DOMAIN 7 GENETICS AND GENETIC TOOLS Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host JAY W. KIM, VEGA BUGATA, GERARDO CORTÉS-CORTÉS, GISELLE QUEVEDO-MARTÍNEZ AND MANEL CAMPS Department of Microbiology and Environmental Toxicology, University of California at Santa Cruz, Santa Cruz, CA, 95064 ABSTRACT Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at oriC, a DnaA-dependent pathway leading to replisome assembly (theta class A), and replication fork restart, a PriA- dependent pathway leading to primosome assembly through primer extension and D-loop formation (theta classes B, C, and D). To ensure autonomy from the host’s replication and to facilitate copy number regulation, theta plasmids have unique mechanisms of replication initiation at the plasmid origin of replication (ori). Tight plasmid copy number regulation is Received: 26 March 2020 essential because of the major and direct impact plasmid gene dosage has on gene Accepted: 07 October 2020 expression. The timing of plasmid replication and segregation are also critical for opti- Posted: 18 November 2020 mizing plasmid gene expression. Therefore, we propose that plasmid replication needs to Editor: James M. Slauch, The School of be understood in its biological context, where complex origins of replication (redundant Molecular and Cellular Biology, University of origins, mosaic and cointegrated replicons), plasmid segregation, and toxin-antitoxin sys- Illinois at Urbana-Champaign, Urbana, IL; Gregory ori Phillips, College of Veterinary Medicine, Iowa tems are often present. Highlighting their tight functional integration with function, we State University, Ames, IA show that both partition and toxin-antitoxin systems tend to be encoded in close physical Citation: EcoSal Plus 2020; doi:10.1128/ proximity to the ori in a large collection of Escherichia coli plasmids. We also propose that ecosalplus.ESP-0026-2019. adaptation of plasmids to their host optimizes their contribution to the host’s fitness while Correspondence: Manel Camps, mcamps@ restricting access to broad genetic diversity, and we argue that this trade-off between ucsc.edu adaptation to host and access to genetic diversity is likely a determinant factor shaping Copyright: © 2020 American Society for the distribution of replicons in populations of enterobacteria. Microbiology. All rights reserved. doi:10.1128/ecosalplus.ESP-0026-2019 INTRODUCTION Plasmids are autonomously replicating sequences. They are found across all kingdoms of life but are particularly abundant in prokaryotes (1). They typically carry nonessential genes that have adaptive value under specific stresses such as heavy metal exposure, antibiotics, or adapting to a host (virulence genes) (2, 3). Here, we review the mechanism of plasmid replication known as theta, which is by far the predominant type in Enterobacteriaceae. This prokaryotic family groups a number of related genera (Escherichia, Shigella, Salmonella, Yersinia) that share the intestinal tract as their central ecological niche and that have been extensively studied due to their clinical relevance (4, 5). ASMScience.org/EcoSalPlus 1 Downloaded from www.asmscience.org by IP: 128.114.255.203 On: Wed, 18 Nov 2020 19:01:02 Kim et al. Enterobacterial plasmids share similar features. Nearly all restricted to replication initiation, with downstream steps of them can be transferred between strains in one way or frequently relying exclusively on host proteins. Having another, a process known as horizontal gene transfer plasmid-specific mechanisms of replication initiation (HGT). This allows plasmids to serve as platforms for the ensures autonomy from the host and facilitates the reg- exchange of genetic material, for capturing new adaptive ulation of plasmid copy number by providing specific genes from the environmental microbial gene pool, and targets for regulation. Plasmid replication initiation for shuffling and spreading these genes among pathogens involves plasmid-encoded replication proteins (Reps) (3, 6, 7). There are three main mechanisms of HGT, and/or plasmid-specific primers. Even these plasmid namely, cell-to-cell contact and formation of a bridge replication initiation factors are often assisted by host (conjugation), direct uptake from the environment proteins (see below). (transformation), and through bacteriophages (transduc- tion) (8). Conjugation is the predominant mechanism in Host proteins participating in plasmid DNA synthesis are Gram-negative bacteria. It involves copying the plasmid part of the machinery that replicates the chromosome at DNA, relaxing it, and moving the DNA through a type IV oriC (recently reviewed in references 8 and 