Characterization of the theta replication plasmid pGR7 from Acetobacter aceti CCM 3610 (2023)

Research in Microbiology

Volume 163, Issues 6–7,

July–August 2012

, Pages 419-426

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A cryptic plasmid of Acetobacter aceti CCM 3610, designated pGR7, was sequenced and characterized. It is a 2446-bp circular molecule with a G+C content of 30%, which is unusual when compared to the already known plasmids isolated from Acetobacter genera. Sequence analysis of pGR7 revealed three putative open reading frames (ORFs). ORF1 displays low similarity with other Acetobacter plasmid replication proteins. The other two ORFs show similarities only to hypothetical proteins and do not encode any important protein. The replication module comprises a DnaA box-like sequence, indirect repeats, a potential prokaryotic promoter and the rep gene. The rep module organization is similar to that found in other theta-replicating plasmids from acetic acid bacteria that stably maintain in both Acetobacter and Escherichia coli, with two repeated sequences containing modules. Nevertheless, the pGR7 plasmid could replicate and be stably maintained only in Acetobacter strains and not in E. coli, another uncommon feature of this plasmid. The Rep protein was cloned into the pET30a+expression vector and purified by high-performance liquid chromatography. The helicase activity was determined and the ability of the protein to bind to the plasmid regulation region was confirmed by an electrophoretic mobility shift assay. The plasmid was stable in the Acetobacter cells after cultivation under nonselective conditions. By real-time polymerase chain reaction, the relative copy number of pGR7 was estimated to be seven copies per host chromosome equivalent.


Plasmids are extrachromosomal, autonomously replicating DNA molecules found in all domains of cellular organisms with characteristic copy numbers within their hosts. Plasmids encode a variety of genetic determinants, allowing their hosts to better survive in adverse environment or to compete with other microorganisms that occupy the same ecological niche. Plasmids code for various functions, including saccharide metabolism, proteinase activity, production of exopolysaccharides and organic acids, DNA restriction–modification, bacteriophage resistance, bacteriocin production or resistance against antibiotics or toxic metals (Thomas, 2000).

Plasmid replication initiation requires specific proteins encoded by the plasmid and its host. Replication starts at a cis site, designated ori, and proceeds by one of the two major DNA replication modes described for circular plasmids, rolling circle (RC) or theta (Thomas, 2000). Replication of RC plasmids is initiated by a site-specific nick that produces single-stranded (ss) DNA replication intermediates (Gruss and Ehrlich, 1989; del Solar etal., 1989; Khan, 2005), whereas theta-type plasmids replicate by unfolding of the two DNA strands at the origin (ori) region, creating double-stranded (ds) DNA replication intermediates by a priming reaction on one or both strands (del Solar etal., 1989; Espinosa etal., 2000). The theta replication mechanism has been studied most extensively on circular plasmids of Gram-negative bacteria and Acetobacter genera (Trček etal., 2000; Grones and Grones, 2011). These studies revealed several steps, such as melting of the parental strands, synthesis of a primer RNA, and initiation of DNA synthesis by covalent extension of the RNA primer. With some exceptions, the plasmid-encoded Rep protein is essential for replication initiation (del Solar etal., 1989).

Molecular biology and functional genomics techniques unveiled new characteristics of acetic acid bacteria and their plasmids, mainly through systematic sequencing (Okumura etal., 1985; Fukaya etal., 1990; Fomenkov etal., 1995; Kretová etal., 2005; Grones and Grones, 2010). Such complete sequences provide insights into plasmid-borne functions, genetic exchange between bacteria, and replicons that are potentially useful for the development of molecular biology tools. The RC-replicating plasmids are widely used as molecular vehicles, but may generate problems, such as segregation instability, production of high-molecular-weight multimers, and decrease in plasmid copy number when the size of the cloned insert increases (Kiewiet etal., 1993). In contrast, theta-replicating plasmids are assumed to be structurally more stable and, thus, tolerate large inserts while maintaining high segregation stability (Kiewiet etal., 1993). The development of Acetobacter theta-replicating vectors may also broaden the range of available compatible cloning vectors for this genus.

