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Design, optimization and validation of genomic DNA microarrays for examining theClostridium acetobutylicum transcriptome

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Abstract

Microarray technology has contributed significantly to the understanding of bacterial genetics and transcriptional regulation. One neglected aspect of this technology has been optimization of microarray-generated signals and quality of generated information. Full genome microarrays were developed forClostridium acetobutylicum through spotting of PCR products that were designed with minimal homology with all other genes within the genome. Using statistical analyses it is demonstrated that signal quality is significantly improved by increasing the hybridization volume, possibly increasing the effective number of transcripts available to bind to a given spot, while changes in labeled probe amounts were found to be less sensitive to improving signal quality. In addition to Q-RT-PCR, array validation was tested by examining the transcriptional program of a mutant (M5) strain lacking the pSOL1 178-gene megaplasmid relative to the wildtype (WT) strain. Under optimal conditions, it is demonstrated that the fraction of false positive genes is 1% when considering differentially expressed genes and 7% when considering all genes with signal above background. To enhance genomic-scale understanding of organismal physiology, using data from these microarrays we estimated that 40–55% of theC. acetobutylicum genome is expressed at any time during batch culture, similar to estimates made forBacillus subtilis.

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References

  1. Ye, R. W., T. Wang, L. Bedzyk, and K. M. Croker (2001) Applications of DNA microarrays in microbial systems.J. Microbiol. Meth. 47: 257–272.

    Article  CAS  Google Scholar 

  2. Conway, T. and G. K. Schoolnik (2003) Microarray expression profiling: capturing a genome-wide portrait of the transcriptome.Mol. Microbiol. 47: 879–889.

    Article  CAS  Google Scholar 

  3. Rhodius, V. A. and R. A. LaRossa (2003) Uses and pitfalls of microarrays for studying transcriptional regulation.Curr. Opin. Microbiol. 6: 114–119.

    Article  CAS  Google Scholar 

  4. Dharmadi, Y. and R. Gonzalez (2004) DNA microarrays: experimental issues, data analysis, and application to bacterial systems.Biotechnol. Progr. 20: 1309–1324.

    Article  CAS  Google Scholar 

  5. Jones, D. T. and D. R. Woods (1986) Acetone-butanol fermentation revisited.Microbiol. Rev. 50: 484–524.

    CAS  Google Scholar 

  6. Tomas, C. A., K. V. Alsaker, H. P. J. Bonarius, W. T. Hendriksen, H. Yang, J. A. Beamish, C. J. Parades, and E. T. Papoutsakis (2003) DNA-array based transcriptional analysis of asporogenous, non-solventogenicClostridium acetobutylicum strains SKO1 and M5.J. Bacteriol. 185: 4539–4547.

    Article  CAS  Google Scholar 

  7. Alsaker, K. V., T. R. Spitzer, and E. T. Papoutsakis (2004) Transcriptional analysis ofspo0A overexpression inClostridium acetobutylicum and its effect on the cell’s response to butanol stress.J. Bacteriol. 186: 1959–1971.

    Article  CAS  Google Scholar 

  8. Tomas, C. A., N. E. Welker, and E. T. Papoutsakis (2003) Overexpression ofgroESL inClostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and large changes in the cell’s transcriptional program.Appl. Environ. Microbiol. 69: 4951–4965.

    Article  CAS  Google Scholar 

  9. Tummala, S. B., S. G. Junne, C. J. Paredes, and E. T. Papoutsakis (2003) Transcriptional analysis of product-concentration driven changes in cellular programs of recombinantClostridium acetobutylicum strains.Biotechnol. Bioeng. 84: 842–854.

    Article  CAS  Google Scholar 

  10. Tomas, C. A., J. A. Beamish, and E. T. Papoutsakis (2004) Transcriptional analysis of butanol stress and tolerance inClostridium acetobutylicum.J. Bacteriol. 186: 2006–2018.

    Article  CAS  Google Scholar 

  11. Hegde, P., R. Qi, K. Abernathy, C. Gay, S. Dharap, R. Gaspard, J. E. Hughes, E. Snesrud, N. Lee, and J. Quackenbush (2000) A concise guide to cDNA microarray analysis.Biotechniques 29: 548–562.

    CAS  Google Scholar 

  12. Yang, H., H. Haddad, C. Tomas, K. Alsaker, and E. T. Papoutsakis (2003) A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis.P.Natl. Acad. Sci. USA 100: 1122–1127.

    Article  CAS  Google Scholar 

  13. Xu, D., G. S. Li, L. Y. Wu, J. Z. Zhou, and Y. Xu (2002) PRIMEGENS: robust and efficient design of gene-specific probes for microarray analysis.Bioinformatics 18: 1432–1437.

    Article  CAS  Google Scholar 

  14. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman (1990) Basic Local Alignment Search Tool.J. Mol. Biol. 215: 403–410.

