amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824

Abstract

Clostridium acetobutylicum produces an extracellular K-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83 013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus K-amylases. Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures. These results are in agreement with the extracellular K-amylase activities indicating that the expression of amyP is regulated at the transcriptional level. amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation. Degeneration of C. acetobutylicum has been associated to the loss of pSOL1. We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon. ß

DNA Array-Based Transcriptional Analysis of Asporogenous, Nonsolventogenic Clostridium acetobutylicum Strains SKO1 and M5

“…In contrast, SKO1 had a greater average peak acid concentration than the WT (256 and 112 mM, respectively). SKO1 samples from the bioreactors were plated on 2ϫ YTGMA (10,20) and shown by iodine staining to degrade starch, indicating that the strain still contained pSOL1. Growth kinetics was also inhibited: the doubling times for the WT and SKO1 were 1.07 and 1.45 h, respectively.…”

SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in Clostridium acetobutylicum ATCC 824

“…amyP, encoding ␣-amylase, is present on pSOL1 and hence would be lost if the cell degrades. This renders the cell unable to metabolize starch (18,32). After 24 h of growth in batch culture, 100-l samples of cell suspension were spread on three agar-solidified 2XYTGMA plates to test for ␣-amylase activity as previously described (18).…”

Expression of a Cloned Cyclopropane Fatty Acid Synthase Gene Reduces Solvent Formation in Clostridium acetobutylicum ATCC 824

“…␣-Amylase activity was detected by the method described previously (22,43). The cells were diluted and plated on 2YTGMA medium, and the plates with colonies (less than 30 on each plate) were overlaid with iodine solution (5% solid iodine, 10% potassium iodine).…”

“…The cells were diluted and plated on 2YTGMA medium, and the plates with colonies (less than 30 on each plate) were overlaid with iodine solution (5% solid iodine, 10% potassium iodine). The colonies with ␣-amylase activity showed starch-hydrolyzing ability, i.e., the colonies were surrounded by a clear halo against a blue background, indicating the presence of the ␣-amylase encoded by a gene on the pSOL1 plasmid, demonstrating that the cells had not degenerated (22,43). Colonies of a degenerated strain, M5 (10), did not show haloes.…”