Abstract
Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Davis, S. C. and Diegel, S. W. (2004), In: Transportation Energy Data Book: Technical Report No. ORNL-6973. Oak Ridge National Laboratory, Oak Ridge, TN.
http://www.ethanolrfa.org/industry/locations/. Accessed March 19, 2007.
Mielenz, J. R. (2001), Curr. Opin. Microbiol. 4, 324–329.
Lynd, L. R., van Zyl, W. H., McBride, J. E., and Laser, M. (2005), Curr. Opin. Biotechnol. 16, 577–583.
Greer, D. (2005), Biocycle 46, 61–65.
Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S. (2002), Microbiol. Mol. Biol. Rev. 66, 506–577.
DeRisi, J., Penland, L., Brown, P. O., et al. (1996), Nat. Genet. 14, 457–460.
Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., and Davis, R. W. (1996), Proc. Natl. Acad. Sci. USA 93, 10,614–10,619.
Sonderegger, M., Jeppsson, M., Hahn-Hagerdal, B., and Sauer, U. (2004), Appl. Environ. Microbiol. 70, 2307–2317.
Tomas, C. A., Welker, N. E., and Papoutsakis, E. T. (2003), Appl. Environ. Microbiol. 69, 4951–4965.
Sedlak, M., Edenberg, H. J., and Ho, N. W. Y. (2003), Enzyme Microbial Technol. 33, 19–28.
Jewett, M. C., Oliveira, A. P., Patil, K. R., and Nielsen, J. (2005), Biotech. Bioprocess Eng. 10, 385–399.
Wahlbom, C. F., Otero, R. R. C., van Zyl, W. H., Hahn-Hägerdal, B., and Johnsson, L. J. (2003), Appl. Environ. Microbiol. 69, 740–746.
Salusjärvi, L., Pitkänen, J.-P., Aristidou, A., Ruohonen, L., and Penttilä, M. (2006), Appl. Biochem. Biotechnol. 128, 237–274.
Zhang, Y.-H. P. and Lynd, L. R. (2005), J. Bacteriol. 187, 99–106.
Li, X., He, Z., and Zhou, J. (2005), Nucleic Acids Res. 33, 6114–6123.
Bozdech, Z., Zhu, J., Joachimiak, M. P., Cohen, F. E., Pulliam, B., and DeRisi, J. L. (2003), Genome Biol. 4, R9.
Tomas, C. A., Alsaker, K. V., Bonarius, H. P. J., et al. (2003), J. Bacteriol. 185, 4539–4547.
Pollack, J. R., Perou, C. M., Alizadeh, A. A., et al. (1999), Nat. Genet. 23, 41–46.
Chhabra, S. R., He, Q., Huang, K. H., et al. (2006), J. Bacteriol. 188, 1817–1828.
Tiquia, S. M., Chong, S. C., Fields, M. W., and Zhou, J. (2004), In: Molecular Microbial Ecology Manual, Kowalchuk, G. A., de Bruijn, F. J., Head, I. M., Lakkennans, A. D., and van Elsas, J. D. (eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 1743–1763.
http://genome.ornl.gov/microbial/cthe/.
Gao, H. C., Wang, Y., Liu, X. D., et al. (2004), J. Bacteriol. 186, 7796–7803.
Verdnik, D., Handran, S., and Pickett, S. (2002), In: DNA image analysis: Nuts & bolts, Kamberova, G. (ed.), DNA Press, Salem, MA, pp. 89–98.
Tiquia, S. M., Wu, L., C. Chong, S. C., et al. (2004), BioTechniques 36, 664–675.
He, Z. L., Wu, L. Y., Li, X. Y., Fields, M. W., and Zhou, J. Z. (2005), Appl. Environ. Microbiol. 71, 3753–3760.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Brown, S.D., Raman, B., McKeown, C.K. et al. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray. Appl Biochem Biotechnol 137, 663–674 (2007). https://doi.org/10.1007/s12010-007-9087-6
Issue Date:
DOI: https://doi.org/10.1007/s12010-007-9087-6