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Research article

Molecular Biology and Omics

Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants

Natalia Kovalskaya 1*, Juli Foster-Frey 2, David M. Donovan 2, Gary Bauchan 3 and Rosemarie W. Hammond 1

1Molecular Plant Pathology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA, 2Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA, 3Electron and Confocal Microscopy Unit, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

Received: May 19, 2015; Accepted: September 23, 2015

J. Microbiol. Biotechnol. 2016; 26(1): 160-170

Published January 28, 2016 https://doi.org/10.4014/jmb.1505.05060

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative antimicrobial treatments. In our study, the functionally active gram-negative bacterium bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a combination of transient expression and vacuole targeting strategies, and its antimicrobial activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and pGR107) resulted in death of the apical portion of experimental plants. To protect plants against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933 to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli (BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our experiments demonstrate that the combination of transient expression and protein targeting to the delta vacuoles is a promising approach to produce functionally active proteins that exhibit toxicity when expressed in plant cells.

Keywords

endolysin, transient expression, vacuolar targeting, Potato virus X, potato proteinase inhibitor I, Nicotiana benthamiana

References

  1. Borisjuk NV, Borisjuk LG, Logendra S, Petersen F, Gleba Y, Raskin I. 1999. Production of recombinant proteins in plant root exudates. Nat. Biotechnol. 17: 466-469.
    Pubmed CrossRef
  2. Borysowski J, Weber-Dabrowska B, Gorski A. 2006. Bacteriophage endolysins as a novel class of antibacterial agent. Exp. Biol. Med. 231: 366-377.
  3. Chapman S, Kavanagh T, Baulcombe D. 1992. Potato virus X as a vector for gene expression in plants. Plant J. 2: 549-557.
    Pubmed
  4. Company N, Nadal A, La Paz J-L, Martínez S, Rasche S, Schillberg S, et al. 2014. The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum. Plant Biotechnol. J. 12: 81-92.
    Pubmed CrossRef
  5. Daniell H, Lee SB, Panchal T, Wiebe PO. 2001. Expression of the native cholera B toxin subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311: 1001-1009.
    Pubmed PMC CrossRef
  6. Donini M, Lico C, Baschieri S, Conti S, Magliani W, Polonelli L, Benvenuto E. 2005. Production of an engineered killer peptide in Nicotiana benthamiana by using a Potato virus X expression system. Appl. Environ. Microbiol. 71: 6360-6367.
    Pubmed PMC CrossRef
  7. Donovan DM. 2007. Bacteriophage and peptidoglycan degrading enzymes with antimicrobial applications. Recent Pat. Biotechnol. 1: 1-10.
    CrossRef
  8. Donovan DM, Becker SC, Dong S, Baker JR, Foster-Frey JA, Pritchard DG. 2009. Peptidoglycan hydrolase enzyme fusions are uniquely suited for treating multi-drug resistant pathogens. Biotech. Int. 21: 6-10.
  9. Drake PM, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P, Ma JK. 2003. Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant Mol. Biol. 52: 233-241.
    Pubmed CrossRef
  10. Düring K, Porsch P, Mahn A, Brinkmann O, Gieffers W. 1999. The non-enzymatic microbicidal activity of lysozymes. FEBS Lett. 449: 93-100.
    CrossRef
  11. Filice GA, Nyman JA, Lexau C. 2010. Excess costs and utilization associated with Staphylococcus aureus infection. Infect. Control Hosp. Epidemiol. 31: 365-367.
    Pubmed CrossRef
  12. Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11: 393-400.
    Pubmed PMC CrossRef
  13. Fischetti VA. 2010. Bacteriophage endolysins: a novel antiinfective to control gram-positive pathogens. Int. J. Med. Microbiol. 300: 357-362.
    Pubmed PMC CrossRef
  14. Haddix AC, Teutsch SM, Corso PS. 2003. Prevention Effectiveness: A Guide to Decision Analysis and Economic Evaluation, pp. 345-357. 2nd Ed. Oxford University Press, New York.
  15. Jackson MA, Nutt KA, Hassall R, Rae AL. 2010. Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane. Funct. Plant Biol. 37: 785-793.
    CrossRef
  16. Jauh G-Y, Fischer AM, Grimes HD, Ryan CA, Rogers JC. 1998. δ-Tonoplast intrinsic protein defines unique plant vacuole functions. Proc. Natl. Acad. Sci. USA 95: 12995-12999.
    Pubmed PMC CrossRef
  17. Jauh G-Y, Phillips TE, Rogers JC. 1999. Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell 11: 1867-1882.
