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Research article

Environmental Microbiology and Biotechnology (EMB)  |  Synthetic Biology and Metabolic Engineering

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency

Yunpeng Shi, Wenyan Cao, Zhiping Zheng, Sha Xu, Lijuan Chai, Shenghu Zhou*, and Yu Deng

School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, P.R. China

Correspondence to:
Shenghu Zhou,        zhoush@jiangnan.edu.cn
Received: February 23, 2024; Revised: June 21, 2024; Accepted: July 1, 2024

J. Microbiol. Biotechnol. 2024; 34(9): 1826-1835

Published September 28, 2024 https://doi.org/10.4014/jmb.2402.02041

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain’s low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

Keywords

Restriction-modification system, Paracoccus denitrificans, DNA methylase, HNH endonuclease, plasmid artificial modification

Graphical Abstract


References

  1. Robertson LA, Kuenen JG. 1983. Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium. Microbiology 129: 2847-2855.
  2. Luria SE, Human ML. 1952. A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol. 64: 557-569.
    Pubmed PMC
  3. Zhao Y, Lu W, Liu Y, Wang J, Zhou S, Mao Y, et al. 2020. Efficient total nitrogen removal from wastewater by Paracoccus denitrificans DYTN‐1. Lett. Appl. Microbiol. 70: 263-273.
    Pubmed
  4. Steinrücke P, Ludwig B. 1993. Genetics of Paracoccus denitrificans. FEMS Microbiol. Lett. 104: 83-117.
    Pubmed
  5. de Vries GE, Harms N, Hoogendijk J, Stouthamer AH. 1989. Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property. Arch. Microbiol. 152: 52-57.
  6. Vasu K, Rao DN, Nagaraja V. 2019. Restriction-Modification Systems, pp. 102-109. In Schmidt TM (ed.), Encyclopedia of Microbiology (Fourth Edition), Ed. Academic Press, Oxford.
  7. Arber W. 1965. Host-controlled modification of bacteriophage. Annu. Rev. Microbiol. 19: 365-378.
    Pubmed
  8. Danna K, Nathans D. 1971. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. USA 68: 2913-2917.
    Pubmed PMC
  9. Kelly TJ, Smith HO. 1970. A restriction enzyme from Hemophilus influenzae: II. base sequence of the recognition site. J. Mol. Biol. 51: 393-409.
    Pubmed
  10. Williams RJ. 2003. Restriction endonuclease. Mol. Biotechnol. 23: 225-243.
    Pubmed
  11. Bujnicki JM. 2004. Molecular Phylogenetics of Restriction Endonucleases, pp. 63-93. In Pingoud AM (ed.), Restriction Endonucleases, Ed. Springer Berlin Heidelberg, Berlin, Heidelberg.
  12. Lynn SP, Cohen LK, Gardner JF, Kaplan S. 1979. Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides. J. Bacteriol. 138: 505-509.
    Pubmed PMC
  13. Lynn SP, Cohen LK, Kaplan S, Gardner JF. 1980. RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides. J. Bacteriol. 142: 380-383.
    Pubmed PMC
  14. Greene PJ, Ballard BT, Stephenson F, Kohr WJ, Rodriguez H, Rosenberg JM, et al. 1988. Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI. Gene 68: 43-51.
    Pubmed
  15. Aiken C, Gumport RI. 1988. Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties. Nucleic Acids Res. 16: 7901-7916.
    Pubmed PMC
  16. Stephenson FH, Ballard BT, Boyer HW, Rosenberg JM, Greene PJ. 1989. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases. Gene 85: 1-13.
    Pubmed
  17. Stephenson FH, Greene PJ. 1989. Nucleotide sequence of the gene encoding the RsrI methyltransferase. Nucleic Acids Res. 17: 10503.
    Pubmed PMC
  18. O'Connor CD, Metcalf E, Wrighton CJ, Harris TJ, Saunders JR. 1984. RsrII--a novel restriction endonuclease with a heptanucleotide recognition site. Nucleic Acids Res. 12: 6701-6708.
    Pubmed PMC
  19. Johnston CD, Cotton SL, Rittling SR, Starr JR, Borisy GG, Dewhirst FE, et al. 2019. Systematic evasion of the restrictionmodification barrier in bacteria. Proc. Natl. Acad. Sci. USA 116: 11454-11459.
    Pubmed PMC
  20. Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF. 2009. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. J. Mol. Biol. 394: 653-680.
    Pubmed
  21. Oliveira PH, Touchon M, Rocha EPC. 2014. The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res. 42: 10618-10631.
    Pubmed PMC
  22. Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, et al. 2003. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31: 1805-1812.
    Pubmed PMC
  23. Roberts RJ, Vincze T, Posfai J, Macelis D. 2014. REBASE - a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 43: D298-D299.
    Pubmed PMC
  24. Tock MR, Dryden DT. 2005. The biology of restriction and anti-restriction. Curr. Opin. Microbiol. 8: 466-472.
    Pubmed
  25. Yasui K, Kano Y, Tanaka K, Watanabe K, Shimizu-Kadota M, Yoshikawa H, et al. 2009. Improvement of bacterial transformation efficiency using plasmid artificial modification. Nucleic Acids Res. 37: e3.
    Pubmed PMC
  26. Naito T, Kusano K, Kobayashi I. 1995. Selfish behavior of restriction-modification systems. Science 267: 897-899.
    Pubmed
  27. Tumuluri VS, Saikrishnan K. 2022. Heterologous expression and high degree purification of the restriction endonuclease SauUSI. Bio Protoc. 12: e4275.
    Pubmed PMC
  28. Mokrishcheva ML, Kertesz-Farkas A, Nikitin DV. 2018. New bifunctional restriction-modification enzyme AloI isoschizomer (PcoI): Bioinformatics analysis, purification and activity confirmation. Gene 660: 8-12.
    Pubmed
  29. Xu SY, Klein P, Degtyarev S, Roberts RJ. 2016. Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes. Sci. Rep. 6: 28579.
    Pubmed PMC
  30. Arber W, Linn S. 1969. DNA modification and restriction. Annu. Rev. Biochem. 38: 467-500.
    Pubmed
  31. Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP. 1997. Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J. Bacteriol. 179: 1998-2005.
    Pubmed PMC
  32. Olaya-Abril A, Luque-Almagro VM, Manso I, Gates AJ, Moreno-Vivián C, Richardson DJ, et al. 2018. Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222. FEMS Microbiol. Lett. 365: fnx251.
  33. Wilkins BM, Chilley PM, Thomas AT, Pocklington MJ. 1996. Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications. J. Mol. Biol. 258: 447-456.
    Pubmed
  34. OHTANI N, SATO M, TOMITA M, ITAYA M. 2008. Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168. Biosci. Biotechnol. Biochem. 72: 2472-2475.
    Pubmed
  35. Purdy D, O'Keeffe TAT, Elmore M, Herbert M, McLeod A, Bokori-Brown M, et al. 2002. Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol. Microbiol. 46: 439-452.
    Pubmed
  36. Ind AC, Porter SL, Brown MT, Byles ED, Beyer JAd, Godfrey SA, et al. 2009. Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans. Appl. Environ. Microbiol. 75: 6613-6615.
    Pubmed PMC

