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A New Method for Gene Deletion to Investigate Cell Wall Biogenesis in Fusobacterium nucleatum

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The Bacterial Cell Wall

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2727))

Abstract

Controlled septal peptidoglycan hydrolysis is vital for bacterial cell division, preserving cellular integrity and facilitating proper daughter cell separation. In Escherichia coli, the FtsEX ABC system governs cell wall hydrolase activation by regulating the EnvC activator. However, the processes underlying cell division in the Gram-negative oral bacterium Fusobacterium nucleatum are poorly understood, mainly due to its well-known genetic intractability. Herein, we provide a step-by-step procedure for a new gene deletion method in F. nucleatum, focusing on the ftsX gene as a target. This novel approach exploits the HicAB toxin-antitoxin system, using HicA as a counter-selective marker to enable efficient and precise gene deletion. By implementing this technique, we successfully demonstrated its applicability in F. nucleatum, providing new insights into this important microorganism’s cell division process. Furthermore, this advanced gene deletion technique offers a valuable resource for future investigation into the functional characterization of genes involved in cell wall biogenesis in F. nucleatum and other genetically intractable microorganisms.

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References

  1. Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE (2001) Bacterial diversity in human subgingival plaque. J Bacteriol 183(12):3770–3783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bolstad A, Jensen HB, Bakken V (1996) Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev 9(1):55–71

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Polak D, Shapira L, Weiss EI, Houri-Haddad Y (2012) The role of coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum on the host response to mixed infection. J Clin Periodontol 39(7):617–625

    Article  CAS  PubMed  Google Scholar 

  4. Zhang Y, Shi W, Song Y, Wang J (2019) Metatranscriptomic analysis of an in vitro biofilm model reveals strain-specific interactions among multiple bacterial species. J Oral Microbiol 11(1):1599670. https://doi.org/10.1080/20002297.2019.1599670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vander Haar EL, So J, Gyamfi-Bannerman C, Han YW (2018) Fusobacterium nucleatum and adverse pregnancy outcomes: epidemiological and mechanistic evidence. Anaerobe 50:55–59

    Article  PubMed  PubMed Central  Google Scholar 

  6. Gauthier S, Tétu A, Himaya E, Morand M, Chandad F, Rallu F, Bujold E (2011) The origin of Fusobacterium nucleatum involved in intra-amniotic infection and preterm birth. J Matern Fetal Neonatal Med 24(11):1329–1332

    Article  CAS  PubMed  Google Scholar 

  7. Yu T, Guo F, Yu Y, Sun T, Ma D, Han J, Qian Y, Kryczek I, Sun D, Nagarsheth N (2017) Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 170(3):548–563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Flanagan L, Schmid J, Ebert M, Soucek P, Kunicka T, Liska V, Bruha J, Neary P, Dezeeuw N, Tommasino M (2014) Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome. Eur J Clin Microbiol Infect Dis 33(8):1381–1390

    Article  CAS  PubMed  Google Scholar 

  9. Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW (2013) Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin. Cell Host Microbe 14(2):195–206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Brennan CA, Garrett WS (2019) Fusobacterium nucleatum—symbiont, opportunist and oncobacterium. Nat Rev Microbiol 17(3):156–166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hummels KR, Berry SP, Li Z, Taguchi A, Min JK, Walker S, Marks DS, Bernhardt TG (2023) Coordination of bacterial cell wall and outer membrane biosynthesis. Nature 615(7951):300–304. https://doi.org/10.1038/s41586-023-05750-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang KC, Mukhopadhyay R, Wen B, Gitai Z, Wingreen NS (2008) Cell shape and cell-wall organization in gram-negative bacteria. Proc Natl Acad Sci U S A 105(49):19282–19287. https://doi.org/10.1073/pnas.0805309105

    Article  PubMed  PubMed Central  Google Scholar 

  13. Huang KC, Mukhopadhyay R, Wen B, Gitai Z, Wingreen NS (2008) Cell shape and cell-wall organization in gram-negative bacteria. Proc Natl Acad Sci 105(49):19282–19287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hummels KR, Berry SP, Li Z, Taguchi A, Min JK, Walker S, Marks DS, Bernhardt TG (2023) Coordination of bacterial cell wall and outer membrane biosynthesis. Nature:1–5

    Google Scholar 

  15. Cook J, Baverstock TC, McAndrew MB, Stansfeld PJ, Roper DI, Crow A (2020) Insights into bacterial cell division from a structure of EnvC bound to the FtsX periplasmic domain. Proc Natl Acad Sci 117(45):28355–28365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wu C, Al Mamun AAM, Luong TT, Hu B, Gu J, Lee JH, D’Amore M, Das A, Ton-That H (2018) Forward genetic dissection of biofilm development by Fusobacterium nucleatum: novel functions of cell division proteins. FtsX and EnvC. mBio 9(2). https://doi.org/10.1128/mBio.00360-18

  17. Vaillancourt K, Bédard N, Bart C, Tessier M, Robitaille G, Turgeon N, Frenette M, Moineau S, Vadeboncoeur C (2008) Role of galK and galM in galactose metabolism by Streptococcus thermophilus. Appl Environ Microbiol 74(4):1264–1267

    Article  CAS  PubMed  Google Scholar 

  18. Neves AR, Pool WA, Solopova A, Kok J, Santos H, Kuipers OP (2010) Towards enhanced galactose utilization by Lactococcus lactis. Appl Environ Microbiol 76(21):7048–7060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bibek GC, Zhou P, Wu C 92023) HicA toxin-based counterselection marker for allelic exchange mutations in Fusobacterium nucleatum. Appl Environ Microbiol: e00091-00023

    Google Scholar 

  20. Peluso EA, Scheible M, Ton-That H, Wu C (2020) Genetic manipulation and virulence assessment of Fusobacterium nucleatum. Curr Protoc Microbiol 57(1):e104. https://doi.org/10.1002/cpmc.104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Canadas IC, Groothuis D, Zygouropoulou M, Rodrigues R, Minton NP (2019) RiboCas: a universal CRISPR-based editing tool for clostridium. ACS Synth Biol 8:1379–1390

    Google Scholar 

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Acknowledgments

This work was supported by a National Institute of Dental and Craniofacial Research (NIDCR) grant (DE030895) to C.W.

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Authors and Affiliations

  1. Department of Microbiology & Molecular Genetics, The University of Texas Health Science Center, Houston, TX, USA

    G. C. Bibek, Peng Zhou & Chenggang Wu

Authors
  1. G. C. Bibek
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  2. Peng Zhou
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  3. Chenggang Wu
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Corresponding author

Correspondence to Chenggang Wu .

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Editors and Affiliations

  1. Division of Oral & Systemic Health Sciences, School of Dentistry, University of California, Los Angeles, Los Angeles, CA, USA

    Hung Ton-That

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Bibek, G.C., Zhou, P., Wu, C. (2024). A New Method for Gene Deletion to Investigate Cell Wall Biogenesis in Fusobacterium nucleatum. In: Ton-That, H. (eds) The Bacterial Cell Wall. Methods in Molecular Biology, vol 2727. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3491-2_6

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  • DOI: https://doi.org/10.1007/978-1-0716-3491-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3490-5

  • Online ISBN: 978-1-0716-3491-2

  • eBook Packages: Springer Protocols

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