that kill cells that have lost the plasmid after division (5).

Since very early in the study of plasmids, attempts were made to develop a classification system based on properties such as copy number or their ability to survive in one or many different bacterial hosts. However, it was soon clear that those criteria were not suitable since unrelated plasmids could be grouped together for instance by their copy number and host range. Furthermore, genes encoding many specialized functions (such as resistance to antibiotics, production of toxins, synthesis or utilization of nutrients) were found to be highly mobile (included in transposable elements and integrons) and therefore plasmids could acquire those functions without changing their inheritance properties. Therefore, these criteria for classification were either too broad, grouping together unrelated plasmids, or were based on properties that could not be ascribed to the biological properties of the plasmid. In the early 1960s a property that is peculiar to plasmids was described: incompatibility. Plasmid incompatibility is the failure of two plasmids to be stably inherited, in the absence of selective pressure, in the same cell line. This phenomenon is a consequence of sharing elements of plasmid inheritance functions such as replication or partition (15, 37, 38). Therefore, incompatibility gives a better idea of closeness among plasmids, which can then be grouped together in what is called incompatibility (Inc) groups. Initially, these incompatibility groups were determined by using biological assays based on plasmid transformation and examination of plasmid stability by determining antibiotic resistance phenotypes and restriction map analysis; however a replicon typing system was recently proposed for plasmid classification (39). This system is more practical and it is based on the identification of sequence homologies between replicons using DNA hybridization techniques. A bank of replicon probes was prepared from replication regions of plasmids belonging to the different Inc groups.

In this review we describe the main plasmid replication systems and their role in plasmid incompatibility. In addition, we describe the structure and replication mechanisms of bacterial linear plasmids.

3. REPLICATION

Several host- and plasmid-encoded functions are required for plasmid replication. Initiation of plasmid replication is molecule specific and of great importance for the propagation process, copy number, and incompatibility properties of plasmids in both Gram-positive and -negative bacteria (40, 41). In general, plasmid replicons contain one or several origins (ori) of replication and one or more regulatory elements, located in a DNA fragment no larger than 4 kb. In addition, most plasmid replicons harbor a gene encoding either a protein or an RNA molecule that functions as a primer for DNA replication. The Rep proteins can often act in trans on a specific ori, but in some cases they may only function in cis. However, in all cases examined so far the preprimer RNA acts in cis with the replication initiation sequences. Composite multi-replicon plasmids were also described. One example is R6K, in which three origins are able to function in vivo independently, although the rate of initiation from each origin is different (42, 43). It was reported that many plasmid origins follow a molecular mechanism similar to oriC, the origin of replication of the E. coli chromosome (44). However, the major difference is that plasmids require an origin-specific plasmid-encoded protein for the initiation step, generally called Rep proteins. These plasmid-encoded Rep proteins act in place of or in combination with DnaA, the replication initiation protein for chromosomal DNA. Some plasmids require additional host-gene products such as dam methylases, integration host factor (IHF), and heat shock proteins to replicate (40, 45). Other plasmids, such as the ColE1-type encode an RNA-specific plasmid molecule and require the host-encoded DNA polymerase I (PolI or PolA), RNA polymerase, and ribonuclease H (RNase H) (46-48). Two types of mechanisms basically control the replication of plasmid DNA. One utilizes a series of repeated sequences, designated iterons, located at ori and capable of interacting with the replicator protein. In the other, small complementary RNA molecules (antisense) hybridize with the transcript responsible for the initiation process, either directly or indirectly by encoding the Rep protein.

3.1. Iteron-regulated plasmids: the R6K plasmid

Plasmid R6K belongs to the group of iteron-regulated replicons encoding an initiator protein that binds to repeated sequences located within ori. This group includes the E. coli oriC (44) and the plasmids F (49), pSC101 (50), P1 (16), pMJ101 (51, and manuscript in preparation), Rts1 (52), the REPI replicon of pColV-K30 (53), and the RK2-and RP-4 related plasmids (54). The conjugative plasmid R6K is a naturally occurring extrachromosomal element that codes for resistance to the antibiotics ampicillin and streptomycin (55) (figure 2A). It is about 38 kb in size and has a copy number of 13 to 40 per cell (56). These features together with a unique mode of replication made R6K an attractive system to study the genetic and molecular mechanisms involved in plasmid DNA replication. In addition, this plasmid and its replication components were among the first used in molecular biology to generate gene fusions, transcription enhancement, protein tagging, and site-specific proteolysis (57-59). Furthermore, R6K recombinant derivatives were instrumental in designing a series of suicide vectors successfully used to generate mutants by allelic exchange or transposition mutagenesis in Gram-negative bacteria (60, 61).

