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. 1991 Apr;57(4):1109–1113. doi: 10.1128/aem.57.4.1109-1113.1991

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Sung Guk Kim 1, Carl A Batt 1,*
PMCID: PMC182853  PMID: 16348458

Abstract

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.

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Selected References

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  1. Andersen J., Forst S. A., Zhao K., Inouye M., Delihas N. The function of micF RNA. micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli. J Biol Chem. 1989 Oct 25;264(30):17961–17970. [PubMed] [Google Scholar]
  2. Anderson D. G., McKay L. L. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. doi: 10.1128/aem.46.3.549-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coleman J., Hirashima A., Inokuchi Y., Green P. J., Inouye M. A novel immune system against bacteriophage infection using complementary RNA (micRNA). Nature. 1985 Jun 13;315(6020):601–603. doi: 10.1038/315601a0. [DOI] [PubMed] [Google Scholar]
  4. Ecker J. R., Davis R. W. Inhibition of gene expression in plant cells by expression of antisense RNA. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5372–5376. doi: 10.1073/pnas.83.15.5372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  6. Hirashima A., Sawaki S., Inokuchi Y., Inouye M. Engineering of the mRNA-interfering complementary RNA immune system against viral infection. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7726–7730. doi: 10.1073/pnas.83.20.7726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  8. Kim S. G., Batt C. A. Identification of a nucleotide sequence conserved in Lactococcus lactis bacteriophages. Gene. 1991 Feb 1;98(1):95–100. doi: 10.1016/0378-1119(91)90109-o. [DOI] [PubMed] [Google Scholar]
  9. Kok J., van der Vossen J. M., Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol. 1984 Oct;48(4):726–731. doi: 10.1128/aem.48.4.726-731.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McKay L. L. Functional properties of plasmids in lactic streptococci. Antonie Van Leeuwenhoek. 1983 Sep;49(3):259–274. doi: 10.1007/BF00399502. [DOI] [PubMed] [Google Scholar]
  11. Mizuno T., Chou M. Y., Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A. 1984 Apr;81(7):1966–1970. doi: 10.1073/pnas.81.7.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pestka S., Daugherty B. L., Jung V., Hotta K., Pestka R. K. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7525–7528. doi: 10.1073/pnas.81.23.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stephenson M. L., Zamecnik P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci U S A. 1978 Jan;75(1):285–288. doi: 10.1073/pnas.75.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Takayama K. M., Houba-Herin N., Inouye M. Overproduction of an antisense RNA containing the oop RNA sequence of bacteriophage lambda induces clear plaque formation. Mol Gen Genet. 1987 Nov;210(1):184–186. doi: 10.1007/BF00337778. [DOI] [PubMed] [Google Scholar]
  15. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  17. Yamamoto K. R., Alberts B. M., Benzinger R., Lawhorne L., Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. doi: 10.1016/0042-6822(70)90218-7. [DOI] [PubMed] [Google Scholar]
  18. van der Vossen J. M., van der Lelie D., Venema G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol. 1987 Oct;53(10):2452–2457. doi: 10.1128/aem.53.10.2452-2457.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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