Abstract
A novel plasmid-based expression strategy, exploiting two features of lytic bacteriophages, was developed in Lactococcus lactis. Components of this system include a phage origin of replication and phage expression signals, which were induced to high efficiency upon phage infection of the host. Phage-specific expression signals were cloned from φ31 in a promoter-screening strategy using the lacZ gene from Streptococcus thermophilus. One clone exhibited a significant induction in β-galactosidase production and concomitant increase in lacZ mRNA during the φ31 infection cycle of the host. Molecular characterization of the cloned insert revealed 888 bp positioned near the φ31 cos site. Primer extension analysis showed that transcription was induced ∼20 min following φ31 infection at four points, apparently organized in two sets of tandem promoters on the cloned phage insert. One of these middle phage promoters also showed a basal level of activity prior to phage infection. The φ31 promoter lacZ cassette was cloned into a low-copy-number vector plasmid containing the φ31 origin of replication (ori31) and the resulting low-copy-number plasmid exhibited negligible β-galactosidase production in L. lactis. However, >2,000 units were detected following a deliberate infection with φ31. A control expression plasmid without ori31 could only be induced to 85 units. The combination of these phage-inducible expression signals together with ori31 functioned synergistically to drive rapid and high efficiency expression of a heterologous gene in L. lactis.
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References
de Vos, W. M. and Simons, G. 1988. Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-β-galactosidase and β-galactosidase genes and their expression products. Biochimie 70: 461–473.
Roy, D. G., Klaenhammer, T. R. and Hassan, H. M. 1993. Cloning and expression of the manganese superoxide dismutase gene of Escherchia coli in Lactococcus lactis and Lactobacillus gasseri. Mol. Gen. Genet. 239: 33–40.
van de Guchte, M., van der Vossen, J. M. B. M., Kok, J. and Venema, G. 1989. Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl. Environ. Microbiol. 55: 224–228
Wells, J. M., Wilson, P. W., Norton, P. M., Gasson, M. J. and Le Page, R. W. 1993. Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol. Microbiol. 8: 1155–1162.
de Vos, W. M., Vos, P., Simons, G. and David, S. 1989. Gene organization and expression in mesophilic lactic acid bacteria. J. Dairy Sci. 72: 3398–3405.
Achen, M. G., Davidson, B. E. and Hillier, A. J. 1986. Construction of plasmid vectors for the detection of streptococcal promoters. Gene 45: 45–49.
Bojovic, B., Djordjevic, G. and Topisirovic, T. 1991. Improved vector for promoter screening in lactococci. Appl. Environ. Microbiol. 57: 385–388.
Van der Vossen, J. M. B. M., van der Lelie, D. and Venema, G. 1987. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl. Environ. Microbiol. 53: 2452–2457.
Nilsson, D. and Johansen, E. 1994. A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis. Biochim. Biophys. Acta 1219: 141–144.
Studier, F. W. and Moffatt, B. A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113–130.
Klaenhammer, T. R. and Fitzgerald, G. F. 1994. Bacteriophage and bacteriophage resistance, p. 106–168. In: Genetics and Biotechnology of Lactic Acid Bacteria. Gasson, M. J. and de Vos, W. M. (Eds.). Blackie Academic and Professional, Glascow, United Kingdom.
Hill, C., Miller, L. and Klaenhammer, T. R. 1990. Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis. J. Bacteriol. 172: 6419–6426.
O'Sullivan, D. J., Hill, C. and Klaenhammer . 1993. Effect of increasing the copy number of bacteriophage origins of replication, in trans, on incoming phage proliferation. Appl. Environ. Microbiol. 59: 2449–2456.
Schroeder, C. J., Robert, C., Lenzen, G., McKay, L. L. and Mercenier, A. 1991. Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactocbacillus bulgaricus β-galactosidase sequences. J. Gen. Microbiol. 137: 369–380.
O'Sullivan, D. J. and Klaenhammer, T. R. 1993. High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 137: 227–231.
Klipper-Balz, R., Fischer, G. and Schleifer, K. H. 1982. Nucleic acid hybridization of group D streptococci. Curr. Microbiol. 7: 245–250.
Powell, I. B. and Davidson, B. E. 1985. Characterization of streptococcal bacteriophage c6a. J. Gen. Virology 66: 2737–2741.