Figure 2. (A) Diagram of the R6K plasmid. The location of the three origins of replication and the genes encoding resistance to streptomycin (Sm) and ampicillin (Amp) are indicated. The arrows mark the in vivo direction of the initial replication from the alpha and beta origins. (B) Diagram of the alpha, beta, and gamma origins of replication (adapted from 74 and 77). Alpha, components of the active alpha origin. The double-headed arrows indicate the DNA fragments containing the alpha origin and the gamma core region required in cis for active replication. The dashed line represents the nonessential intervening sequences. The location of the long inverted repeat is indicated by the hairpin followed by the genes encoding the DDP1 and DDP2 proteins. The thick arrow and the large rectangle indicate the position of an iteron and a DnaG binding site, respectively. The circle indicates the replication protein Pi bound to the iteron. Beta, components of the active beta origin. The double-headed arrows indicate the DNA fragment containing the gamma core region and beta origin required in cis for active replication. The beta origin includes the pir and bis genes, a half iteron (half-thick arrow) bound by Pi (circle), the ori beta long inverted repeat (hairpin), and the gene encoding the DDP3 protein. Gamma, components of the enhancer and core regions of the gamma origin containing binding sites for the host-encoded proteins DnaA, IHF, and RNA polymerase. Ppir and the open arrow represent the promoter site and the direction of transcription of the pir gene, respectively. The thick arrows represent the seven iterons that bind the Pi replication protein (circles). stb represents the locus involved in plasmid maintenance. The Fis-binding sites are indicated by the arrowheads.

The alpha, beta, and gamma origins of R6K are all clustered within a 4-kb DNA fragment that contains also the pir and bis genes encoding the Pi and Bis replication proteins, respectively (figure 2). The main active origins in vivo are alpha and beta, while ori gamma tends to remain inactive (42) due to the synthesis of a silencer RNA encoded immediately downstream ori gamma (62). However, ori gamma can replicate autonomously when the other two origins are deleted and the pir gene is provided either in cis or in trans (63-67). Thus, ori gamma has served as the simplest model system derived from R6K to study the replication of an iteron-containing DNA molecule.

The gamma origin is the major binding-site for the Pi replication protein (57, 68, 69) and is required in cis for initiation of plasmid replication from the other two ori sites (70-72). Thus, the gamma origin behaves as a prokaryotic enhancer-type element since DNA-protein interactions at this site induce significant changes in DNA structure that facilitate initiation of DNA replication from the two other distantly located origins (73). The molecular organization of this origin is similar to other plasmid origins although it contains two functionally distinct segments: the enhancer and the core regions (figure 2B) (71, 74). The core is a 277-bp segment, which is common to all three origins and is essential for replication. It consists of three distinct regions: (i) the AT-rich region, bound by Pi and IHF, (ii) seven 22-bp repeats bound by Pi, and (iii) a region which interacts with DnaA, IHF, and RNA polymerase host proteins. The 106-bp enhancer lies immediately to the left of the core and includes a DnaA box and a small segment containing the stb locus (75). The enhancer region is required for stable maintenance of R6K derivatives containing only the gamma-origin harboring plasmids. The stb locus has some similarities with the par locus of pSC101, however the partition systems of these two plasmids differ from each other in several aspects (75). It has been proposed (75) that a host-encoded protein binds to the stb repeats, a hypothesis that is supported by the fact that R6K derivatives carrying all three origins bind in vitro to both inner and outer membrane fractions of E. coli (76). An alternative explanation is that stb mediates plasmid partition by altering the structure of gamma-origin containing plasmids (75). The host-encoded protein Fis binds to 10 sites in the gamma-origin that overlap the binding sites for the R6K-encoded Pi protein and the host-encoded DNA binding proteins DnaA, IHF, and RNA polymerase (77). The Fis protein appears to be required for plasmid replication only when Pi copy-up variants and the penicillin resistant marker are simultaneously used. This is an interesting observation since it demonstrates that plasmid genes encoding antibiotic resistance play a role not only in the ability of bacterial cells to survive in adverse environments, but also are important in the replication of plasmid DNA.

The functional alpha replicon contains two elements, separated by a nonessential 3-kb fragment, that must be present in cis and oriented as in the intact R6K: (i) a 580-bp fragment containing the alpha ori and a long 98-bp palindrome which serves as the recognition signal for initiation of DNA replication from this ori, and (ii) the 277-bp core segment located within the gamma ori (figure 2B). DNA homology analysis revealed the presence of a 23-bp sequence which resembles the seven 22-bp iterons found in the core segment (78) and plays a role in the Pi protein-mediated looping between the gamma and alpha sequences (73). In addition, this fragment contains a DnaG-binding site that can serve as a DnaB loading site by DnaB-DnaG interaction (79).