DNA of several plasmids from acetic acid bacteria of various sizes has been purified and characterized. The first identified cryptic plasmid from Acetobacter was used for the construction of cloning vectors (Fukaya etal., 1990; Tamaki etal., 1991; Fujiwara etal., 1992). Later on, several plasmids were isolated from Acetobacter pasteurianus as pAC1 (19kb) (Grones etal., 1989, 1993; Grones and Turňa, 1992), pAP12875 (Fomenkov etal., 1995) and pAP1, pAP2, pAP3 and pAP4 (Krahulec etal., 2003). Plasmid pJK2-1 was isolated from Gluconobacter (Trček etal., 2000), pAG20 from Acetobacter aceti (Kretová etal., 2005) and pGP2 from Acetobacter estunensis (Grones and Grones, 2011). These plasmids can be divided into two groups based on their replication mechanism. One group uses a Rep protein for replication initiation and the other, including pAP1 to pAP4, uses an RNA primer as do ColE1-like plasmids.

Here, we isolated, sequenced and characterized the novel A. aceti plasmid pGR7 that possesses unique properties never described before in plasmids of acetic acid bacteria. We identified three open reading frames (ORFs), one of which encodes the replication protein Rep7, which was overproduced and purified by high-performance liquid chromatography (HPLC). This protein was found to be essential for plasmid replication. Its helicase activity and ability to bind the plasmid regulation region were verified.

Section snippets

Bacterial strains, plasmids and growth conditions

Acetobacter aceti CCM 3610, A. estunensis CCM 3613 and A. pasteurianus CCM 3614 were obtained from the Czech Collection of Microorganisms (CCM; Brno, Czech Republic). The cells were grown in YPG medium (5% yeast extract, 3% peptone, 20% d-glucose) at 28°C under aerobic conditions. Escherichia coli XL1 Blue (endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 [F':Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+) (Stratagene, La Jolla, CA, USA) used as a cloning host, and E. coli BL21 (DE3) (F ompT gal

Sequence analysis of plasmid pGR7

Plasmid pGR7 was originally isolated from A. aceti CCM 3610. Analysis of the restriction endonuclease profile revealed a single EcoRI site. EcoRI-digested pGR7 DNA was cloned into the pUK21 vector to determine the plasmid nucleotide sequence. Sequence analysis showed that pGR7 was 2464bp in length with a G+C content of 30%. Compared to the G+C content of previously known plasmids from Acetobacteriaceae, namely 49.2% for pGP2 (Grones and Grones, 2010), 51.6% for pAG20 (Kretová etal., 2005


Plasmid pGR7, isolated from A. aceti CCM 3610, features several major differences when compared to the other known plasmids from Acetobacter. pGR7 contains all features typical for theta-replicating plasmids, including the presence of DnaA-like boxes and an AT-rich region with inverted repeats in the proposed ori region. We showed that Rep7 is an essential molecular player during pGR7 plasmid replication initiation. The protein binds to the plasmid regulation region and successfully unfolds the


We thank Martine De Cock for help in preparing the manuscript. This work was supported by the long-term research program of the Ministry of Education of the Slovak Republic (projects VEGA 1/2332/05 and VEGA 1/0233/08).

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      Cryptic plasmids usually encode proteins that are involved in plasmid replication, mobilization, and/or additional functions that are not phenotypically expressed. Although large cryptic plasmids have been reported (Li et al., 2004), they are usually small and replicate via theta (Grones and Grones, 2012; Hefford et al., 1993) or rolling-circle mechanisms (Rozhon et al., 2011). Furthermore, our recent research has shown that it is possible to separate plasmid modules that possess different characteristics (Smorawinska et al., 2012), indicating that they may have evolved separately.