Ludwig, W. E., Seewaldt, E., Klipper-balz, R., Schleifer, K. H., Magrum, L., Woese, C. R., Fox, G. E. and Stackebrandt, E. 1985. The phylogenetic position of Streptococcus and Enterococcus. J. Gen Microbiol. 131: 543–551.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.
Birkeland, N. -K. and Lonneborg, A. -M. 1993. The cos region of lactococcal bacteriophage φLC3. DNA Sequence-J. 4: 211–214.
Caulcott, C. A., Lilley, G., Wright, E. M., Robinson, M. K. and Yarranton, G. T. 1985. Investigation of the instability of plasmids directing the expression of met-prochymosin in Escherichia coli. J. Gen. Microbiol. 131: 3355–3365.
Wright, E. M., Humphreys, G. O. and Yarranton, G. T. 1986. Dual-origin plasmids containing an ampliflable ColEl on; temperature-controlled expression of cloned genes. Gene. 49: 311–321.
Yarranton, G. T., Wright, E., Robinson, M. K. and Humphreys, G. O. 1984. Dual-origin plasmid vectors whose origin of replication is controlled by the coliphage lambda promoter PL . Gene. 28: 293–300.
Chandry, P. S., Davidson, B. E. and Hillier, A. J. 1994. Temporal transcription map of the Lactococcus lactis bacteriophage ski. Microbiol. 140: 2251–2261.
Adachi, T., Yamagata, Y., Tsukagoshi, N. and Udaka, S. 1989. Multiple and tandemly arranged promoters of the cell wall protein gene operon in Bacillus brevis 47. J. Bacteriol. 171: 1010–1016.
Fischer, M., Fytlovitch, S., Amit, B., Wortzel, A. and Beck, Y. 1990. A constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli. Appl. Microbiol. Biotechnol. 33: 424–428.
Vidgren, G., Palva, I., Pakkanen, R., Lounatmaa, K. and Palva, A. 1992. S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence. J. Bacteriol. 174: 7419–7427.
Calander, R. 1988. The Bacteriophages I. Plenum Press, New York.
McClure, W. R. 1985. Mechanism and control of transcription in prokaryotes. Ann. Rev. Biochem. 54: 171–204.
Alatossava, T. and Klaenhammer, T. R. 1991. Molecular characterization of three small isometric-headed bacteriophages which vary in their sensitivity to the lactococcal phage resistance plasmid pTR2030. Appl. Environ. Microbiol. 57: 1346–1353.
Moineau, S., Fortier, J., Ackermann, H. W. and Pandian, S. 1992. Characterization of lactococcal bacteriophages in Quebec cheese plants. Can. J. Microbiol. 38: 875–882.
Dao, M. L. and Ferretti, J. J. 1985. Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptoccal genes. Appl. Environ. Microbiol. 49: 115–119.
Hill, C., Pierce, K. and Klaenhammer, T. R. 1989. The conjugative plasmid pTR2030 encodes two bacteriophage defense mechanisms in lactococci, restriction modification and abortive infection. Appl. Environ. Microbiol. 55: 2416–2419.
Casadaban, N. and Cohen, S. N. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherchia coli. J. Mol. Biol. 138: 179–210.
Bullock, W. O., Fernandez, J. M. and Short, J. M. 1987. XLl-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5: 376.
Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
O'Sullivan, D.J. and Klaenhammer, T. R. 1993. Rapid mini-prep isolation of high quality plasmid DNA from Lactococcus spp. and Lactobacillus spp. Appl. Environ. Microbiol. 59: 2730–2733.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
O'Sullivan, D. J., Zagula, K. and Klaenhammer, T. R. 1995. In vivo restriction by LlaI is encoded by three genes, arranged in an operon with llaIM, on the conjugative Lactococcus plasmid pTR2030. J. Bacteriol. 177: 134–143.
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O'Sullivan, D., Walker, S., West, S. et al. Development of an Expression Strategy Using a Lytic Phage to Trigger Explosive Plasmid Amplification and Gene Expression†. Nat Biotechnol 14, 82–87 (1996). https://doi.org/10.1038/nbt0196-82
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DOI: https://doi.org/10.1038/nbt0196-82
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