The minimal beta-replicon is a 2-kb fragment that encompasses: (i) the gamma core region, (ii) the pir gene encoding the Pi initiator protein, (iii) the bis gene encoding the 17.2 kDa Bis protein, and (iiii) the beta ori (72, 80-82) (figure 2B). The Bis protein is required only by this origin and its synthesis is coupled in cis to the expression of Pi protein from an unaltered pir gene (81). This origin also contains a half iteron and a 98-bp palindrome which has high homology with the hairpin located in the alpha origin (78). These two elements are required for the Pi-mediated looping between the beta and gamma sequences (73), and the initiation of DNA replication at the ori beta, respectively.

The Pi initiator protein is a homodimer with a 36-kDa molecular weight for the monomeric form that is lysine rich and weakly basic (63). It binds to the seven iterons in the gamma origin and to an eighth iteron and a smaller inverted pair of repeats located in the operator-promoter region of the pir gene (57, 59, 68, 69, 83) (figure 2B). In addition, Pi interacts with the iterons located in the alpha and beta iterons, however, these contacts are weak and require the enhancing effect of Pi already bound to the seven gamma iterons (73, 84). This protein is essential for replication from each R6K origin and although it can be provided in cis or trans to regulate the activity of the gamma and alpha origins, it is required in cis for activation of the beta origin (65, 72, 78, 82, 85). Thus, the Pi protein has a positive role (86) in the replication of R6K, which is displayed by its ability to enhance replication of ori alpha and ori beta. This activity is mediated by promoting DNA unwinding and bending and looping out of intervening sequences located between the ori gamma core region and the alpha and beta origins (84, 87). These DNA conformational changes lead to the activation of these two ori sites. Site-directed mutagenesis showed that the Pi protein is necessary but not sufficient for activation of ori beta and probably ori alpha (73). The DNA looping process is also required for the transfer of a multiprotein complex capable of initiating DNA replication. It was recently reported that Pi specifically interacts with the host-encoded helicase DnaB replication protein (73, 79). This observation indicates that DnaB is initially recruited by Pi bound to ori gamma and then delivered by the Pi-induced DNA looping to the alpha and beta origins.

Three additional proteins required for the distortion of the DNA structure of the R6K origins were recently described (88). Two of them, designated DDP1 and DDP2, are encoded by two tandem genes located at the 5 end of the long inverted repeat of the alpha origin. The other protein, designated DDP3, is encoded by a gene mapped at the 3 end of the beta origin long inverted repeat (figure 2B). Although the distortions caused by these proteins are potentially linked to R6K replication, they are not equivalent to those described in other replication regions previously characterized (44). It was also suggested (88) that this distortion system serves to synchronize the initiation of replication and establishes the direction of replication from the alpha and beta origins.

3.2. RNA-regulated plasmids: the ColE1-type plasmids

A large number of naturally occurring plasmids as well as many of the most commonly used cloning vehicles replicate their DNA using a common mechanism: the synthesis of an RNA molecule that forms a double stranded structure with the template DNA followed by digestion with RNase H and initiation of replication by DNA polymerase I (5, 40, 89-95). These plasmids include the naturally occurring ColE1, pMB1, p15A, pJHCMW1, as well as cloning vehicles such as pBR322 and related vectors, the pUC plasmids, the pET series, the pBluescript series and several others (91-93, 96-103). Of all these plasmids, ColE1 initiation of replication and its regulation has been the most thoroughly studied (47, 101, 102, 104-139).

Figure 3. Mechanism of initiation of replication of ColE1 and its regulation. The diagram in the top shows a genetic map of the initiation of replication region of ColE1. The left portion of the diagram depicts the mechanism of initiation of replication. The right portion shows the action of RNA I and the Rom protein. The arrows inside black or white circles indicate initiation of transcription locations for RNA II and RNA I. The origin of replication (ori) site (nucleotide 555 of RNA II) and the site of action of RNase H are indicated. A through D represents different steps in the initiation of replication. The proposed mechanism of the succession of events involved in the regulation of the initiation of replication of ColE1 by RNA I and Rom (adapted from 40) are shown on the right portion. The interaction between RNA I and RNA II leads to the inhibition of DNA synthesis is shown. The first interaction between RNA I and RNA II (kissing) is reversible and stabilized by the Rom protein. A more detailed scheme of the possible interactions between the RNA and Rom species has been reported elsewhere (40).