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    What is the mechanism of theta plasmid replication? ›

    All modalities of theta plasmid replication initiate synthesis with the leading-strand at a pre-determined site and complete replication through recruitment of the host's replisome, which extends the leading-strand continuously while synthesizing the lagging-strand discontinuously.

    What is the conclusion of theta replication? ›

    We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.

    What is theta type of replication? ›

    The theta mode of replication is a type of replication that is seen in circular DNA molecules. A circular chromosome is a form of circular DNA found in bacteria and archaea that has no free ends, unlike the linear DNA strands seen in most eukaryotes.

    Why is theta replication unidirectional? ›

    Theta replication is unidirectional, meaning that replication occurs in one direction from the origin of replication (the point at which replication begins) to the terminus (the point at which replication ends).

    Is theta replication semi-conservative? ›

    An intermediate Theta structure is formed due to the replication eye. The replication is semi-conservative type as the replication seems to be occurring at one or two moving Y-junction in the circle replication forks.

    What are the three types of plasmid replication? ›

    Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer.

    What is the rolling circle mechanism of plasmid replication? ›

    Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming ...

    What is the solution to the end replication problem? ›

    In most eukaryotes, this problem is solved by 3′ extension of telomeres by a reverse transcriptase called telomerase, and subsequent fill in by conventional DNA replication machinery (Wellinger, 2014). Figure 1. The “Unusual” telomeric chromatin and the “classical” End Replication Problem.

    What are the two main types of replication? ›

    Transactional replication and merge replication provide options for these types of applications.

    What is the definition of 3 end in biology? ›

    The 3' end is that end of the molecule which terminates in a 3' phosphate group. The 3' direction is the direction toward the 3' end.

    What determines the direction of replication? ›

    A DNA strand does, in fact, have an orientation. The configuration of the phosphate and deoxyribose sugar units all along the DNA backbone determines it.

    Is theta model of replication unidirectional? ›

    Mechanism of Theta Plasmid Replication:

    The process gets initiated by the RNA primer. Then deoxyribonucleotides are added which extends the process. The replication process may proceed in one (unidirectional) or both directions (bi-directional).

    Why does DNA replicate in opposite directions? ›

    Answer and Explanation: DNA polymerase moves in opposite directions because it is only able to attach to a free 3' end. DNA polymerase is a three dimensional enzyme and can only attach to a specific configuration of substrate, which is the free 3' OH group on a nucleotide.

    What are the three modes of replication? ›

    There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive.

    What 2 enzymes are used during DNA replication? ›

    Helicase (separate double-stranded DNA into single strands allowing each strand to be copied) DNA gyrase (reduces supercoiling which builds up during DNA unwinding)

    What would a chromosome look like after DNA replication? ›

    After DNA replication, each chromosome now consists of two physically attached sister chromatids. After chromosome condensation, the chromosomes condense to form compact structures (still made up of two chromatids).

    What are 4 important characteristics of a plasmid? ›

    Characteristics of plasmids
    • extrachromosomal circular DNA molecules which are not part of the bacterial genome.
    • size range: 1-200 kb.
    • carry functions advantageous to the host such as: produce enzymes which degrade antibiotics or heavy metals. ...
    • Replication is coupled to host replication in a:

    What are the 5 types of plasmids? ›

    5 types of plasmids are given below.
    • Resistance Plasmids.
    • Virulence Plasmids.
    • Degradative Plasmids.
    • Col Plasmids.
    • Fertility F Plasmids.

    What is the difference between plasmid and R plasmid? ›

    The key difference between F plasmid and R plasmid is that F plasmid is an extrachromosomal DNA that contains genes coding for fertility factor. Meanwhile, R plasmid is an extrachromosomal DNA that contains genes coding for the resistance against antibiotics.

