al. 2018). ChrI and ChrII each possess their own ParAB‐parS systems and use these for efficient segregation of chromosome copies at cell division (Fogel and Waldor 2005; Yamaichi et al. 2007, 2011).
1.13 Plasmids
In many bacteria, autonomously replicating and segregating genetic elements called plasmids accompany the chromosome in the cell. Like most bacterial chromosomes, plasmids are usually covalently closed, circular DNA molecules, but this is not always the case: some are linear. Certain plasmids are categorised as additional chromosomes (or ‘chromids’) due to their size, their carriage of genes normally found on bona fide chromosomes, their unitary copy number, and/or the coordination of their replication and segregation with the main chromosome (Barloy‐Hubler and Jebbar 2008; Fournes et al. 2018). Other very big plasmids are called ‘mega‐plasmids’ and can encode functions required for symbiosis or virulence (Schwartz 2008). In general, plasmids carry genes that are useful rather than essential, so their loss is not usually fatal to the cell; in contrast, loss of the chromosome is fatal.
Plasmids came to attention due to their involvement in bacterial sex (the Fertility, or F factor) and when it was discovered that they carried genes for resistance to antimicrobial agents, including antibiotics (R factors). Investigations of these phenomena led to the discovery of plasmid conjugation and the existence of other mobile genetic elements such as transposons and integrons. Plasmid studies revealed a wealth of information about plasmid replication processes, segregation systems, and copy number control mechanisms. This field also provided cloning vectors to support the emergence of the recombinant DNA technology that, in part, underpins biotechnology. Plasmid research has provided important insights into gene regulation mechanisms, including the provision of early examples of the regulatory roles of small RNA molecules.
1.14 Plasmid Replication
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