Replication of ColE1-type plasmids is initiated at a unique ori site (140, 141) (figure 3) and unlike other plasmid families, a plasmid-encoded protein does not mediate initiation of replication. However, ColE1 requires the host's DNA Polymerase I (PolI) enzyme (46), a host-encoded RNA polymerase and RNase H (46-48). The replication process is initiated by the synthesis of an RNA molecule, called RNA II, which is initiated 555 nucleotides upstream of ori (figure 3, step A). This RNA II extends about 700 nucleotides from its initiation (figure 3, steps B and C) and its 3' end forms a duplex with the template plasmid DNA location close to ori (figure 3, step C) (47, 130). This process, known as coupling, is strongly dependent on the formation of a specific secondary structure at the 5' end of the RNA II molecule that results in an interaction between the RNA portion on the ori and an upstream region of this molecule with the template DNA (see figure 3, step C) (111, 113, 114, 116, 142). RNase H, which digests the RNA II at the replication origin, recognizes this RNA II-DNA duplex. As a consequence a free 3'-hydroxyl group is generated that serves as primer for DNA synthesis catalyzed by PolI (figure 3, steps C and D) (47). Mutagenesis analysis on RNA II showed that some point mutations prevent this molecule from adopting the right spatial conformation that in turn affects hybridization with the template DNA (111, 113, 114). Once PolI begins the addition of deoxynucleotides, the remaining portion of RNA II which is still hybridized to the template DNA is digested at other sites by RNase H and by the 5'-3' exonuclease activity of PolI (figure 3, step D) (125). ColE1 DNA replication proceeds unidirectionally with the initiation of the lagging strand synthesis at specific ColE1 sites.

Two alternative mechanisms that seem not to be used in wild-type cells and that may be adaptations to specific host mutations were described for replication in the absence of either RNase H or RNase H and PolI (109, 115, 143, 144). In these two mechanisms, RNA II is required to be in the correct three dimensional configuration to act as the primer for DNA replication (109, 144, 145). In the case of a double mutant lacking PolI and RNase H, RNA II synthesis is extended because of the lack of RNase H. This fact allows the formation of a single stranded DNA region that can extend to a length that is adequate for assembly of a replisome and initiation of synthesis on the opposite DNA strand (lagging strand) (40, 115). In the case of RNase H deficient mutants, a mechanism dependent of PolI was described. The extended RNA II species can be recognized by PolI and used as a primer (109). However, in vitro experiments suggested that this mechanism of initiation is rather inefficient (47).

3.3. Rolling-circle replicating plasmids: the pT181-type plasmids

A number of small, high copy number plasmids from Gram-positive bacteria have been initially shown to replicate using the rolling circle mechanism of replication and later some plasmids of Gram-negative bacteria were identified that use this mechanism of replication (41, 146-153). The molecular mechanisms of replication of plasmids pT181, pC221, pUB110, pC194 and pMV158 are the best known to date (43, 53, 154-158).

Based on sequence comparisons and genetic organization of the replication regions of plasmids of Gram-positives, five replication systems have been defined (5, 41, 147, 154), the rolling circle, the theta mechanism (two different types: those using a short RNA primer and those using a long RNA primer), and the Streptomyces and Borrelia linear plasmids (discussed in section 3.4). The most common replication system among the Gram-positives plasmids is the rolling circle (5). They replicate using an asymmetric rolling circle pathway similar to that of the single-stranded filamentous bacteriophages (5, 41, 159). Four groups of rolling circle replicating plasmids have been recognized. The pT181, the pMV158, the pC194, and the pSN2 groups (41, 43, 53, 146, 147, 150, 154-156). Here we will review the replication mechanism of pT181, one of the best understood rolling circle replication mechanisms.

The pT181 plasmid was isolated from Staphylococcus aureus (160) and a diagram showing its replication region and the mechanism of initiation of replication is shown in figure 4. The plasmid pT181 encodes a 38-kDa initiator protein, RepC, that has sequence-specific endonuclease and topoisomerase I-like activities (161). This protein induces a nick in one of the pT181 DNA strands (leading strand) at a specific site signaling the initiation of replication. The nick generates a free 3'-OH end that is used as primer for DNA synthesis (162). The functional conformation of the RepC protein is a homodimer that recognizes and binds to a specific site (Rep binding site) that encompasses an inverted repeat (IR III in figure 4) (161). A domain of six amino acids in RepC plays an important role in the recognition and interaction of the Rep binding site (figure 4) (162). The binding efficiency of RepC is increased by the presence of cmp, a 100-bp cis-acting replication enhancer located about 1 kb from the nicking site (163, 164). It has been recently demonstrated that a S. aureus protein, CBF1, binds cmp and increases distortion of the already bent cmp locus (165). Whether this binding is associated with the enhancing activity of cmp is still not known.