    Is theta replication found in eukaryotes? ›

    Theta replication is found in prokaryotes, mitochondria and chloroplast as they have circular DNA but it does not occur in eukaryotic chromosomes present in nucleus because they have linear double stranded DNA.

    What is the function of the θ subunit of E coli DNA polymerase III? ›

    The θ Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the ɛ Proofreading Subunit.

    What are the three types of sequences within bacterial origins of replication? ›

    Ori Sequences

    DNA replication origins are characterized primarily be three types of structures: (1) sites for binding of proteins, mainly initiation and auxiliary proteins, (2) a characteristically AT-rich region that is unwound, and (3) sites and structural properties involved in regulating initiation events.

    What is the difference between plasmid and circular DNA? ›

    Plasmid DNA is linear in shape whereas chromosomal DNA can be linear or circular in shape. Naturally, plasmid DNA is present as a tightly supercoiled circle to allow itself to fit inside the cell. Chromosomal DNA is often observed as X-shaped structures.

    Why does plasmid DNA need to be circular? ›

    They are circular.

    That's because it can be cut open without falling apart, then snap back together once new DNA has been incorporated.

    What is antibiotic resistance gene in plasmid? ›

    Antibiotic resistance genes are often located on plasmids or transposons and can be transferred from cell to cell by conjugation, transformation, or transduction. This gene exchange allows the resistance to rapidly spread throughout a population of bacteria and among different species of bacteria.

    What happens to the DNA when the process of replication ends? ›

    DNA replication finishes when converging replication forks meet. During this process, called replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved.

    Is DNA lost during replication? ›

    After each round of DNA replication, some telomeric sequences are lost at the 5′ end of the newly synthesized strand on each daughter DNA, but because these are noncoding sequences, their loss does not adversely affect the cell. However, even these sequences are not unlimited.

    What are the steps of the DNA replication process? ›

    Replication occurs in three major steps: the opening of the double helix and separation of the DNA strands, the priming of the template strand, and the assembly of the new DNA segment.

    What are the 4 substances needed for DNA replication? ›

    There are four basic components required to initiate and propagate DNA synthesis. They are: substrates, template, primer and enzymes.

    What are the 4 key players in DNA replication? ›

    DNA replication involves a certain sequence of events. For each event, there is a specific enzyme which facilitates the process. There are four main enzymes that facilitate DNA replication: helicase, primase, DNA polymerase, and ligase.

    What are two ways in which DNA replication is important? ›

    The process of DNA replication helps in the inheritance process by transfer of the genetic material from one generation to another. Therefore it is required for the growth, repair, and regeneration of tissues in living organisms.

    What are the different types of DNA replication in bacteria? ›

    In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair.

    What are the two DNA molecules after replication? ›

    The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. This is why DNA replication is described as semi-conservative, half of the chain is part of the original DNA molecule, half is brand new.

    What does 5 mean in DNA? ›

    Each end of DNA molecule has a number. One end is referred to as 5' (five prime) and the other end is referred to as 3' (three prime). The 5' and 3' designations refer to the number of carbon atom in a deoxyribose sugar molecule to which a phosphate group bonds.

    What is the 3 end of DNA replication? ›

    The 3′-end (three prime end) of a strand is so named due to it terminating at the hydroxyl group of the third carbon in the sugar-ring, and is known as the tail end.

    Which enzyme removes primer in DNA replication? ›

    Because of its 5′ to 3′ exonuclease activity, DNA polymerase I removes RNA primers and fills the gaps between Okazaki fragments with DNA.

    What direction is DNA made in replication? ›

    New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5' to 3' direction. During DNA replication, one new strand (the leading strand) is made as a continuous piece.

    What is theta type plasmid replication? ›

    All modalities of theta plasmid replication initiate synthesis with the leading-strand at a pre-determined site and complete replication through recruitment of the host's replisome, which extends the leading-strand continuously while synthesizing the lagging-strand discontinuously.