    Pubmed PMC CrossRef
  18. Jørgensen CS, Ryder LR, Steinø A, Højrup P, Hansen J, Beyer NH, et al. 2003. Dimerization and oligomerization of the chaperone calreticulin. Eur. J. Biochem. 270: 4140-4148.
    Pubmed CrossRef
  19. Komarnytsky S, Borisjuk NV, Borisjuk LG, Alam MZ, Raskin I. 2000. Production of recombinant proteins in tobacco guttation fluid. Plant Physiol. 124: 927-933.
    Pubmed PMC CrossRef
  20. Kovalskaya N, Hammond RW. 2009. Expression and functional characterization of the plant antimicrobial snakin1 and defensin recombinant proteins. Protein Expr. Purif. 63: 12-17.
    Pubmed CrossRef
  21. Kovalskaya N, Zhao Y, Hammond RW. 2011. Antibacterial and antifungal activity of a snakin-defensin hybrid protein expressed in tobacco and potato plants. Open Plant Sci. J. 5: 29-42.
  22. Lacomme C, Chapman S. 2008. Use of Potato virus X (PVX)based vectors for gene expression and virus-induced gene silencing (VIGS). Curr. Protoc. Microbiol. 8: 16I.1.1-16I.1.13.
  23. Lico C, Chen Q, Santi L. 2008. Viral vectors for production of recombinant proteins in plants. J. Cell. Physiol. 216: 366-377.
    Pubmed CrossRef
  24. Loeffler JM, Fischetti VA. 2003. Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrob. Agents Chemother. 47: 375-377.
    Pubmed PMC CrossRef
  25. Loeffler JM, Nelson D, Fischetti VA. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294: 2170-2172.
    Pubmed CrossRef
  26. Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK, Christeller JT. 2002. Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgenic Res. 11: 199-214.
    Pubmed CrossRef
  27. Oliveira H, Melo LDR, Santos SB, Nóbrega FL, Ferreira EC, Cerca N, et al. 2013. Molecular aspects and comparative genomics of bacteriophage endolysins. J. Virol. 87: 4558-4570.
    Pubmed PMC CrossRef
  28. Orito Y, Morita M, Hori K, Unno H, Tanji Y. 2004. Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis. Appl. Microbiol. Biotechnol. 65: 105-109.
    Pubmed CrossRef
  29. Pang T, Fleming TC, Pogliano K, Young R. 2013. Visualization of pinholin lesions in vivo. Proc. Natl. Acad. Sci. USA 110:E2054-E2063.
    Pubmed PMC CrossRef
  30. Pang T, Savva CG, Fleming KG, Struck DK, Young R. 2009. Structure of the lethal phage pinhole. Proc. Natl. Acad. Sci. USA 106: 18966-18971.
    Pubmed PMC CrossRef
  31. Park T, Struck DK, Dankenbring CA, Young R. 2007. The pinholin of lambdoid phage 21: control of lysis by membrane depolarization. J. Bacteriol. 189: 9135-9139.
    Pubmed PMC CrossRef
  32. Perna NT, Plunkett GIII, Burland V, Mau B, Glasner JD, Rose DJ, et al. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529-533.
    Pubmed CrossRef
  33. Rashel M, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, et al. 2007. Efficient elimination of multidrugresistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11. J. Infect. Dis. 196: 1237-1247.
    Pubmed CrossRef
  34. Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R. 2001. Production of antimicrobial defensin in Nicotiana benthamiana with a Potato virus X vector. Mol. Plant Microbe Interact. 14: 111-115.
    Pubmed CrossRef
  35. São-José C, Parreira R, Vieira G, Santos MA. 2000. The Nterminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells. J. Bacteriol. 182: 5823-5831.
    Pubmed PMC CrossRef
  36. Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7: 1147-1171.
    Pubmed PMC CrossRef
  37. Shen Y, Mitchell MS, Donovan DM, Nelson DC. 2012. Phage-based enzybiotics, pp. 217-239. In Hyman P, Abedon ST (eds.). Bacteriophages in Health and Disease. CAB International, Wallingford, UK.
    CrossRef
  38. Tan M, Hegde RS, Jiang X. 2004. The P domain of novovirus capsid protein forms dimer and binds to histoblood group antigen receptors. J. Virol. 78: 6233-6242.
    Pubmed PMC CrossRef
  39. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, et al. 2003. Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res. 31: 1174-1179.
    Pubmed PMC CrossRef
  40. Wang I-N, Deaton J, Young R. 2003. Sizing the holin lesion with an endolysin-beta-galactosidase fusion. J. Bacteriol. 185: 779-787.
    Pubmed PMC CrossRef
  41. Wang I-N, Smith DL, Young R. 2000. Holins: the protein clocks of bacteriophage infections. Annu. Rev. Microbiol. 54: 799-825.
    Pubmed CrossRef
  42. Xu M, Struck DK, Deaton J, Wang I-N, Young RY. 2004. A signal-arrest release sequence mediates export and control of the phage P1 endolysin. Proc. Natl. Acad. Sci. USA 101:6415-6420.
    Pubmed PMC CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(1): 160-170