Figure 4. Mechanism of initiation of replication of the plasmid pT181 and its regulation (adapted from 5, 162, 170). (A) Diagram of the pT181 replication region showing the two origins of replication (leading and lagging strands). The broken line arrow represents the RepC coding region. The six amino acid region of RepC recognized by ori are indicated as a black square. The location of the cop region is indicated by a box. The three RNA species are indicated showing the inverted repeats I, II, III, and IV. For the sake of clarity, these inverted repeats are not at scale. (B) Model for initiation of replication of pT181. The first diagram shows the region encompassing the origin of replication of the leading strand and the three inverted repeats. This region is also referred to as the double stranded origin (DSO). The second diagram shows the formation of the cruciform structure after binding of the RepC homodimer, the bending of the DNA at the binding region, and the change in structure of RepC. The AT-rich region that includes IR I facilitates the melting process. The third diagram shows the assembly of a replisome after the nicking took place. Presumably DNA polymerase III initiates replication in the presence of the helicase PcrA and single-strand binding protein (ssb). (C) Mechanism of regulation of expression of RepC by antisense RNA (adapted from 41, 218). The early RNA III transcript can interact with antisense RNA (RNA I). This interaction leads to the formation of a stem-loop between inverted repeats III and IV that results in a transcription termination signal. In the absence of RNA I, the inverted repeat III is sequestered by inverted repeat I, inhibiting formation of the transcription termination signal and leading to completion of the repC mRNA.

Binding of the RepC homodimer to IR III triggers bending of DNA in this region (166) followed by a change in structure of RepC, DNA melting, and formation of a cruciform structure at the IR II region (figure 4) (162). The melting step is facilitated by the presence of an AT-rich inverted repeat region (IR I) located upstream of IR II (5). This induces the formation of a cruciform structure that may help in approximating the nicking site of the leading strand to the active site of RepC. This process involves a tyrosine residue that appears to facilitate the generation of the nick. After the endonuclease attack, the RepC protein remains covalently bound to the 5' end of the DNA by a phosphotyrosine bond (155). It is probable that RepC remains covalently attached to the DNA throughout the replication of the leading strand. However, although this seems to be the case for

pT181, it was recently shown that in a derivative of pMV158 the initiator protein does not remain covalently bound to the DNA after nicking (156). After generation of the 3'-OH terminal end by RepC nicking of pT181 DNA, an initiation complex is formed with DNA polymerase III, the helicase PcrA and single strand binding protein (figure 4) (5, 167, 168). Following (or during) replication, there is an addition of a 10-12-mer oligodeoxynucleotide identical to the sequence located immediately 3' to the origin of the leading strand to the RepC molecule resulting in a modified protein known as RepC*. This modification process leads to the loss of the two enzymatic activities of RepC (169, 170). As a consequence, the active initiator RepC/RepC homodimer becomes the inactive RepC/RepC* heterodimer after it has been used for replication of pT181 (171).

Replication of the leading strand does not require any protein encoded by the plasmid (172). Initiation of replication of the lagging strand is initiated at a different location than the leading strand known as SSO (single-strand origin) or palA (173). This locus comprises a stretch of about 160-bp palindromic DNA sequence (5, 41). As a consequence of the synthesis of the leading strand a displaced single-stranded DNA is generated which allows the initiation of replication region of the lagging strand to adopt the appropriate conformation to serve as a priming site. It has been previously shown that some rolling circle replicating plasmids have different origins of replication for their lagging strands when replicating in different hosts, which suggests that lagging strand origins of replication may be important in determining the host range of plasmids (5).

Termination of pT181 synthesis of the leading strand occurs at the nick site by a strand transfer RepC-mediated mechanism (174). Once the nick site has been replicated and extended a few nucleotides beyond this site, one of the subunits of the RepC dimer contacts the growing strand. This interaction initiates a strand transfer reaction resulting in the formation of a single stranded monomer (old strand), a double stranded molecule where one of the strands is newly synthesized, and a dimer in which one of the monomers is attached to the oligonucleotide resulting from the extension of replication beyond the nick site (5, 41).

3.4. Replication of linear plasmids: the Streptomyces and Borrelia plasmids

It was proposed initially that the Streptomyces linear plasmids replicate by a protein-primed replication mechanism (18, 175), where the telomere is the origin of replication. This region is recognized by specific DNA binding proteins that promote the unwinding of the double helix and serve as the primer for a specific DNA polymerase. However, it was latter observed that the pSCL S. clavuligerus linear plasmid can replicate as a circular DNA molecule when the telomeres are removed and the ends are ligated (176). This observation suggested that linear plasmids such as pSLA2 must contain an internal site capable of promoting DNA replication. Agarose gel electrophoresis analysis showed that linear plasmids are indeed replicated primarily bidirectionally from an internal origin, located near the center of the plasmid, toward the ends, rather than by full-length strand displacement initiated at the telomeres. However, pSLA2 still requires a protein-primed strand displacing mechanism to complete the replication of a 280-nucleotide segment at both plasmid termini. It was postulated (175) that the synthesis of the 5 terminal segment of the lagging strand of this linear plasmid uses the 3 overhang of the leading strand as a template and is primed by the protein covalently attached to the 5 end of the mature plasmid.