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Article

Research article

J. Microbiol. Biotechnol. 2024; 34(9): 1826-1835

Published online September 28, 2024 https://doi.org/10.4014/jmb.2402.02041

Copyright © The Korean Society for Microbiology and Biotechnology.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency

Yunpeng Shi, Wenyan Cao, Zhiping Zheng, Sha Xu, Lijuan Chai, Shenghu Zhou*, and Yu Deng

School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, P.R. China

Correspondence to:Shenghu Zhou,        zhoush@jiangnan.edu.cn

Received: February 23, 2024; Revised: June 21, 2024; Accepted: July 1, 2024

Abstract

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain’s low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

Keywords: Restriction-modification system, Paracoccus denitrificans, DNA methylase, HNH endonuclease, plasmid artificial modification

References

  1. Robertson LA, Kuenen JG. 1983. Thiosphaera pantotropha gen. nov. sp. nov., a facultatively anaerobic, facultatively autotrophic sulphur bacterium. Microbiology 129: 2847-2855.
  2. Luria SE, Human ML. 1952. A nonhereditary, host-induced variation of bacterial viruses. J. Bacteriol. 64: 557-569.
    Pubmed KoreaMed
  3. Zhao Y, Lu W, Liu Y, Wang J, Zhou S, Mao Y, et al. 2020. Efficient total nitrogen removal from wastewater by Paracoccus denitrificans DYTN‐1. Lett. Appl. Microbiol. 70: 263-273.
    Pubmed
  4. Steinrücke P, Ludwig B. 1993. Genetics of Paracoccus denitrificans. FEMS Microbiol. Lett. 104: 83-117.
    Pubmed
  5. de Vries GE, Harms N, Hoogendijk J, Stouthamer AH. 1989. Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property. Arch. Microbiol. 152: 52-57.
  6. Vasu K, Rao DN, Nagaraja V. 2019. Restriction-Modification Systems, pp. 102-109. In Schmidt TM (ed.), Encyclopedia of Microbiology (Fourth Edition), Ed. Academic Press, Oxford.
  7. Arber W. 1965. Host-controlled modification of bacteriophage. Annu. Rev. Microbiol. 19: 365-378.
    Pubmed
  8. Danna K, Nathans D. 1971. Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. USA 68: 2913-2917.
    Pubmed KoreaMed
  9. Kelly TJ, Smith HO. 1970. A restriction enzyme from Hemophilus influenzae: II. base sequence of the recognition site. J. Mol. Biol. 51: 393-409.
    Pubmed
  10. Williams RJ. 2003. Restriction endonuclease. Mol. Biotechnol. 23: 225-243.
    Pubmed
  11. Bujnicki JM. 2004. Molecular Phylogenetics of Restriction Endonucleases, pp. 63-93. In Pingoud AM (ed.), Restriction Endonucleases, Ed. Springer Berlin Heidelberg, Berlin, Heidelberg.
  12. Lynn SP, Cohen LK, Gardner JF, Kaplan S. 1979. Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides. J. Bacteriol. 138: 505-509.
    Pubmed KoreaMed
  13. Lynn SP, Cohen LK, Kaplan S, Gardner JF. 1980. RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides. J. Bacteriol. 142: 380-383.
    Pubmed KoreaMed
  14. Greene PJ, Ballard BT, Stephenson F, Kohr WJ, Rodriguez H, Rosenberg JM, et al. 1988. Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI. Gene 68: 43-51.
    Pubmed
  15. Aiken C, Gumport RI. 1988. Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties. Nucleic Acids Res. 16: 7901-7916.
    Pubmed KoreaMed
  16. Stephenson FH, Ballard BT, Boyer HW, Rosenberg JM, Greene PJ. 1989. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases. Gene 85: 1-13.
    Pubmed
