Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The genome and transcriptomes of the anti-tumor agent Clostridium novyi-NT

Nature Biotechnology volume 24, pages 1573–1580 (2006)Cite this article

Abstract

Bacteriolytic anti-cancer therapies employ attenuated bacterial strains that selectively proliferate within tumors. Clostridium novyi-NT spores represent one of the most promising of these agents, as they generate potent anti-tumor effects in experimental animals. We have determined the 2.55-Mb genomic sequence of C. novyi-NT, identifying a new type of transposition and 139 genes that do not have homologs in other bacteria. The genomic sequence was used to facilitate the detection of transcripts expressed at various stages of the life cycle of this bacterium in vitro as well as in infections of tumors in vivo. Through this analysis, we found that C. novyi-NT spores contained mRNA and that the spore transcripts were distinct from those in vegetative forms of the bacterium.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Circular representation of C. novyi-NT genome and transcriptomes.
Figure 2: Transcriptomes of C. novyi-NT in various growth states.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

Gene Expression Omnibus

References

  1. Ryan, R.M., Green, J. & Lewis, C.E. Use of bacteria in anti-cancer therapies. Bioessays 28, 84–94 (2006).

    CAS  PubMed  Google Scholar 

  2. Minton, N.P. Clostridia in cancer therapy. Nat. Rev. Microbiol. 1, 237–242 (2003).

    CAS  PubMed  Google Scholar 

  3. Pawelek, J.M., Low, K.B. & Bermudes, D. Bacteria as tumour-targeting vectors. Lancet Oncol. 4, 548–556 (2003).

    PubMed  Google Scholar 

  4. Barbe, S., Van Mellaert, L. & Anne, J. The use of clostridial spores for cancer treatment. J. Appl. Microbiol. 101, 571–578 (2006).

    CAS  PubMed  Google Scholar 

  5. Theys, J. et al. Tumor-specific gene delivery using genetically engineered bacteria. Curr. Gene Ther. 3, 207–221 (2003).

    CAS  PubMed  Google Scholar 

  6. Jain, R.K. & Forbes, N.S. Can engineered bacteria help control cancer? Proc. Natl. Acad. Sci. USA 98, 14748–14750 (2001).

    CAS  PubMed  Google Scholar 

  7. Cerar, A., Zidar, N. & Vodopivec, B. Colorectal carcinoma in endoscopic biopsies; additional histologic criteria for the diagnosis. Pathol. Res. Pract. 200, 657–662 (2004).

    PubMed  Google Scholar 

  8. Dang, L.H., Bettegowda, C., Huso, D.L., Kinzler, K.W. & Vogelstein, B. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc. Natl. Acad. Sci. USA 98, 15155–15160 (2001).

    CAS  PubMed  Google Scholar 

  9. Agrawal, N. et al. Bacteriolytic therapy can generate a potent immune response against experimental tumors. Proc. Natl. Acad. Sci. USA 101, 15172–15177 (2004).

    CAS  PubMed  Google Scholar 

  10. Bettegowda, C. et al. Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria. Proc. Natl. Acad. Sci. USA 100, 15083–15088 (2003).

    CAS  PubMed  Google Scholar 

  11. Diaz, L.A., Jr et al. Pharmacologic and toxicologic evaluation of C. novyi-NT spores. Toxicol. Sci. 88, 562–575 (2005).

    CAS  PubMed  Google Scholar 

  12. Nolling, J. et al. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183, 4823–4838 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Sebaihia, M. et al. The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet. 38, 779–786 (2006).

    PubMed  Google Scholar 

  14. Shimizu, T. et al. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc. Natl. Acad. Sci. USA 99, 996–1001 (2002).

    CAS  PubMed  Google Scholar 

  15. Bruggemann, H. et al. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc. Natl. Acad. Sci. USA 100, 1316–1321 (2003).

    CAS  PubMed  Google Scholar 

  16. Moriya, S., Imai, Y., Hassan, A.K. & Ogasawara, N. Regulation of initiation of Bacillus subtilis chromosome replication. Plasmid 41, 17–29 (1999).

    CAS  PubMed  Google Scholar 

  17. Segall, A.M. & Craig, N.L. New wrinkles and folds in site-specific recombination. Mol. Cell 19, 433–435 (2005).

    CAS  PubMed  Google Scholar 

  18. Songer, J.G. Bacterial phospholipases and their role in virulence. Trends Microbiol. 5, 156–161 (1997).

    CAS  PubMed  Google Scholar 

  19. Titball, R.W., Naylor, C.E. & Basak, A.K. The Clostridium perfringens α-toxin. Anaerobe 5, 51–64 (1999).

    CAS  PubMed  Google Scholar 

  20. Chambon, P., Deutscher, M.P. & Kornberg, A. Biochemical studies of bacterial sporulation and germination. X. Ribosomes and nucleic acids of vegetative cells and spores of Bacillus megaterium. J. Biol. Chem. 243, 5110–5116 (1968).

