LAUNS VI Neoh Leal? Plasmid incompatibili maintained in oe ae fe coe as the inability of different plasmids to be The replication problem Plasmids wit i eo ean the same replicon are incompatible because they compete for the nee ol machinery within the cel. Most plasmids encode a negative involving antisense RNAs or iterons that inhibits replication when copy number in the cell is hi licatic See igh, but allows replication when copy number in the cell an me eid ies nde on the plaid either indirectly inhibit replication through iting n achinery proteins, or directly inhibit by binding to the origin of repli ing repl Y snety (al Soke plication and blocking replication machit 1998). As the copy number of the plas inareased amourte ofthese Q mid antisense RNAS inhib plasmid repleation soeu amouns of hese Compatible Incompatible ° oOo CO%G ° 0° of o Level of negative conve reflect copy number Lovel of negative conzel does na elect copy number Normal replication frequency ‘Abnormally low replication frequency Plasmids maintained Plasmids lost ver generations. Figure 1: The replication problem. Iterons are repeated sequences within the origin of replication and are required for replication in plasmids that contain them (del Solar et al, 1998). There are two models for iteron-based plasmid replication control that are not necessarily mutually éxclusive. In the first, the replication initiation factor RepA binds to the iteron to initiation replication. However, in iteron-containing plasmids, RepA can also bind to its own promoter and inhibit its own transcription. As copy number increases, Rep transoription is repressed and replication is downregulated, In the second model, termed the “handcuff” model, iterons of two separate plasmids bind to the same RepA molecules, forming a “handcuff” structure that physically blocks replication machinery when plasmid copy number is high (del Solar et al., 1998). In cases of compatible plasmids, different negative regulation systems control plasmid replication of each plasmid using unique replication machinery. However, when plasmids have the same origin of replic ition, the negative regulation system = acannot distinguish between the different plasmids. The concentration of negative regulation system elements in the cell is artificially high for both plasmids, so each Plasmid “thinks” it has a higher copy number than it actually does, and neither plasmid is maintained (Figure 1) The partitioning problem Plasmid incompatibility also occurs because of plasmid partitioning (Schumacher, 2012). This concept is a litle more complex than simply competing for replication There ate several different versions of the plasmid partitioning system in the bacteria, but generally it consists of a centromere-ke region on the plasmid, a centromere-like region binding protein (the CBP), and a partition NTPase (Schumacher, 2012). During cell division, CBPs bind to the centromere-like region on each plasmid and pair the plasmids together (akin to sister chromatids coming together in eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When plasmids are compatible, different CBPs bind to each plasmid type, and different NTPases separate the plasmid pairs into the new daughter cells. For high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding region compete for the same CBPs and NTPase to correctly x partition nid loss NTPase (Diaz et Figure 2: The partitioning problem.USS UT oh -7- WaltA? Cannot distinguish between the different plasmids. The concentration of negative regulation system elements in the cell is artificially high for both plasmids, so each plasmid “thinks’ it has a higher copy number than it actually does, and neither plasmid is maintained (Figure 1), The partitioning problem Plasmid incompatibility also occurs because of plasmid partitioning (Schumacher, 2012). This concept is a little more complex than simply competing for replication Control factors and tends to be more of a concern with low copy plasmids, though the Partitioning problem does occur in high copy plasmids (Diaz et al, 2015). Plasmids contain a sy: ‘stem to partition themselves to each daughter cell during cell division, They don't simply rely on chance (or random diffusion) to make this happen. There are several different versions of the plasmid partitioning system in the bacteria, but generally it consists of a centromere-like region on the plasmid, a centromere-like region binding protein (the CBP), and a partition NTPase (Schumacher, 2012). During cell division, CBPs bind to the centromere-like region On each plasmid and pair the plasmids together (akin to sister chromatids coming together in eukaryotic cells). The partition NTPase is recruited and “walks” each plasmid to a separate daughter cell. When plasmids are compatible, different CBPs bind to each plasmid type, and different NTPases separate the plasmid pairs into the new daughter cells For high copy plasmids, incompatibility due to partitioning is similar to incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding region compete for the same CBPs and NTPase to correctly partition plasmids to each daughter cell. When there are not enough CBPs and NTPase to go around, the plasmids are randomly positioned, leading to plasmid loss (Diaz et al., 2015). Figure 2: The partitioning problem.PLASIVID INCOMPATIBILITY, For low copy plasmids. an identification issue: the CBPs in the cell cen’ a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However, studies that are more recent suggest that the late replication that occurs for some types low copy plasmids during cell division simply does not allow for enough time for correct Partitioning to occur (Diaz et al., 2015). It is also worth noting that plasmids can be symmetrically incompatible, meaning that both plasmids are lost from the cell with the same probability, or asymmetrical, where one plasmid lost from the cell at a higher rate than the coresident plasmid. Asymmetric plasmid loss occurs for several reasons, including one plasmid blocking replication of another and one plasmid outcompeting the other plasmid for replication or partitioning machinery (Novick, 1987). In cases where one plasmid outcompetes the other, usually a smaller, higher copy plasmid can more effectively titrate replication or partitioning machinery away fro|'1 a larger, low copy plasmid and the larger, low copy plasmid is more likely to be lust over time.