Friday, April 16, 2010

An efficient stress-free strategy to displace stable bacterial plasmids

Lisa Hale, Orestis Lazos, Anthony S. Haines, and Christopher M. Thomas

Plasmid curing is the process of displacing a plasmid from a plasmid-bearing strain. This is useful for studying phenotypic effects of plasmids on their bacterial hosts. Curing is easy to do when plasmids are unstable and easily lost, but more challenging when dealing with stable plasmids, which can be maintained for a long time in the host even in the absence of selection. Traditionally, plasmid curing is achieved by growing the bacterial host under stressors such as high temperature, detergents or mutagens [1]. This process has the disadvantage of inducing mutations in the bacterial host, which is undesirable. The authors propose a method of plasmid curing based on plasmid incompatibility that can avoid chromosomal mutations. Plasmid incompatibility arises when two plasmids having related replication functions find themselves in the same bacterial cell. This results in the displacement of one by the other that has a second, unrelated replicon [2]. However some plasmids have post-seggregational-killing (psk) genes [3], which produce toxins that kill plasmid-free bacteria in the absence of the anti-toxin, which is another hindrance in plasmid curing. To overcome this, the authors included an anti-toxin gene in their displacing plasmid, which can counter the toxin produced in plasmid-free cells and prevent host killing during plasmid curing. They constructed three vectors having similar replication and stability functions to the plasmids that were being displaced. Plasmid pCURE1, which was constructed to cure plasmid pO157 from E. coli O157:H7, had a pO157-related replicon repF1B and an unrelated replicon from pMB1. To prevent psk, anti-toxin genes related to corresponding genes of pO157 were cloned into pCURE1. Plasmid pCURE1 was introduced into the pO157-bearing strain and successfully cured E. coli O157:H7 of plasmid pO157. Thus, while repF1B replicon disrupted replication of pO157, the anti-toxin produced by pCURE1 prevented host lysis. This produced E. coli O157:H7 bearing only pCURE1, which being unstable was lost rapidly in the absence of selection. Similarly two more vectors (pCURE2 and pCURE11) were constructed for displacing an F plasmid and an IncP-1 plasmid. This approach was thus successful in displacing two different kinds of F plasmids as well as those of the IncP-1 family. The authors also ruled out the possibility of chromosomal integration of pO157 through PCR using pO157-specific primers.
In summary, although plasmid curing through incompatibility has been used before [4], this paper presents a new method, which overcomes psk during plasmid curing. The only drawback is that the authors do not mention how frequent psk is and if the inefficiency of the previously used method was shown to be linked to a plasmid-encoded psk system. To show a direct link between plasmid curing and psk, they could have compared the displacing capacity of a vector with a psk system and another without a psk system. Also, while the authors suggest their method to be more efficient than the previously used method, they do not quantitatively compare the two.

1. Stanisich, V.A. 1984. Identification and analysis of plasmids at the genetic level, pp. 5-32. In P.M. Bennett and J. Grinsted (Eds.), Plasmid Technology. Academic Press, London.
2. Novick, R.P. 1987. Plasmid Incompatibility. Microbiol. Rev. 51:381-395.
3. Gerdes, K., S. Ayora, I. Canosa, P. Ceglowski, R. Diaz-Orejas, T. Franch,
A.P. Gultyaev, R. Bugge Jensen, et al. 2000. Plasmid maintenance systems, pp. 49-85. In
C.M. Thomas (Ed.), The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread.
Harwoord Academic Press, Amsterdam.
4. Tatsuno, I., M. Horie, H. Abe, T. Miki, K. Makino, H. Shinagawa, H. Taguchi,
S. Kamiya, et al. 2001. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157: H7 is required for full epithelial cell adherence phenotype. Infect. Immun. 69:6660-6669

Diya Sen
Graduate Student
University of Idaho


Anonymous said...


Guy said...
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Guy said...

The reference would be useful:
BioTechniques, Vol. 48, No. 3, March 2010, pp. 223–228

(sorry -- 1st comment had malformed html)

Virtual Genome Project (VGP) said...

Thanks for pointing that out. Hope the link works now....