Читать книгу Structure and Function of the Bacterial Genome - Charles J. Dorman - Страница 25

The term plasmid was introduced in the mid‐twentieth century to describe self‐replicating extrachromosomal DNA elements (Lederberg 1952). Self‐transmissible plasmids are important contributors to HGT; indeed, some plasmids can even participate in DNA transfer between different domains of life (Suzuki et al. 2008; Zambryski et al. 1989). Limits to the host range of a plasmid include barriers to mating bridge establishment, to successful DNA transfer, and to successful plasmid replication (Jain and Srivastava 2013). Plasmid size is a poor predictor of host range: while many broad‐host‐range plasmids are large, many others are quite small. For example, pBC1 can function in both Gram‐negative and Gram‐positive bacteria, yet it is only 1.6 kb in size (De Rossi et al. 1992). At the other end of the scale, the intensively studied RK2/RP4 plasmid group is in the 60‐kb‐size range but is confined to Gram‐negative hosts, albeit a wide selection of these (Thomas et al. 1982). It is an advantage to have several origins of plasmid replication as this improves the chances of being able to replicate in a given host. However, the presence of multiple origins is not in itself a reliable indicator of broad host range: the F plasmid has a narrow range yet it has three origins of replication. Instead, it is the structure of the origin(s) that seems to be important in determining host range. Plasmids with a minimum dependence on host‐encoded factors for replication are likely to have a broad host range; for example, RSF1010 from incompatibility group Q (IncQ) uses a strand‐displacement mode of replication that relies on no host‐encoded factors for the initiation of DNA synthesis (Loftie‐Eaton and Rawlings 2012).

The RK2 plasmid (IncP) has a broad host range and can replicate in E. coli or Pseudomonas aeruginosa (Shah et al. 1995). It can replicate in E. coli using just a minimal origin, oriV (‘vegetative’ origin), containing five iterons (directly repeated sequences) and four binding sites for DnaA (Doran et al. 1999) (Figure 1.12). In P. aeruginosa, RK2 needs an additional three iterons but can dispense with the DnaA boxes (Schmidhauser et al. 1983). RK2 is also capable of replicating without DnaA when in C. crescentus (Wegrzyn et al. 2013).

Figure 1.12 Theta model of plasmid replication. (a) The structure of the origin of replication in the broad‐host‐range, single‐copy, IncP plasmid RK2, showing the relative positions and numbers of the DnaA boxes, the iterons (binding sites for the replication protein, TrfA), the AT‐rich DNA unwinding element (DUE), and the adjacent GC‐rich sequence. (b) A replication cycle is shown for an idealised plasmid using theta replication. Replication begins with the binding of the replication protein to the iterons and the recruitment of the host‐encoded DnaA to the DnaA boxes. (c) The DNA in the DUE becomes single‐stranded, creating a replication bubble to which host‐encoded replication proteins are recruited. (d, e) Depending on the plasmid, DNA synthesis proceeds either uni‐ (e.g. ColE1) or bidirectionally (e.g. RK2). (f) The products of plasmid replication are catenated, double‐stranded circles and these are unlinked by Topo IV. (g, h) The unlinked plasmids are negatively supercoiled by DNA gyrase, recreating the substrate for another round of replication.

Iterons are the binding sites for plasmid‐encoded Rep (replication) proteins. The Rep proteins are needed to initiate plasmid DNA replication, but they can also inhibit this process. In addition, Rep proteins have roles as transcription regulators, acting as auto‐repressors. They are also subject to turnover by host‐encoded proteases (Konieczny et al. 2014). The positions, numbers, orientations, lengths, DNA helical phasing, and spacer lengths of iterons vary from plasmid to plasmid and making alterations to any of these details within a particular plasmid typically has a negative effect on replication (Konieczny et al. 2014). In RK2, the Rep protein (called TrfA) and DnaA both target the origin, which has an A+T‐rich DUE adjacent to the iteron arrays. Some iteron plasmids (but not RK2, which requires HU) have a requirement for IHF binding to the origin for efficient initiation of replication (Shah et al. 1995). Sites for Dam methylation (5′‐GATC‐3′) and SeqA binding are also features of some iteron‐dependent origins (Brendler et al. 1995). SeqA binding blocks replication initiation by excluding the replication proteins. A GC‐rich sequence motif is located adjacent to the DUE in RK2, but its significance is unclear (Figure 1.12). Other iteron origins have requirements for the FIS NAP and the IciA protein, an inhibitor of DNA unwinding at the DUE.

Many of the cis or trans‐acting components of iteron origins, and their architectures, are reminiscent of oriC on the chromosome. The TrfA replication protein of RK2 interacts with, and recruits, the DnaB helicase. The ability of a plasmid replication protein to recruit a host helicase may be a determining factor limiting plasmid host range (Zhong et al. 2005). TrfA also acts with Hda, the inhibitor of DnaA activity, to prevent over‐initiation of RK2 replication (Kim et al. 2003). It has been suggested that TrfA has a motif that is suitable for interaction with the β‐clamp of E. coli DNA Pol III (Kongsuwan et al. 2006). In addition to DnaB, iteron‐based initiation also requires DnaC (in E. coli), the DnaG primase, DNA gyrase, the Pol III holoenzyme, and the SSB, as is also seen at oriC (Section 1.3). Unlike initiation of chromosome replication at oriC, initiation of plasmid DNA replication at iteron origins is ATP‐independent; there is no requirement for the DnaA‐ATP form of DnaA (Konieczny et al. 2014).