Published online January 28, 2016 https://doi.org/10.4014/jmb.1505.05060

Copyright © The Korean Society for Microbiology and Biotechnology.

Antimicrobial Activity of Bacteriophage Endolysin Produced in Nicotiana benthamiana Plants

Natalia Kovalskaya 1*, Juli Foster-Frey 2, David M. Donovan 2, Gary Bauchan 3 and Rosemarie W. Hammond 1

1Molecular Plant Pathology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA, 2Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA, 3Electron and Confocal Microscopy Unit, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

Received: May 19, 2015; Accepted: September 23, 2015

Abstract

The increasing spread of antibiotic-resistant pathogens has raised the interest in alternative
antimicrobial treatments. In our study, the functionally active gram-negative bacterium
bacteriophage CP933 endolysin was produced in Nicotiana benthamiana plants by a
combination of transient expression and vacuole targeting strategies, and its antimicrobial
activity was investigated. Expression of the cp933 gene in E. coli led to growth inhibition and
lysis of the host cells or production of trace amounts of CP933. Cytoplasmic expression of the
cp933 gene in plants using Potato virus X-based transient expression vectors (pP2C2S and
pGR107) resulted in death of the apical portion of experimental plants. To protect plants
against the toxic effects of the CP933 protein, the cp933 coding region was fused at its Nterminus
to an N-terminal signal peptide from the potato proteinase inhibitor I to direct CP933
to the delta-type vacuoles. Plants producing the CP933 fusion protein did not exhibit the
severe toxic effects seen with the unfused protein and the level of expression was 0.16 mg/g of
plant tissue. Antimicrobial assays revealed that, in contrast to gram-negative bacterium E. coli
(BL21(DE3)), the gram-positive plant pathogenic bacterium Clavibacter michiganensis was more
susceptible to the plant-produced CP933, showing 18% growth inhibition. The results of our
experiments demonstrate that the combination of transient expression and protein targeting to
the delta vacuoles is a promising approach to produce functionally active proteins that exhibit
toxicity when expressed in plant cells.

Keywords: endolysin, transient expression, vacuolar targeting, Potato virus X, potato proteinase inhibitor I, Nicotiana benthamiana

