This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Harris, L. M.
Right arrow Articles by Papoutsakis, E. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Harris, L. M.
Right arrow Articles by Papoutsakis, E. T.

 Previous Article  |  Next Article 

Journal of Bacteriology, July 2002, p. 3586-3597, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3586-3597.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Northern, Morphological, and Fermentation Analysis of spo0A Inactivation and Overexpression in Clostridium acetobutylicum ATCC 824

Latonia M. Harris,1,{dagger} Neil E. Welker,2 and Eleftherios T. Papoutsakis1*

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

Received 3 January 2002/ Accepted 1 April 2002

The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration—a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.

* Corresponding author. Mailing address: Department of Chemical Engineering, Northwestern University, Evanston, IL 60208. Phone: (847) 491-7455. Fax: (847) 491-3728. E-mail: e-paps{at}northwestern.edu.

{dagger} Present address: Schering-Plough Research Institute, Union, NJ 07083.

Journal of Bacteriology, July 2002, p. 3586-3597, Vol. 184, No. 13
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.13.3586-3597.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Harry, K. H., Zhou, R., Kroos, L., Melville, S. B. (2009). Sporulation and Enterotoxin (CPE) Synthesis Are Controlled by the Sporulation-Specific Sigma Factors SigE and SigK in Clostridium perfringens. J. Bacteriol. 191: 2728-2742 [Abstract] [Full Text]  
  • Shi, Z., Blaschek, H. P. (2008). Transcriptional Analysis of Clostridium beijerinckii NCIMB 8052 and the Hyper-Butanol-Producing Mutant BA101 during the Shift from Acidogenesis to Solventogenesis. Appl. Environ. Microbiol. 74: 7709-7714 [Abstract] [Full Text]  
  • Tracy, B. P., Gaida, S. M., Papoutsakis, E. T. (2008). Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia. Appl. Environ. Microbiol. 74: 7497-7506 [Abstract] [Full Text]  
  • Borden, J. R., Papoutsakis, E. T. (2007). Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum. Appl. Environ. Microbiol. 73: 3061-3068 [Abstract] [Full Text]  
  • Bongiorni, C., Stoessel, R., Perego, M. (2007). Negative Regulation of Bacillus anthracis Sporulation by the Spo0E Family of Phosphatases. J. Bacteriol. 189: 2637-2645 [Abstract] [Full Text]  
  • Huang, I-H., Sarker, M. R. (2006). Complementation of a Clostridium perfringens spo0A Mutant with Wild-Type spo0A from Other Clostridium Species. Appl. Environ. Microbiol. 72: 6388-6393 [Abstract] [Full Text]  
  • Alsaker, K. V., Papoutsakis, E. T. (2005). Transcriptional Program of Early Sporulation and Stationary-Phase Events in Clostridium acetobutylicum. J. Bacteriol. 187: 7103-7118 [Abstract] [Full Text]  
  • Scotcher, M. C., Rudolph, F. B., Bennett, G. N. (2005). Expression of abrB310 and sinR, and Effects of Decreased abrB310 Expression on the Transition from Acidogenesis to Solventogenesis, in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 71: 1987-1995 [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]  
  • Zhao, Y., Tomas, C. A., Rudolph, F. B., Papoutsakis, E. T., Bennett, G. N. (2005). Intracellular Butyryl Phosphate and Acetyl Phosphate Concentrations in Clostridium acetobutylicum and Their Implications for Solvent Formation. Appl. Environ. Microbiol. 71: 530-537 [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., Beamish, J., Papoutsakis, E. T. (2004). Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum. J. Bacteriol. 186: 2006-2018 [Abstract] [Full Text]  
  • Paredes, C. J., Rigoutsos, I., Papoutsakis, E. T. (2004). Transcriptional organization of the Clostridium acetobutylicum genome. Nucleic Acids Res 32: 1973-1981 [Abstract] [Full Text]  
  • Liu, J., Tan, K., Stormo, G. D. (2003). Computational identification of the Spo0A-phosphate regulon that is essential for the cellular differentiation and development in Gram-positive spore-forming bacteria. Nucleic Acids Res 31: 6891-6903 [Abstract] [Full Text]  
  • Tomas, C. A., Welker, N. E., Papoutsakis, E. T. (2003). Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program. Appl. Environ. Microbiol. 69: 4951-4965 [Abstract] [Full Text]  
  • Girbal, L., Mortier-Barriere, I., Raynaud, F., Rouanet, C., Croux, C., Soucaille, P. (2003). Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum. Appl. Environ. Microbiol. 69: 4985-4988 [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]  
  • Zhao, Y., Hindorff, L. A., Chuang, A., Monroe-Augustus, M., Lyristis, M., Harrison, M. L., Rudolph, F. B., Bennett, G. N. (2003). Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 69: 2831-2841 [Abstract] [Full Text]  
  • Tummala, S. B., Welker, N. E., Papoutsakis, E. T. (2003). Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum. J. Bacteriol. 185: 1923-1934 [Abstract] [Full Text]  
  • Yang, H., Haddad, H., Tomas, C., Alsaker, K., Papoutsakis, E. T. (2003). A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis. Proc. Natl. Acad. Sci. USA 100: 1122-1127 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»