An important mechanism of copy number control in iteron plasmids is ‘handcuffing’, where the monomeric Rep proteins bound to iterons on two plasmids dimerise, bridging the two replicons (Das and Chattoraj 2004). Handcuffing may be counteracted by the DnaJ‐DnaK‐GrpE protein chaperone triad, which can convert the Rep dimers to monomers (Toukdarian and Helinski 1998). The Rep proteins downregulate replication initiation through the auto‐repression of their own genes (Kelley and Bastia 1985). The level of active Rep proteins in the cell is controlled by proteolysis and protein chaperones: monomers are active and dimers are inactive in promoting replication (Konieczny et al. 2014). Active Rep proteins are a proxy for the number of plasmid copies in the cell and allow feedback through dimer formation to downregulate Rep activity, and hence to inhibit the synthesis of new plasmid copies. Antisense RNAs also play important roles in modulating plasmid replication through their ability to interfere with the expression of plasmid‐encoded replication factors (Brantl 2014).

RK2 and its plasmid relatives use a theta model of DNA replication (Figure 1.12). Plasmid ColE1, the backbone for many cloning vectors used in recombinant DNA technology, also uses theta replication, but differs from RK2‐like plasmids in relying on host factors to open the double‐stranded origin and to prime synthesis (Lilly and Camps 2015; Wang et al. 2004). DNA duplex unwinding is driven by transcription of a stable RNA pre‐primer that forms an R‐loop in the ori region of ColE1. This process is driven by negative supercoiling of the plasmid DNA. RNase H then processes the bound RNA to generate the primer RNA that is then extended by Pol I. This marks the beginning of leading strand DNA synthesis. As the newly synthesised DNA strand makes progress through the plasmid DNA duplex, it base pairs with the template to create a D‐loop that recruits PriA. Pol III takes over leading strand synthesis and initiates the synthesis of the lagging strand; the converging replisomes continue moving until they are at or near the termination site terH (Nakasu and Tomizawa 1992). Gaps between the strands are then filled in by Pol I (Troll et al. 2014).

Rolling circle replication allows plasmids to replicate independently of chromosomal DNA (Khan 2005). The process relies on a nick made by a plasmid‐encoded initiator protein in one plasmid DNA strand, providing a primer for leading strand initiation and a lagging strand origin (Figure 1.13). No RNA primer is required. Rolling circle replication is chiefly found in plasmids from Gram‐positive bacteria, although it does occur in replicons from Gram‐negatives and archaea (del Solar et al. 1993; Ruiz‐Masó et al. 2015). The process replicates the leading strand and the lagging strand in two separate steps. Leading strand replication begins with the nicking of the double‐strand origin (dso) by a plasmid‐encoded replication protein, Rep. This is a member of the HuH superfamily of DNA endonucleases (Chandler et al. 2013) and it has a binding site located adjacent to the dso that positions it appropriately to cut the DNA. The DNA to be cleaved is presented to Rep in a single‐stranded form within a stem‐loop structure that extrudes from the negatively supercoiled plasmid. This extrusion event is Rep‐binding‐dependent (Ruiz‐Masó et al. 2007). Rep forms a covalent bond with the cleaved DNA through an active site tyrosine (Noirot‐Gros et al. 1994; Thomas et al. 1990). Host DNA polymerases use the intact template strand to guide DNA synthesis while simultaneously displacing the non‐template strand. The displaced strand is coated with SSB and is ejected as a covalently closed, single‐stranded circle at the end of leading strand synthesis. This single‐stranded circle is then used as the template for lagging strand synthesis, a process that involves only host‐encoded proteins (especially RNA polymerase and DNA polymerase I) and initiates at a structured region in the circle known as the single‐strand origin, sso (del Solar et al. 1987; Gruss et al. 1987; Kramer et al. 1997). Control of rolling circle replication is achieved principally through the control of Rep protein production. For this reason, the expression of the rep gene is strictly regulated, typically via mechanisms that employ an antisense RNA or an antisense RNA working with a DNA‐binding regulatory protein. The first type operates through transcriptional attenuation while the second involves protein‐mediated transcriptional repression backed up by translational inhibition using a trans‐acting RNA (Brantl 2014; del Solar and Espinosa 2000; Novick et al. 1989).

Figure 1.13 Rolling circle plasmid replication. A circular plasmid using the rolling circle mechanism of replication is shown at top left. The double‐stranded circle is shown in a topologically relaxed state, but it would be negatively supercoiled in the bacterium, a state that encourages extrusion of a cruciform that contains the double‐strand origin (dso), the site of replication initiation. The dso is represented by a slightly thicker line in the drawing. Extrusion of the cruciform presents the dso in single‐stranded form to the plasmid‐encoded replication protein, Rep. The Rep protein is positioned appropriately by binding to a recognition site on the plasmid adjacent to the dso. The bacterial DNA polymerase use the 3′‐OH at the nick to prime DNA synthesis; no RNA primer is required. A dotted line represents the newly synthesised DNA and an arrow next to this line shows the direction of DNA synthesis. The plasmid duplex unwinds as DNA synthesis proceeds, displacing the non‐template DNA strand, which is then coated by the single‐stranded DNA‐binding protein, SSB. A full round of replication displaces the non‐template strand completely, producing a double‐stranded plasmid (with one newly synthesised strand) and a single‐stranded circle. This circle is used as the template for the synthesis of the lagging strand. Host proteins exclusively conduct lagging strand synthesis (especially RNA polymerase and DNA polymerase I), a process that begins with priming by RNA polymerase via RNA synthesis at the single‐strand origin, sso.