    CAS  PubMed  Google Scholar 

  21. Jeng, Y.H. & Doi, R.H. Messenger ribonucleic acid of dormant spores of Bacillus subtilis. J. Bacteriol. 119, 514–521 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu, H. et al. Formation and composition of the Bacillus anthracis endospore. J. Bacteriol. 186, 164–178 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Setlow, P. Spore germination. Curr. Opin. Microbiol. 6, 550–556 (2003).

    CAS  PubMed  Google Scholar 

  24. Fawcett, P., Eichenberger, P., Losick, R. & Youngman, P. The transcriptional profile of early to middle sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 97, 8063–8068 (2000).

    CAS  PubMed  Google Scholar 

  25. Alsaker, K.V. & Papoutsakis, E.T. Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum. J. Bacteriol. 187, 7103–7118 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang, S.T. et al. The forespore line of gene expression in Bacillus subtilis. J. Mol. Biol. 358, 16–37 (2006).

    CAS  PubMed  Google Scholar 

  27. Evguenieva-Hackenberg, E. Bacterial ribosomal RNA in pieces. Mol. Microbiol. 57, 318–325 (2005).

    CAS  PubMed  Google Scholar 

  28. Arthur, J.R. The glutathione peroxidases. Cell. Mol. Life Sci. 57, 1825–1835 (2000).

    CAS  PubMed  Google Scholar 

  29. Moore, T.D. & Sparling, P.F. Interruption of the gpxA gene increases the sensitivity of Neisseria meningitidis to paraquat. J. Bacteriol. 178, 4301–4305 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Weeks, G., Shapiro, M., Burns, R.O. & Wakil, S.J. Control of fatty acid metabolism. I. Induction of the enzymes of fatty acid oxidation in Escherichia coli. J. Bacteriol. 97, 827–836 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kunau, W.H., Dommes, V. & Schulz, H. beta-oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog. Lipid Res. 34, 267–342 (1995).

    CAS  PubMed  Google Scholar 

  32. Ward, N. & Fraser, C.M. How genomics has affected the concept of microbiology. Curr. Opin. Microbiol. 8, 564–571 (2005).

    CAS  PubMed  Google Scholar 

  33. Coenye, T., Gevers, D., Van de Peer, Y., Vandamme, P. & Swings, J. Towards a prokaryotic genomic taxonomy. FEMS Microbiol. Rev. 29, 147–167 (2005).

    CAS  PubMed  Google Scholar 

  34. Subramanian, G., Mural, R., Hoffman, S.L., Venter, J.C. & Broder, S. Microbial disease in humans: a genomic perspective. Mol. Diagn. 6, 243–252 (2001).

    CAS  PubMed  Google Scholar 

  35. Dang, L.H. et al. Targeting vascular and avascular compartments of tumors with C. novyi-NT and anti-microtubule agents. Cancer Biol. Ther. 3, 326–337 (2004).

    CAS  PubMed  Google Scholar 

  36. Jaeger, K.E., Dijkstra, B.W. & Reetz, M.T. Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu. Rev. Microbiol. 53, 315–351 (1999).

    CAS  PubMed  Google Scholar 

  37. Forsdahl, K. & Larsen, T.S. Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus. J. Mol. Cell. Cardiol. 27, 893–900 (1995).

    CAS  PubMed  Google Scholar 

  38. Craig, N.L. Target site selection in transposition. Annu. Rev. Biochem. 66, 437–474 (1997).

    CAS  PubMed  Google Scholar 

  39. Cano, R.J. & Borucki, M.K. Revival and identification of bacterial spores in 25- to 40-million-year-old Dominican amber. Science 268, 1060–1064 (1995).

    CAS  PubMed  Google Scholar 

  40. Vreeland, R.H., Rosenzweig, W.D. & Powers, D.W. Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal. Nature 407, 897–900 (2000).

    CAS  PubMed  Google Scholar 

  41. Ewing, B., Hillier, L., Wendl, M.C. & Green, P. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185 (1998).

    CAS  PubMed  Google Scholar 

  42. Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8, 186–194 (1998).

    CAS  PubMed  Google Scholar 

  43. Gordon, D., Abajian, C. & Green, P. Consed: a graphical tool for sequence finishing. Genome Res. 8, 195–202 (1998).

    CAS  PubMed  Google Scholar 

  44. Carraro, D.M. et al. PCR-assisted contig extension: stepwise strategy for bacterial genome closure. Biotechniques 34, 626–628, 630–622 (2003).