References

  1. Borisjuk NV, Borisjuk LG, Logendra S, Petersen F, Gleba Y, Raskin I. 1999. Production of recombinant proteins in plant root exudates. Nat. Biotechnol. 17: 466-469.
    Pubmed CrossRef
  2. Borysowski J, Weber-Dabrowska B, Gorski A. 2006. Bacteriophage endolysins as a novel class of antibacterial agent. Exp. Biol. Med. 231: 366-377.
  3. Chapman S, Kavanagh T, Baulcombe D. 1992. Potato virus X as a vector for gene expression in plants. Plant J. 2: 549-557.
    Pubmed
  4. Company N, Nadal A, La Paz J-L, Martínez S, Rasche S, Schillberg S, et al. 2014. The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum. Plant Biotechnol. J. 12: 81-92.
    Pubmed CrossRef
  5. Daniell H, Lee SB, Panchal T, Wiebe PO. 2001. Expression of the native cholera B toxin subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311: 1001-1009.
    Pubmed KoreaMed CrossRef
  6. Donini M, Lico C, Baschieri S, Conti S, Magliani W, Polonelli L, Benvenuto E. 2005. Production of an engineered killer peptide in Nicotiana benthamiana by using a Potato virus X expression system. Appl. Environ. Microbiol. 71: 6360-6367.
    Pubmed KoreaMed CrossRef
  7. Donovan DM. 2007. Bacteriophage and peptidoglycan degrading enzymes with antimicrobial applications. Recent Pat. Biotechnol. 1: 1-10.
    CrossRef
  8. Donovan DM, Becker SC, Dong S, Baker JR, Foster-Frey JA, Pritchard DG. 2009. Peptidoglycan hydrolase enzyme fusions are uniquely suited for treating multi-drug resistant pathogens. Biotech. Int. 21: 6-10.
  9. Drake PM, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P, Ma JK. 2003. Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco roots. Plant Mol. Biol. 52: 233-241.
    Pubmed CrossRef
  10. Düring K, Porsch P, Mahn A, Brinkmann O, Gieffers W. 1999. The non-enzymatic microbicidal activity of lysozymes. FEBS Lett. 449: 93-100.
    CrossRef
  11. Filice GA, Nyman JA, Lexau C. 2010. Excess costs and utilization associated with Staphylococcus aureus infection. Infect. Control Hosp. Epidemiol. 31: 365-367.
    Pubmed CrossRef
  12. Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11: 393-400.
    Pubmed KoreaMed CrossRef
  13. Fischetti VA. 2010. Bacteriophage endolysins: a novel antiinfective to control gram-positive pathogens. Int. J. Med. Microbiol. 300: 357-362.
    Pubmed KoreaMed CrossRef
  14. Haddix AC, Teutsch SM, Corso PS. 2003. Prevention Effectiveness: A Guide to Decision Analysis and Economic Evaluation, pp. 345-357. 2nd Ed. Oxford University Press, New York.
  15. Jackson MA, Nutt KA, Hassall R, Rae AL. 2010. Comparative efficiency of subcellular targeting signals for expression of a toxic protein in sugarcane. Funct. Plant Biol. 37: 785-793.
    CrossRef
  16. Jauh G-Y, Fischer AM, Grimes HD, Ryan CA, Rogers JC. 1998. δ-Tonoplast intrinsic protein defines unique plant vacuole functions. Proc. Natl. Acad. Sci. USA 95: 12995-12999.
    Pubmed KoreaMed CrossRef
  17. Jauh G-Y, Phillips TE, Rogers JC. 1999. Tonoplast intrinsic protein isoforms as markers for vacuolar functions. Plant Cell 11: 1867-1882.
    Pubmed KoreaMed CrossRef
  18. Jørgensen CS, Ryder LR, Steinø A, Højrup P, Hansen J, Beyer NH, et al. 2003. Dimerization and oligomerization of the chaperone calreticulin. Eur. J. Biochem. 270: 4140-4148.
    Pubmed CrossRef
  19. Komarnytsky S, Borisjuk NV, Borisjuk LG, Alam MZ, Raskin I. 2000. Production of recombinant proteins in tobacco guttation fluid. Plant Physiol. 124: 927-933.
    Pubmed KoreaMed CrossRef
  20. Kovalskaya N, Hammond RW. 2009. Expression and functional characterization of the plant antimicrobial snakin1 and defensin recombinant proteins. Protein Expr. Purif. 63: 12-17.
    Pubmed CrossRef
  21. Kovalskaya N, Zhao Y, Hammond RW. 2011. Antibacterial and antifungal activity of a snakin-defensin hybrid protein expressed in tobacco and potato plants. Open Plant Sci. J. 5: 29-42.