The knowledge on the replication mechanisms of linear plasmids in Borrelia has been extended by a series of very recent publications (24, 177, 178). Some of the Borrelia linear plasmids can exist and replicate either as a linear or a monomeric circular element (24), a process that involves circular intermediates. These intermediates are formed by a head-to-tail junction, after a nick introduced in the inverted repeat opens the plasmid termini (figure 5, steps A and B), which can replicate as any other well characterized circular replicon. The linear double-stranded copies are generated from the circular intermediate by a second nick within the terminal inverted repeats (figure 5, steps C and D). More recently, it was found that B. burgdorferi sensu lato contains atypical large linear plasmids ranging from 92 to 105 kb (178). These plasmids carry p27 and ospAB, genes that were also detected in other isolates on the 50-kb linear plasmid pAB50. A more detailed analysis of the larger plasmids demonstrated that they are formed by tail-to-tail dimerization of pAB50. The presence of such a dimers can explain the unusual plasmid variability observed among different isolates and may provide new information regarding the replication mechanism of these linear replicons. It was postulated (178) that these linear dimers are the result of failed segregation after DNA replication by a mechanism similar to that described for vaccinia viruses (179). In this model proposed by Marconi et al. (178), initiation of plasmid replication proceeds from one of the termini, after the head hairpin loop in this particular case is nicked, allowing the formation of tail-to-tail dimer intermediates. Alternatively, these dimers can arise by DNA replication initiated from an ori located within a linear monomer that results in a circular replication intermediate. In the normal replication process, the circular intermediate is resolved into two linear monomers by independent cleavage events at each telomere junction. Cleavage failure at the tail hairpin by a specific DNA cleavage system results in two monomers linked tail to tail, a possibility that was confirmed experimentally by restriction analysis and Southern blot DNA hybridization experiments (178). However, this same analysis demonstrated that not all Borrelia large plasmids were originated via dimer formation and alternative DNA replication systems still uncharacterized must exist.

Figure 5. Replication model for linear plasmids in Borrelia (adapted from 24). (A) The replication process begins by the nicking (open-arrowhead arrows) of the initiation site located within the telomeric inverted repeats (shaded arrows). (B) The open linear plasmid circularizes due to the presence of complementary sequences at its termini and replicates as a circular replicon. (C) The newly replicated plasmids are nicked within the telomeric inverted repeats. (D) The free single-stranded ends pair back with their complementary copies located in the same monomer, reconstituting the hairpins at the plasmid termini. Two copies of the original linear plasmid are generated after DNA ligation.

Some of the proteins involved in the replication of the linear plasmids in Borrelia were identified by determining the complete nucleotide sequence of the 16-kb plasmid lp16.9 isolated from B. burgdorferi B31 (177). This study revealed the presence of 15 open reading frames (Orf), named A to O. The predicted proteins encoded by OrfM and N showed homology to proteins involved in plasmid replication and cell division. OrfM is homologous to MinD, a cytoplasmic membrane protein with ATPase activity required for the correct placement of the division site (180, 181). OrfM has also homology with plasmid partition proteins, such as ParA or SopA (182, 183), and the RepB protein encoded by the pheromone-responsive plasmid pAD1 in Enterococcus faecalis and involved in the control of the copy number of this extrachromosomal element (184). The predicted features and the primary sequence of the OrfN hypothetical polypeptide are similar to those of the CopB DNA-binding proteins, also known as RepB or RepA2, involved in the control of plasmid copy number in Gram-negative bacteria (185). This sequence analysis also led to the hypothesis that OrfN may interact with the short repeated sequence located within the promoter region of OrfM and, thus, controlling the expression of the latter (177). In summary, sequence analysis of Borrelia plasmids showed that the few proteins identified are similar to prokaryotic proteins involved in plasmid replication and maintenance in Gram-positive and -negative bacteria.