13–15) and/or secretion system to a recipient cell in an ATP-dependent that restart replication following replication fork collapse fashion (reviewed in references 3, 7,and9). The majority (reviewed in references 16–19). These include proteins of enterobacterial plasmids carry sets of conjugation facilitating the melting of the DNA duplex (DnaA), genes, although these are not always active or complete helicases (PriA, DnaB), proteins that catalyze primer (10). Some virulence plasmids do not have a conjugation synthesis (DnaG) and primer extension (Pol I, Pol III machinery but can be transferred along with a conjugative holoenzyme), proteins that process primers and gaps plasmid, which acts as a helper; this process is known as (Pol I, ligase), proteins that control the local supercoiling plasmid mobilization (10). Transduction is also frequent status of DNA (topoisomerases), and proteins that bind in enterobacteria (11). It occurs when a bacteriophage and stabilize single-stranded DNA (ssDNA) (SSB). Note packages bacterial genetic material along with its own that replication proceeds in the 5′ to 3′ direction. The genetic material and infects another bacterium (general- synchronous replication of leading and lagging strands ized transduction) or when a phage that has integrated involves two core Pol III enzymes, with the enzyme into the bacterial chromosome is improperly excised and replicating the leading strand doing so continuously and packages bacterial DNA (specialized transduction) (12). the enzyme replicating the lagging strand doing so dis- continuously, i.e., in segments. The Pol III core enzymes Here, we present our current mechanistic understanding in the Pol III holoenzyme are joined by two τ (tau) of theta plasmid replication and its regulation. We also subunits and assisted by β units that act as mobile sliding present concepts that are emerging from genomic stud- clamps, which ensures the coordination and processivity ies, which help place the replication of these plasmids in of the replication in the two strands. its broader biological context. Given that plasmids iso- lated from a variety of Enterobacteriaceae share similar features (5), we use virulence plasmids in extraintestinal MECHANISMS OF PLASMID REPLICATION pathogenic Escherichia coli (ExPEC) as representative of Replication initiation determines the mechanics of plas- enterobacteria. mid replication. Within theta plasmids, we can distinguish two broad mechanisms of replication initiation: oriC-like (which parallels replication initiation at oriC)andPriA- PLASMID REPLICATION INITIATION AT ORIGINS mediated (which is similar to replication restart after OF REPLICATION replication fork collapse). The mechanisms involved in Plasmid replication starts at a specific location known as these two categories of plasmid replication (as well as those the plasmid origin of replication, or ori. The origin of of nontheta plasmids) are systematically compared in replication contains nucleotide sequence motifs that are Table 1 and discussed in detail below. recognized in trans by the replication machinery. This machinery typically involves a combination of plasmid oriC-like plasmids correspond to class A theta plasmid and host elements. Since borrowing genes from the replication. PriA-mediated plasmids correspond to theta host minimizes the metabolic cost of plasmid mainte- classes B, C, and D. Both categories converge at the DnaB nance, plasmid-encoded replicative elements are typically loading step and rely on the hosts’ Pol III holoenzyme to 2 ASMScience.org/EcoSalPlus Downloaded from www.asmscience.org by IP: 128.114.255.203 On: Wed, 18 Nov 2020 19:01:02 ASMScience.org/EcoSalPlus Table 1 Comparison of the different modes of plasmid replicationa Characteristic Theta A Theta Bb Theta C Theta D Strand Rolling-circle displacement Directionality of Bi/uni Uni Uni Uni Bi Uni replication (unidirectional vs bi-directional) Coupling of leading- Coupled Coupled Coupled Coupled Uncoupled Uncoupled and lagging-strand (replisome) (replisome) (replisome) (replisome) synthesis Simultaneous Yes Yes Yes Yes Yes No, with generation of leading- and lagging- single-stranded inter- strand synthesis mediate Leading strand synthesis Continuous vs Continuous Continuous Continuous Continuous Continuous Continuous discontinuous Initiation factor Rep melts duplex NA RepA (also primase Rep E melts duplex only Rep C (DnaA-like) Rep (generates initial and recruits DnaB activity) and has active role in nick and seals it) helicase, primer generation often assisted by DnaA and by NAPs Initiation factor Multiple iterons NA Iteron with two functional No iterons, no bending ssi sites