  17. Stephenson FH, Greene PJ. 1989. Nucleotide sequence of the gene encoding the RsrI methyltransferase. Nucleic Acids Res. 17: 10503.
    Pubmed KoreaMed
  18. O'Connor CD, Metcalf E, Wrighton CJ, Harris TJ, Saunders JR. 1984. RsrII--a novel restriction endonuclease with a heptanucleotide recognition site. Nucleic Acids Res. 12: 6701-6708.
    Pubmed KoreaMed
  19. Johnston CD, Cotton SL, Rittling SR, Starr JR, Borisy GG, Dewhirst FE, et al. 2019. Systematic evasion of the restrictionmodification barrier in bacteria. Proc. Natl. Acad. Sci. USA 116: 11454-11459.
    Pubmed KoreaMed
  20. Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF. 2009. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes. J. Mol. Biol. 394: 653-680.
    Pubmed
  21. Oliveira PH, Touchon M, Rocha EPC. 2014. The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res. 42: 10618-10631.
    Pubmed KoreaMed
  22. Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, et al. 2003. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31: 1805-1812.
    Pubmed KoreaMed
  23. Roberts RJ, Vincze T, Posfai J, Macelis D. 2014. REBASE - a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 43: D298-D299.
    Pubmed KoreaMed
  24. Tock MR, Dryden DT. 2005. The biology of restriction and anti-restriction. Curr. Opin. Microbiol. 8: 466-472.
    Pubmed
  25. Yasui K, Kano Y, Tanaka K, Watanabe K, Shimizu-Kadota M, Yoshikawa H, et al. 2009. Improvement of bacterial transformation efficiency using plasmid artificial modification. Nucleic Acids Res. 37: e3.
    Pubmed KoreaMed
  26. Naito T, Kusano K, Kobayashi I. 1995. Selfish behavior of restriction-modification systems. Science 267: 897-899.
    Pubmed
  27. Tumuluri VS, Saikrishnan K. 2022. Heterologous expression and high degree purification of the restriction endonuclease SauUSI. Bio Protoc. 12: e4275.
    Pubmed KoreaMed
  28. Mokrishcheva ML, Kertesz-Farkas A, Nikitin DV. 2018. New bifunctional restriction-modification enzyme AloI isoschizomer (PcoI): Bioinformatics analysis, purification and activity confirmation. Gene 660: 8-12.
    Pubmed
  29. Xu SY, Klein P, Degtyarev S, Roberts RJ. 2016. Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes. Sci. Rep. 6: 28579.
    Pubmed KoreaMed
  30. Arber W, Linn S. 1969. DNA modification and restriction. Annu. Rev. Biochem. 38: 467-500.
    Pubmed
  31. Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP. 1997. Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J. Bacteriol. 179: 1998-2005.
    Pubmed KoreaMed
  32. Olaya-Abril A, Luque-Almagro VM, Manso I, Gates AJ, Moreno-Vivián C, Richardson DJ, et al. 2018. Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222. FEMS Microbiol. Lett. 365: fnx251.
  33. Wilkins BM, Chilley PM, Thomas AT, Pocklington MJ. 1996. Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications. J. Mol. Biol. 258: 447-456.
    Pubmed
  34. OHTANI N, SATO M, TOMITA M, ITAYA M. 2008. Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168. Biosci. Biotechnol. Biochem. 72: 2472-2475.
    Pubmed
  35. Purdy D, O'Keeffe TAT, Elmore M, Herbert M, McLeod A, Bokori-Brown M, et al. 2002. Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol. Microbiol. 46: 439-452.
    Pubmed
  36. Ind AC, Porter SL, Brown MT, Byles ED, Beyer JAd, Godfrey SA, et al. 2009. Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans. Appl. Environ. Microbiol. 75: 6613-6615.
    Pubmed KoreaMed