4. PLASMID COPY NUMBER AND INCOMPATIBILITY

Several of the plasmid replication elements described above are also involved in the expression of functions controlling the initiation of plasmid DNA replication. These control mechanisms are ultimately responsible for the copy number and incompatibility properties of different plasmids. The two main mechanisms controlling plasmid replication and incompatibility are based on antisense RNAs and repeated sequences located near the origin of replication. There are also plasmids such as R1, pMV158 and pIP501 in which the control of copy number involves both, antisense RNA and protein. In the following sections we will concentrate on the description of some antisense RNA- and iteron-mediated control of copy number mechanisms. Control of copy number of other plasmids not presented here have been thoroughly reviewed recently (5, 15, 37, 40, 147, 154, 158, 186-188). 4.1. Antisense RNAs

Antisense RNA molecules are responsible for regulation of replication of several plasmids by different mechanisms: in some cases they control the synthesis of a replication protein and in some others they inhibit the activity of the RNA primer (186, 189). In the case of IncFII plasmids, which include R1, R100 or R6-5 as well as other related elements belonging to the IncFIIa, IncFIFc, IncFIII, and IncFVII group, an antisense RNA of about 90 nucleotides regulates initiation of replication (186). The best known of these plasmids is R1 that requires the RepA protein for initiation of replication (190, 191). The RepA mRNA is known as CopT (Cop target) and the regulator antisense RNA is called CopA. CopA controls initiation of R1 replication by interacting with CopT resulting in posttranscriptional inhibition of RepA synthesis (192-200). The concentration of CopA in the cytosol is influenced by the PcnB and RNase E proteins (201). On the other hand, in the case of ColE1-type plasmids the antisense RNA acts by interacting with the RNA primer.

The mechanism of regulation of initiation of replication of ColE1 is among the best understood (5, 37, 40, 117, 187, 188, 202-204). It is worthy to mention that the study of replication of the ColEl plasmid yielded the discovery of antisense RNA and the first demonstration that antisense RNA can control gene expression. Regulation of initiation of replication of ColE1 is mediated by the action of a 108-nucleotide antisense RNA transcript, called RNA I. This RNA specie is encoded in a region that overlaps the coding region for RNA II (figure 3) (47, 205). Therefore, RNA I is an antisense molecule with respect to RNA II. RNA I inhibits initiation of replication by binding to nascent RNA II by complementary base pairing. This process results in a conformational change in RNA II that prevents coupling with DNA (101, 114, 130, 133, 206). There are several stem-loops within the secondary structures of both RNA species (figure 3). The first interaction between both RNA species occurs at complementary loops. This is a reversible process denominated "kissing" that results in the generation of an unstable initial complex (figure 3) (110, ,131, 132, 135, 207). After this initial contact takes place, an irreversible process leads to the formation of an RNA duplex between RNA I and RNA II (figure 3) (102, 132, 133). For RNA I to exert its inhibitory effect it must be present during a short, specific interval in the synthesis of RNA II (133, 208). However, this situation occurs very often because RNA I initiation of transcription proceeds as much as five times more often than transcription of RNA II. As a consequence only one out of about 20 RNA II molecules that are initiated results in a productive DNA replication. Some other factors can have an influence in the available amount of RNA I in the cell: polyadenylation (209, 210) and bacterial RNase E which inactivates RNA I by cleavage of its 5' end (211).

Another factor involved in the control of initiation of replication of some, but not all, ColE1-type plasmids is a 63 amino acid protein called Rom (RNA one modulator) or Rop (Repressor of primer) (figure 3) (5, 131-133, 205, 212). The gene encoding this short protein is located downstream of ori. Rom binds to the stem-loop portions of the unstable initial complex (110, 207), reducing the dissociation constant of the complex. Therefore, its presence results in the creation of a pathway for a stable binding of RNA I and RNA II which reduces the number of productive initiation of replication rounds (figure 3) (132, 136). As a consequence, although the presence of Rom is not needed for viability of the plasmid, ColE1 deletion derivatives that lack rom present a higher copy number than plasmids that have the complete ColE1 replication region (5, 131, 213). RNA I is the main incompatibility determinant in ColE1-type plasmids. Two plasmids that depend on the same RNA I species for regulation of initiation of replication can not coexist in the same cell (15, 37, 38). It has been shown that even single nucleotide changes can have profound effects in the incompatibility properties of ColE1-type plasmids (101, 206).

Regulation of the synthesis of the RepC protein is the main mechanism of control replication of pT181 (5, 214-216). The organization of pT181 replication regulation region (cop) is shown in figure 4. The repC mRNA (RNA III) includes a leader sequence typical of genes subjected to regulation by attenuation (217). This leader sequence has four interacting sequences (I - IV). Sequence I, a 9-nucleotide sequence called "preemptor" can form duplexes with sequences II and III (figure 4). In addition to RNA III, there are two antisense RNA species (RNA I and RNA II). These RNAs are complementary to the leader sequence of RNA III. In the presence of antisense RNA, a stem-loop structure between inverted repeats III and IV, which is followed by an AUUUUUU sequence, is formed in the leader portion of RNA III. This structure acts as a transcription termination signal (figure 4) (218). In the absence of antisense RNA, the interaction between inverted repeats I (the preemptor) and III generates a structure that prevents the formation of the III-IV stem-loop allowing transcription of repC to proceed (figure 4) (215, 218). Thus, the main incompatibility elements in pT181-type plasmids are the antisense RNA and the origin of replication of the leading strand (5, 215, 218). The origin of replication of the leading strand directly binds RepC, titrating the protein that is present at limiting levels in the cytosol due to the controlling action of the antisense RNAs.

