The RK2 Plasmid is a broad-host-range

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; howev ...

belonging to the incP incompatibility group It is notable for its ability to replicate in a wide variety of

single-celled organism A unicellular organism, also known as a single-celled organism, is an organism that consists of a single cell, unlike a multicellular organism that consists of multiple cells. Organisms fall into two general categories: prokaryotic organisms and ...

s, which makes it suitable as a

genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including ...

tool. It is capable of transfer, replication, and maintenance in most

genera Genus ( plural genera ) is a taxonomic rank used in the biological classification of living and fossil organisms as well as viruses. In the hierarchy of biological classification, genus comes above species and below family. In binomial ...

Gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wa ...

bacteria. RK2 may sometimes be referred to as pRK2, which is also the name of another, unrelated plasmid. The IncP-1 plasmid group (IncP plasmids in ''Escherichia coli'') of which RK2 is a part has been described as "highly potent, self-transmissible, selfish DNA molecules with a complicated regulatory circuit"

Discovery

RK2 was first isolated in connection with an outbreak of

antibiotic-resistant Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. P ...

Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common encapsulated, gram-negative, aerobic– facultatively anaerobic, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, ''P. aer ...

'' and ''

Klebsiella aerogenes ''Klebsiella aerogenes'', previously known as ''Enterobacter aerogenes,'' is a Gram-negative, oxidase negative, catalase positive, citrate positive, indole negative, rod-shaped bacterium. The bacterium is approximately 1-3 microns in length, a ...

'' in

Birmingham Birmingham ( ) is a city and metropolitan borough in the metropolitan county of West Midlands in England. It is the second-largest city in the United Kingdom with a population of 1.145 million in the city proper, 2.92 million in the We ...

in 1969, as one of a family of plasmids implicated in transfer of

Ampicillin Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B stre ...

resistance between bacterial strains.LEWIS C. INGRAM, M. H. RICHMOND, AND R. B. SYKES: "Molecular Characterization of the R Factors Implicated in the Carbenicillin Resistance of a Sequence of Pseudomonas aeruginosa Strains Isolated from Burns", ''ANTIMICROBIAL AGENTS AND CHEMOTHERAPY'', Feb. 1973, p. 279-288 Plasmids in the IncP-1 subgroup has been isolated from wastewater, agricultural soil, and hospitals.

Structure

RK2 is approximately 60 kbp long and contains

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

s for

replication Replication may refer to: Science * Replication (scientific method), one of the main principles of the scientific method, a.k.a. reproducibility ** Replication (statistics), the repetition of a test or complete experiment ** Replication crisi ...

, maintenance,

conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form * Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change ...

and

antibiotic resistance Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistanc ...

. The resistance genes confer resistance to the antibiotics kanamycin, ampicillin and tetracycline. In addition, RK2 contains a set of potentially lethal (to the cell) genes, called ''kil'' genes, and a set of complementary

transcriptional repressor Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules calle ...

genes, called ''kor'' (short for "kil-override") genes, which inactivate the ''kil'' genes. The ''kil'' and ''kor'' genes together are suspected to play a role in the broad host range of RK2.

Replication

The essential replication system in RK2 consists of an origin of replication, ''oriV'', and a gene, ''trfA'', whose gene product, the TrfA protein, binds to and activates ''oriV''. In ''Escherichia coli'', replication proceeds unidirectionally from ''oriV'' after activation by TrfA. In E. coli, multiple plasmid copies appear to cluster together, creating a few multiplasmid clusters in each cell. The copy number of RK2 is about 4-7 per cell in ''E. coli'' and 3 in ''P. aeruginosa''.

Minimal derivatives

Several minimal derivatives of RK2 have been prepared. In these plasmids most of the genes have been removed, leaving only genes essential for replication and one or more

selectable marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and g ...

s. One such "mini-replicon" is the plasmid PFF1, which is 5873 basepairs long. PFF1 consists of an

origin of replication The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by se ...

, oriV, an

origin of transfer An origin of transfer (''oriT'') is a short sequence ranging from 40-500 base pairs in length that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation. The transfer of DNA is a critica ...

, oriT, a gene coding for plasmid replication proteins, trfA, and two

genes, ''bla'' and ''cat'', which confer resistance to

and

Chloramphenicol Chloramphenicol is an antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medica ...

, respectively. Minimal plasmids such as PFF1 are useful for studying the basic mechanisms of plasmid replication and copy number regulation, as there are less superfluous genetic elements which might affect the processes being studied. Several mutants of PFF1 which affect the copy number of the plasmid have been identified. Two such mutants, PFF1cop254D and PFF1cop271C, increase the copy number of PFF1 in ''E. coli'' from approximately 39-40 to about 501 and 113 plasmids per cell, respectively. An increase in copy number is useful for genetic engineering applications to increase the production yield of

recombinant protein Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...

.Janet Martha Blatny, Trygve Brautaset, Hanne C. Winther-Larsen, Ponniah Karunakaran, Svein Valla: "Improved Broad-Host-Range RK2 Vectors Useful for High and Low Regulated Gene Expression Levels in Gram-Negative Bacteria", ''Plasmid'' Volume 38, Issue 1, July 1997, Pages 35-51

Notes

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Discovery

Structure

Replication

Minimal derivatives

Notes

Further reading