    What is meant by theta replication? ›

    Theta Model of Replication

    A common type of replication that takes place in circular DNA, such as that found in E. coli and other bacteria, is called theta replication because it generates a structure that resembles the Greek letter theta (θ).

    Can DNA be copied in both directions? ›

    As shown in Figure 1, when the double helix unwinds, replication proceeds along the two single strands at the same time but in opposite directions (i.e., left to right on one strand, and right to left on the other). This forms two replication forks that move along the DNA, replicating as they go.

    What protein helps stabilize the DNA strands during replication? ›

    DNA Helicases - These proteins bind to the double stranded DNA and stimulate the separation of the two strands. DNA single-stranded binding proteins - These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases.

    How do you know if a strand is leading or lagging? ›

    The strand that opens in the 3' to 5' direction towards the replication fork is referred to as the lagging strand. The strand that runs in the 5' to 3' direction in the replication fork is referred to as the leading strand.

    What is theta mode of replication in bacteriophage? ›

    There are two modes of bacteriophage lambda DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (theta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode.

    What does DNA polymerase theta do? ›

    DNA Polymerase theta (Pol θ) is an error-prone A-family polymerase that is highly conserved among multicellular eukaryotes and plays multiple roles in DNA repair and the regulation of genome integrity.

    What is the mechanism of plasmid transmission? ›

    Plasmids can be transmitted by any of the three HGT mechanisms: transformation (29, 30), phage-mediated transduction (31), and conjugation (3). Conjugation is considered the most important mechanism of plasmid transmission, and the conjugative process has been extensively studied (discussed below).

    What are 2 mechanisms for bacterial exchange of plasmids? ›

    In transduction, DNA is accidentally moved from one bacterium to another by a virus. In conjugation, DNA is transferred between bacteria through a tube between cells.

    What are the 2 types of replication of bacteriophages? ›

    Once a bacteriophage attaches to a susceptible host, it pursues one of two replication strategies: lytic or lysogenic.

    What are the 5 stages of a bacteriophages lytic replication cycle? ›

    The lytic cycle, which is also commonly referred to as the "reproductive cycle" of the bacteriophage, is a six-stage cycle. The six stages are: attachment, penetration, transcription, biosynthesis, maturation, and lysis.

    What is DNA polymerase III subunit Theta? ›

    theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta.

    What are the characteristics of a plasmid? ›

    Plasmid is small in size, circular in shape and it is a piece of DNA that is not the same as chromosomal DNA. Its ability to replicate is independent of chromosomal DNA. They are usually found in bacteria, but they are also present in multicellular organisms.

    What is the difference between a vector and a plasmid? ›

    Difference between vector and plasmid

    Vector is an artificially synthesized/ manipulated DNA whereas a plasmid naturally occurs in bacterial cells. There are several vectors, which can be used in recombinant DNA, whereas all plasmids may not be used directly in recombinant DNA technology.

    What are the different types of plasmids and their functions? ›

    Broadly put, there are two types of plasmids – conjugative and non-conjugative. A conjugative plasmid can promote sexual conjugation between cells through the transfer of genes. On the other hand, non-conjugative plasmids are incapable of conjugation and can only be transferred through conjugative plasmids.

    What are the 3 methods of genetic transfer in bacteria? ›

    There are three “classical" methods of DNA transfer in nature: bacterial conjugation, natural transformation, and transduction (von Wintersdorff et al., 2016).

    How are plasmids transferred between bacteria? ›

    Plasmids can be transferred through direct physical contact between bacteria in a process known as conjugation, which helps bacteria share their antibiotic resistance genes with their neighbors.

    What are 3 ways antibiotics become resistant? ›

    Abstract. The three fundamental mechanisms of antimicrobial resistance are (1) enzymatic degradation of antibacterial drugs, (2) alteration of bacterial proteins that are antimicrobial targets, and (3) changes in membrane permeability to antibiotics.


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