    CAS  Google Scholar 

  15. Rozen, S. and H. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. pp 365–386. In: S. Krawetz, S. Misener, (eds.).Methods in Molecular Biology. Humana Press, Totowa, NJ.

    Google Scholar 

  16. Richmond, C. S., J. D. Glasner, R. Mau, H. F. Jin, and F. R. Blattner (1999) Genome-wide expression profiling inEscherichia coli K-12.Nucleic Acids Res. 27: 3821–3833.

    Article  CAS  Google Scholar 

  17. Raghavachari, N., Y. P. Bao, G. S. Li, X. Y. Xie, and U. R. Müller (2003) Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.Anal. Biochem. 312: 101–105.

    Article  CAS  Google Scholar 

  18. Eisen, M. B., P. T. Spellman, P. O. Brown, and D. Botstein (1998) Cluster analysis and display of genome-wide expression patterns.Proc. Natl. Acad. Sci. USA. 95: 14863–14868.

    Article  CAS  Google Scholar 

  19. Clark, S. W., G. N. Bennett, and F. B. Rudolph (1989) Isolation and characterization of mutants ofClostridium acetobutylicum ATCC 824 deficient in acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A transferase (EC 2.8.3.9) and in other solvent pathway enzymes.Appl. Environ. Microbiol. 55: 970–976.

    CAS  Google Scholar 

  20. Wiesenborn, D. P., F. B. Rudolph, and E. T. Papoutsakis (1988) Thiolase fromClostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents.Appl. Environ. Microbiol. 54: 2717–2722.

    CAS  Google Scholar 

  21. Tummala, S. B., N. E. Welker, and E. T. Papoutsakis (2003) Design of antisense RNA constructs for downregulation of the acetone formation pathway ofClostridium acetobutylicum.J. Bacteriol. 185: 1923–1934.

    Article  CAS  Google Scholar 

  22. Buday, Z., J. C. Linden, and M. N. Karim (1990) Improved acetone butanol fermentation analysis using subambient HPLC column temperature.Enzyme Microb. Tech. 12: 24–27.

    Article  CAS  Google Scholar 

  23. Wei, Y., J. M. Lee, C. Richmond, F. R. Blattner, J. A. Rafalski, and R. A. LaRossa (2001) High-density microarray-mediated gene expression profiling ofEscherichia coli.J. Bacteriol. 183: 545–556.

    Article  CAS  Google Scholar 

  24. Schena, M., D. Shalon, R. Heller, A. Chai, P. O. Brown, and R. W. Davis (1996) Parallel human genome analysis: microarray-based expression monitoring of 1,000 genes.Proc. Natl. Acad. Sci. USA 93: 10614–10619.

    Article  CAS  Google Scholar 

  25. Chhabra, S. R., K. R. Shockley, S. B. Conners, K. L. Scott, R. D. Wolfinger, and R. M. Kelly (2003) Carbohydrate-induced differential gene expression patterns in the hyperthermophilic bacteriumThermotoga maritima.J. Biol. Chem. 278: 7540–7552.

    Article  CAS  Google Scholar 

  26. Worley, J., K. Bechtol, S. Penn, D. Roach, D. Hanzel, M. Trounstine, and D. Barker (2000) A systems approach to fabricating and analyzing DNA microarrays. In: M. Schena, editor.Microarray Biochip Technology. Eaton Publishing, Natick, MA, USA.

    Google Scholar 

  27. Yuen, P. K., G. Li, Y. Bao, and U. R. Muller (2003) Microfluidic devices for fluidic circulation and mixing improve hybridization signal intensity on DNA arrays.Lab Chip 3: 46–50.

    Article  CAS  Google Scholar 

  28. Adey, N. B., M. Lei, M. T. Howard, J. D. Jensen, D. A. Mayo, D. L. Butel, S. C. Coffin, T. C. Moyer, D. E. Slade, M. K. Spute, A. M. Hancock, G. T. Eisenhoffer, B. K. Dalley, and M. R. McNeely (2002) Gains in sensitivity with a device that mixes microarray hybridization solution in a 25-micron-thick chamber.Anal. Chem. 74: 6413–6417.

    Article  CAS  Google Scholar 

  29. Gadgil, C., A. Yeckel, J. J. Derby, and W. S. Hu (2004) A diffusion-reaction model for DNA microarray assays.J. Biotechnol. 114: 31–45.

    Article  CAS  Google Scholar 

  30. Borden, J. R., C. J. Paredes, and E. T. Papoutsakis (2005) Diffusion, mixing, and associated dye effects in DNA-microarray hybridizations.Biophys. J. In press.

  31. Nölling, J., G. Breton, M. V. Omelchenko, K. S. Makarova, Q. Zeng, R. Gibson, H. M. Lee, J. Dubois, D. Qiu, J. Hitti, Y. Wolf, R. L. Tatusov, F. Sabathe, L. Doucette-Stamm, P. Soucaille, M. J. Daly, G. N. Bennett, E. V. Koonin, and D. R. Smith (2001) Genome sequence and comparative analysis of the solvent-producing bacteriumClostridium acetobutylicum.J. Bacteriol. 183: 4823–4838.

    Article  Google Scholar 

  32. Cornillot, E., R. V. Nair, E. T. Papoutsakis, and P. Soucaille (1997) The genes for butanol and acetone formation inClostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain.J. Bacteriol. 179: 5442–5447.

    CAS  Google Scholar 

  33. Stim-Herndon, K. P., R. Nair, E. T. Papoutsakis, and G. N. Bennett (1996) Analysis of degenerate variants ofClostridium acetobutylicum ATCC 824.Anaerobe 2: 11–18.

    Article  CAS  Google Scholar 

  34. Tseng, G. C., M. K. Oh, L. Rohlin, J. C. Liao, and W. H. Wong (2001) Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects.Nucleic Acids Res. 29: 2549–2557.

    Article  CAS  Google Scholar 

  35. Harris, L. M., N. E. Welker, and E. T. Papoutsakis (2002) Northern, morphological, and fermentation analysis ofspo0A inactivation and overexpression inClostridium acetobutylicum ATCC 824.J. Bacteriol. 184: 3586–3597.

    Article  CAS  Google Scholar 

  36. Strauch, M. A., G. B. Spiegelman, M. Perego, W. C. Johnson, D. Burbulys, and J. A. Hoch (1989) The transition state transcription regulatorabrB ofBacillus subtilis is a DNA-binding protein.EMBO J. 8: 1615–1621.

    CAS  Google Scholar 

  37. Strauch, M., V. Webb, G. Spiegelman, and J. A. Hoch (1990) The Spo0A protein ofBacillus subtilis is a repressor of theabrB gene.Proc. Natl. Acad. Sci. USA 87: 1801–1805.

    Article  CAS  Google Scholar 

  38. Molle, V., M. Fujita, S. T. Jensen, P. Eichenberger, J. E. Gonzalez-Pastor, J. S. Liu, and R. Losick (2003) The Spo0A regulon ofBacillus subtilis.Mol. Microbiol. 50: 1683–1701.

    Article  CAS  Google Scholar 

  39. Scotcher, M. C., F. B. Rudolph, and G. N. Bennett (2005) Expression ofabrB310 andsinR, and effects of decreasedabrB310 expression on the transition from acidogenesis to solventogenesis, inClostridium acetobutylicum ATCC 824.Appl. Environ. Microbiol. 71: 1987–1995.

    Article  CAS  Google Scholar 

  40. Ravagnani, A., K. C. Jennert, E. Steiner, R. Grunberg, J. R. Jefferies, S. R. Wilkinson, D. I. Young, E. C. Tidswell, D. P. Brown, P. Youngman, J. G. Morris, and M. Young (2000) Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia.Mol. Microbiol. 37: 1172–1185.

    Article  CAS  Google Scholar 

  41. Helmann, J. D. and C. P. Moran Jr (2002) RNA polymerase and sigma factors. pp 289–312. In: A. L. Sonenshein, J. A. Hoch, R. Losick, (eds.),Bacillus subtilis and Its Closest Relatives: From Genes to Cells. ASM Press, Washington, D.C., USA.

    Google Scholar 

  42. Schaffer, S., N. Isci, B. Zickner, and P. Dürre (2002) Changes in protein synthesis and identification of proteins specifically induced during solventogenesis inClostridium acetobutylicum.Electrophoresis 23: 110–121.

    Article  CAS  Google Scholar 

  43. Eymann, C., A. Dreisbach, D. Albrecht, J. Bernhardt, D. Becher, S. Gentner, L. T. Tam, K. Büttner, G. Buurman, C. Scharf, S. Venz, U. Völker, and M. Hecker (2004) A comprehensive proteome map of growingBacillus subtilis cells.Proteomics 4: 2849–2876.

    Article  CAS  Google Scholar 

  44. Kanehisa, M., S. Goto, S. Kawashima, Y. Okuno, and M. Hattori (2004) The KEGG resource for deciphering the genome.Nucleic Acids Res. 32: D277-D280.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Northwestern University, 60208, Evanston, IL, USA

    Keith V. Alsaker, Carlos J. Paredes & Eleftherios T. Papoutsakis

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  1. Keith V. Alsaker
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  2. Carlos J. Paredes
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  3. Eleftherios T. Papoutsakis
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Correspondence to Eleftherios T. Papoutsakis.

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Alsaker, K.V., Paredes, C.J. & Papoutsakis, E.T. Design, optimization and validation of genomic DNA microarrays for examining theClostridium acetobutylicum transcriptome. Biotechnol. Bioprocess Eng. 10, 432–443 (2005). https://doi.org/10.1007/BF02989826

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