4.2. DNA iterons

In the case of the iteron-regulated plasmids, the clusters of repeated sequences located at ori, and the copy control and autoregulation regions are the main incompatibility determinants (5). Initially, it was proposed that the replication protein was titrated by the iterons, limiting the rounds of initiation, however, further studies on plasmid replication suggested the existence of other mechanisms involved in copy number control and incompatibility (38, 219). The structural analysis of P1 RepA protein-DNA complexes revealed that the ori and copy control iterons can compete with the autoregulation iterons for RepA binding. A close examination of those RepA-DNA complexes showed the formation of a DNA loop between the incA determinant and the origin of replication (220). This DNA looping between incA and ori, mediated by RepA, can occur intra- as well as intermolecularly (220, 221), producing a steric blockage of replication initiation that causes plasmid incompatibility and affects negatively the plasmid copy number.

More recently, a similar negative control mechanism was proposed for P1 (221, 222), RK2 (54), and R6K (223). This model, known as "handcuffing" or coupling, which is based on the interaction between the replication protein and the origin of replication, proposes two alternative pathways for the Rep-bound iterons depending on the intracellular concentration of these complexes (figure 6). At low concentration, the Rep-iteron complexes stimulate the initiation of replication until the regular copy number is achieved. However, when the copy number is increased beyond the normal levels or a second plasmid harboring the same replication system is introduced into the cell, the Rep-iteron concentration is high enough to produce the binding of almost all protein-bound iterons. This direct interaction between the plasmid molecules produces the coupling or handcuffing of the origins, making them unavailable for the initiation of replication. Thus, iteron-containing plasmids regulate their copy number and express incompatibility by sensing the formation of coupled origin sequences mediated specifically by their cognate initiation proteins (224).

Figure 6. Model for control of plasmid replication by the handcuffing mechanism (adapted from 221, 222). (A) At low copy number replication is initiated by the interaction of the Rep protein with the iterons located within ori. (B) Further plasmid replication is blocked by antiparallel pairing when the plasmid reaches the appropriate copy number or a different plasmid harboring the same replication system is introduced into the cell. (C) The plasmid copies are pulled apart into the new daughter cells by a partition and segregation mechanism (D), where the replication cycle is then reiterated. The curved arrows depict the rep gene while the open and closed circles represent the Rep protein bound to the inc and ori determinants, respectively.

In the case of R6K, the control of copy number and incompatibility functions are due to the two negative activities of the Pi replication protein. One of them is as an autorepressor at the level of transcription and involves its binding to iteron sequences located within the operator-promoter site of the pir gene (figure 2B). These interactions act by either preventing the binding of the RNA polymerase to the promoter or displacing the RNA polymerase from promoter-enzyme complexes (66, 83, 225) leading to a reduction in the synthesis of Pi. The other negative role was detected when increased intracellular levels of Pi either lowered the plasmid copy number or completely prevented its replication (225). This negative regulatory activity of Pi was explained by molecular models involving either direct Pi-DNA interactions or association of Pi with either other Pi molecules or with host encoded proteins (86, 223). The R6K "handcuffing" model is based on the observation that Pi has the ability to associate two DNA molecules containing gamma ori sequences and to enhance the DNA ligase-catalyzed multimerization of a single DNA fragment carrying this origin of replication (223). In addition, it was shown that the negative domain of Pi is located in the N-terminal region of this protein and that the Pi-mediate inhibition of R6K replication does not require direct binding to DNA (226). Consequently, the origins located within these DNA-protein complexes are unable to initiate replication most likely because Pi-induced DNA structure alterations in the origins or potential Pi-host protein interactions are prevented. In summary, all these observations proved that Pi has positive and negative functions in the replication of R6K. This well controlled replication process is the result of a competition between positive gamma-alpha and gamma-beta interactions and the inhibitory aggregation of gamma-containing molecules, all mediated by the Pi replication protein.

6. ACKNOWLEDGMENTS

Some of the experiments reported in this review that were conducted in the laboratories of the authors were supported by Public Health Service grants AI37781 (LAA), AI39738 (MET) and AI19018 (JHC) from the National Institutes of Health, and research funds from Miami University (LAA) and California State University Fullerton (MET).

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