The RK2 Plasmid is a broad-host-range plasmid belonging to the incP incompatibility group[1] It is notable for its ability to replicate in a wide variety of single-celled organisms, which makes it suitable as a genetic engineering tool.[2] It is capable of transfer, replication, and maintenance in most genera of Gram-negative bacteria. RK2 may sometimes be referred to as pRK2, which is also the name of another, unrelated plasmid.[3][4][5] Other names for RK2 include R18, R68, RP1, and RP4. These were all separate isolates, and later found to be identical plasmids.[6] 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"[7]

Discovery

RK2 was first isolated in connection with an outbreak of antibiotic-resistant Pseudomonas aeruginosa and Klebsiella aerogenes in Birmingham in 1969, as one of a family of plasmids implicated in transfer of Ampicillin resistance between bacterial strains.[8] Plasmids in the IncP-1 subgroup has been isolated from wastewater, agricultural soil, and hospitals.[9]

Structure

RK2 is approximately 60 kbp long and contains genes for replication, maintenance, conjugation and antibiotic resistance. The resistance genes confer resistance to the antibiotics kanamycin, ampicillin and tetracycline.[8] In addition, RK2 contains a set of potentially lethal (to the cell) genes, called kil genes, and a set of complementary transcriptional repressor 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.[10]

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.[11][12] In Escherichia coli, replication proceeds unidirectionally from oriV after activation by TrfA.[13] In E. coli, multiple plasmid copies appear to cluster together, creating a few multiplasmid clusters in each cell.[14][15] The copy number of RK2 is about 4-7 per cell in E. coli and 3 in P. aeruginosa.[16]

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 markers. One such "mini-replicon" is the plasmid PFF1, which is 5873 basepairs long.

PFF1 consists of an origin of replication, oriV, an origin of transfer, oriT, a gene coding for plasmid replication proteins, trfA, and two antibiotic resistance genes, bla and cat, which confer resistance to Ampicillin and Chloramphenicol, 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.[17] An increase in copy number is useful for genetic engineering applications to increase the production yield of recombinant protein.[18]

Notes

  1. ^ David H. Figurski, Robert F. Pohlman, David H. Bechhofer, Alice S. Prince and Christie A. Kelton: "Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells", Genetics, Volume 79. March 1982, pp. 1935-1939
  2. ^ J M Blatny, T Brautaset, C H Winther-Larsen, K Haugan and S Valla: "Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon", Appl. Environ. Microbiol. 1997, Volume 63, Issue 2, p. 370
  3. ^ National Center for Biotechnology Information: "Escherichia coli W plasmid pRK2, complete sequence". https://www.ncbi.nlm.nih.gov/nuccore/315063834
  4. ^ Colin T Archer ,Jihyun F Kim ,Haeyoung Jeong ,Jin Hwan Park ,Claudia E Vickers ,Sang Yup Lee, Lars K Nielsen: "The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli", BMC Genomics, 2011, http://www.biomedcentral.com/1471-2164/12/9
  5. ^ Stepánek V, Valesová R, Kyslík P.: "Cryptic plasmid pRK2 from Escherichia coli W: sequence analysis and segregational stability.", Plasmid, 2005 Jul;54(1):86-91.
  6. ^ Pansegrau W, Lanka E, Barth P, Figurski D, Guiney D, Haas D, Helinski D, Schwab H, Stanisich V, Thomas C: "Complete Nucleotide Sequence of Birmingham IncPα plasmids: Compilation and Comparative Analysis", Journal of Molecular Biology, 1994
  7. ^ Malgorzata Adamczyk and Grazyna Jagura-Burdzy: "Spread and survival of promiscuous IncP-1 plasmids", Acta Biochimica Polonica, Vol 50, no. 2/2003, p. 425-453
  8. ^ a b 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
  9. ^ Thomas CM et al.: "Broad-Host-Range Plasmids from Agricultural Soils Have IncP-1 Backbones with Diverse Accessory Genes", Appl Environ Microbiol. 2011 Nov;77(22):7975-83. Epub 2011 Sep 23.
  10. ^ J A Kornacki, C H Chang, and D H Figurski: "kil-kor regulon of promiscuous plasmid RK2: structure, products, and regulation of two operons that constitute the kilE locus.", J Bacteriol. 1993 August; 175(16): 5078–5090.
  11. ^ ROSS H. DURLAND, ARESA TOUKDARIAN, FERRIC FANG AND DONALD R. HELINSKI:"Mutations in the trfA Replication Gene of the Broad-Host-Range Plasmid RK2 Result in Elevated Plasmid Copy Numbers", Journal of Bacteriology, Vol. 174, No. 12, June 1992, p. 4110-4119
  12. ^ CHRISTOPHER M. THOMAS, RICHARD MEYER,* AND DONALD R. HELINSKI: "Regions of Broad-Host-Range Plasmid RK2 Which Are Essential for Replication and Maintenance", Journal of Bacteriology, Vol. 172, No. 7 JUlY 1990, p. 3859-3867
  13. ^ Richard J. Meyer and Donald R Helinski: "Unidirectional replication of the P-group plasmid RK2", Biochimica et Biophysica Acta, 478 (1977) pp. 109-113
  14. ^ Joe Pogliano, Thanh Quoc Ho, Zhenping Zhong, and Donald R. Helinski: "Multicopy plasmids are clustered and localized in Escherichia coli", PNAS April 2001, vol. 98 issue. 8, pp. 4486-4491
  15. ^ Kolatka K, Witosinska M, Pierechod M, Konieczny I.: "Bacterial partitioning proteins affect the subcellular location of broad-host-range plasmid RK2", Plasmid, 2010 Nov;64(3):119-34
  16. ^ U Kües and U Stahl: "Replication of plasmids in gram-negative bacteria", Microbiol Rev. 1989 December; 53(4): 491–516
  17. ^ Ferric C. Fang, R. H. D., Donald R. Helinski (1993). "Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria." 133(1): 1-8
  18. ^ 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

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