    CAS  PubMed  Google Scholar 

  45. Seshadri, R. et al. Genome sequence of the PCE-dechlorinating bacterium Dehalococcoides ethenogenes. Science 307, 105–108 (2005).

    CAS  PubMed  Google Scholar 

  46. El-Deiry, W.S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993).

    CAS  PubMed  Google Scholar 

  47. Nuwaysir, E.F. et al. Gene expression analysis using oligonucleotide arrays produced by maskless photolithography. Genome Res. 12, 1749–1755 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Parmigiani, G., Garrett, E.S., Irizarry, R. & Zeger, S.L. The analysis of gene expression data: methods and software. (Springer, New York, 2003).

    Google Scholar 

  49. Scharpf, R., Iacobuzio-Donahue, C.A., Sneddon, J.B. & Parmigiani, G. When should one subtract background fluorescence in cDNA microarrays? Biostatistics (in the press).

  50. Smyth, G.K. . Limma in Bioinformatics and Computational Biology Solutions Using R and Bioconductor (eds. Gentleman, V., Carey, S., Dudoit, R. & Irizarry, W.H.) 397–420 (Springer, New York, 2005).

    Google Scholar 

  51. Cheong, I. et al. A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science, in press (2006).

    Google Scholar 

Download references

Acknowledgements

The authors thank Peter Setlow for his insightful suggestions about the nature of spore mRNA, Rachel Green for her comments on rRNA fragmentation, Anca Segall for her comments on transposition, and Tanja Davidsen, Michelle Gwinn Giglio and Nikhat Zafar of TIGR for their expert assistance with the genomic bioinformatic analysis. The authors also thank Brent Ewing, Phil Green and David Gordon for kindly providing the Phred/Phrap and Consed software package. The B. subtilis strains were generously provided by Peter Mullany. This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research, the Commonwealth Foundation, the Miracle Foundation, National Science Foundation grant DMS034211 and National Institutes of Health grant CA062924.

Author information

Author notes

  1. Chetan Bettegowda

    Present address: Department of Neurology and Neurosurgery, Johns Hopkins Medical Institutions, 600 N. Wolfe Street, Baltimore, Maryland, 21287, USA

  2. Chetan Bettegowda, Xin Huang and Jimmy Lin: These authors contributed equally to this work.

Authors and Affiliations

  1. The Howard Hughes Medical Institute, The Ludwig Center for Cancer Genetics & Therapeutics at The Sidney Kimmel Comprehensive Cancer Center, and Department of Pharmacology and Molecular Sciences, Johns Hopkins Medical Institutions, 1650 Orleans Street, Baltimore, 21231, Maryland, USA

    Chetan Bettegowda, Xin Huang, Jimmy Lin, Ian Cheong, Manu Kohli, Stephen A Szabo, Xiaosong Zhang, Luis A Diaz Jr, Victor E Velculescu, Kenneth W Kinzler, Bert Vogelstein & Shibin Zhou

  2. Departments of Oncology, Biostatistics and Pathology, Johns Hopkins Medical Institutions, 550 North Broadway, Baltimore, 21205, Maryland, USA

    Giovanni Parmigiani

Authors
  1. Chetan Bettegowda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jimmy Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ian Cheong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Manu Kohli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stephen A Szabo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaosong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Luis A Diaz Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Victor E Velculescu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Giovanni Parmigiani
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Kenneth W Kinzler
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Bert Vogelstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Shibin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shibin Zhou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

rRNA species. (PDF 110 kb)

Supplementary Fig. 2

RT-PCR confirmation of selected microarray data. (PDF 143 kb)

Supplementary Fig. 3

Transcript abundance in denuded spores. (PDF 134 kb)

Supplementary Table 1

Integrated database of C. novyi-NT genome and transcriptomes. (XLS 1009 kb)

Supplementary Table 2

Coding sequences related to mobile elements. (XLS 38 kb)

Supplementary Table 3

Hypothetical CDS unique to C. novyi-NT. (PDF 54 kb)

Supplementary Table 4

Functional classification of C. novyi-NT genes. (PDF 75 kb)

Supplementary Table 5

Other bacteria harboring homologs of the C. novyi-NT transposase. (PDF 82 kb)

Supplementary Table 6

Functional classification of C. novyi-NT genes expressed in specific growth phases. (PDF 68 kb)

Supplementary Table 7

Genes expressed preferentially in different growth phases. (XLS 104 kb)

Supplementary Table 8

Fifthy genes with transcripts most abundant in spores. (XLS 328 kb)

Supplementary Table 9

Genes transcribed during the final stages of sporulation. (XLS 214 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bettegowda, C., Huang, X., Lin, J. et al. The genome and transcriptomes of the anti-tumor agent Clostridium novyi-NT. Nat Biotechnol 24, 1573–1580 (2006). https://doi.org/10.1038/nbt1256

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1256

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing