This Article Full Text Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tummala, S. B. Articles by Papoutsakis, E. T. Search for Related Content PubMed PubMed Citation Articles by Tummala, S. B. Articles by Papoutsakis, E. T.

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

Department of Chemical Engineering,¹ Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208²

Received 5 August 2002/ Accepted 30 December 2002

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

This article has been cited by other articles:

• Kedar, G. C., Brown-Driver, V., Reyes, D. R., Hilgers, M. T., Stidham, M. A., Shaw, K. J., Finn, J., Haselbeck, R. J. (2007). Evaluation of the metS and murB Loci for Antibiotic Discovery Using Targeted Antisense RNA Expression Analysis in Bacillus anthracis. Antimicrob. Agents Chemother. 51: 1708-1718 [Abstract] [Full Text]  
• Raju, D., Setlow, P., Sarker, M. R. (2007). Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation. Appl. Environ. Microbiol. 73: 2048-2053 [Abstract] [Full Text]  
• Nakashima, N., Tamura, T., Good, L. (2006). Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res 34: e138-e138 [Abstract] [Full Text]  
• Scotcher, M. C., Bennett, G. N. (2005). SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in Clostridium acetobutylicum ATCC 824. J. Bacteriol. 187: 1930-1936 [Abstract] [Full Text]  
• Alsaker, K. V., Spitzer, T. R., Papoutsakis, E. T. (2004). Transcriptional Analysis of spo0A Overexpression in Clostridium acetobutylicum and Its Effect on the Cell's Response to Butanol Stress. J. Bacteriol. 186: 1959-1971 [Abstract] [Full Text]  
• Tomas, C. A., Alsaker, K. V., Bonarius, H. P. J., Hendriksen, W. T., Yang, H., Beamish, J. A., Paredes, C. J., Papoutsakis, E. T. (2003). DNA Array-Based Transcriptional Analysis of Asporogenous, Nonsolventogenic Clostridium acetobutylicum Strains SKO1 and M5. J. Bacteriol. 185: 4539-4547 [Abstract] [Full Text]  
• Tummala, S. B., Junne, S. G., Papoutsakis, E. T. (2003). Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations. J. Bacteriol. 185: 3644-3653 [Abstract] [Full Text]