  22. Lacomme C, Chapman S. 2008. Use of Potato virus X (PVX)based vectors for gene expression and virus-induced gene silencing (VIGS). Curr. Protoc. Microbiol. 8: 16I.1.1-16I.1.13.
  23. Lico C, Chen Q, Santi L. 2008. Viral vectors for production of recombinant proteins in plants. J. Cell. Physiol. 216: 366-377.
    Pubmed CrossRef
  24. Loeffler JM, Fischetti VA. 2003. Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrob. Agents Chemother. 47: 375-377.
    Pubmed KoreaMed CrossRef
  25. Loeffler JM, Nelson D, Fischetti VA. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294: 2170-2172.
    Pubmed CrossRef
  26. Murray C, Sutherland PW, Phung MM, Lester MT, Marshall RK, Christeller JT. 2002. Expression of biotin-binding proteins, avidin and streptavidin, in plant tissues using plant vacuolar targeting sequences. Transgenic Res. 11: 199-214.
    Pubmed CrossRef
  27. Oliveira H, Melo LDR, Santos SB, Nóbrega FL, Ferreira EC, Cerca N, et al. 2013. Molecular aspects and comparative genomics of bacteriophage endolysins. J. Virol. 87: 4558-4570.
    Pubmed KoreaMed CrossRef
  28. Orito Y, Morita M, Hori K, Unno H, Tanji Y. 2004. Bacillus amyloliquefaciens phage endolysin can enhance permeability of Pseudomonas aeruginosa outer membrane and induce cell lysis. Appl. Microbiol. Biotechnol. 65: 105-109.
    Pubmed CrossRef
  29. Pang T, Fleming TC, Pogliano K, Young R. 2013. Visualization of pinholin lesions in vivo. Proc. Natl. Acad. Sci. USA 110:E2054-E2063.
    Pubmed KoreaMed CrossRef
  30. Pang T, Savva CG, Fleming KG, Struck DK, Young R. 2009. Structure of the lethal phage pinhole. Proc. Natl. Acad. Sci. USA 106: 18966-18971.
    Pubmed KoreaMed CrossRef
  31. Park T, Struck DK, Dankenbring CA, Young R. 2007. The pinholin of lambdoid phage 21: control of lysis by membrane depolarization. J. Bacteriol. 189: 9135-9139.
    Pubmed KoreaMed CrossRef
  32. Perna NT, Plunkett GIII, Burland V, Mau B, Glasner JD, Rose DJ, et al. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529-533.
    Pubmed CrossRef
  33. Rashel M, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, et al. 2007. Efficient elimination of multidrugresistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11. J. Infect. Dis. 196: 1237-1247.
    Pubmed CrossRef
  34. Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R. 2001. Production of antimicrobial defensin in Nicotiana benthamiana with a Potato virus X vector. Mol. Plant Microbe Interact. 14: 111-115.
    Pubmed CrossRef
  35. São-José C, Parreira R, Vieira G, Santos MA. 2000. The Nterminal region of the Oenococcus oeni bacteriophage fOg44 lysin behaves as a bona fide signal peptide in Escherichia coli and as a cis-inhibitory element, preventing lytic activity on oenococcal cells. J. Bacteriol. 182: 5823-5831.
    Pubmed KoreaMed CrossRef
  36. Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7: 1147-1171.
    Pubmed KoreaMed CrossRef
  37. Shen Y, Mitchell MS, Donovan DM, Nelson DC. 2012. Phage-based enzybiotics, pp. 217-239. In Hyman P, Abedon ST (eds.). Bacteriophages in Health and Disease. CAB International, Wallingford, UK.
    CrossRef
  38. Tan M, Hegde RS, Jiang X. 2004. The P domain of novovirus capsid protein forms dimer and binds to histoblood group antigen receptors. J. Virol. 78: 6233-6242.
    Pubmed KoreaMed CrossRef
  39. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, et al. 2003. Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res. 31: 1174-1179.
    Pubmed KoreaMed CrossRef
  40. Wang I-N, Deaton J, Young R. 2003. Sizing the holin lesion with an endolysin-beta-galactosidase fusion. J. Bacteriol. 185: 779-787.
    Pubmed KoreaMed CrossRef
  41. Wang I-N, Smith DL, Young R. 2000. Holins: the protein clocks of bacteriophage infections. Annu. Rev. Microbiol. 54: 799-825.
    Pubmed CrossRef
  42. Xu M, Struck DK, Deaton J, Wang I-N, Young RY. 2004. A signal-arrest release sequence mediates export and control of the phage P1 endolysin. Proc. Natl. Acad. Sci. USA 101:6415-6420.
    Pubmed KoreaMed CrossRef