Structure and Function of the Bacterial Genome. Charles J. Dorman

Structure and Function of the Bacterial Genome - Charles J. Dorman


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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).

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      High copy number plasmids, i.e. those with 10 or more copies per cell, lack genes that are capable of encoding active partitioning machinery (Million‐Weaver and Camps 2014). Random distribution of the multicopy plasmid through the cytosol seems to account for the faithful inheritance of plasmids such as ColE1 (Durkacz and Sherratt 1983). The formation of plasmid multimers poses a risk because this process reduces the copy number of independently segregating units, but multimer resolution systems such as cer/XerCD in ColE1 provide a potent antidote (Summers and Sherratt 1984). This resolution mechanism is a close relative of the chromosomal dif/XerCD system, albeit with additional co‐factors (Section 1.8). Plasmid distribution in the cytosol is likely to be influenced by the presence of other molecules and structures, not least the nucleoid, and nucleoid exclusion does seem to be a factor in confining plasmids to the space just inside the cytoplasmic membrane (Reyes‐Lamothe et al. 2014; Wang et al. 2016; Yao et al. 2007). The plasmids occur in clusters and frequently these clusters are seen at the poles of the cell; clusters are dynamic, they can divide, with some sub‐clusters relocating to the mid‐cell (Yao et al. 2007). The introduction of another type of plasmid produces an even more complex clustering pattern (Diaz et al. 2015; Yao et al. 2007). It appears that multicopy plasmids move between existing in clusters and being alone, and that these forms diffuse randomly within the confines imposed by the nucleoid and other cell structures (Wang 2017).

      Low‐copy number plasmids cannot rely on strategies based on random spatial distribution to ensure their segregation to the daughter cells at division. These plasmids have active partitioning systems, systems that have counterparts in the chromosomes of many bacteria (but not E. coli). These partitioning (Par) systems consist of two proteins, ParA and ParB, and a centromere‐like DNA site called parS (Baxter and Funnell 2014; Gerdes et al. 2010). The ParB protein binds to parS and ParA interacts with ParB, hydrolysing ATP or GTP to provide the energy needed to drive the partitioning process.

      The R1 drug‐resistance single‐copy plasmid has a ParMRC partitioning system that consists of a centromere‐like parC site, an adaptor protein ParR that binds to parC and an actin‐like ATPase, ParM. ParM forms filaments that grow bidirectionally, with a ParR‐parC complex one either end. As the filament


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