| The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned and two recombinant strains were generated during this study, a spo0A inactivation strain (SKO1) and a spo0A overexpression strain (824(pMPSOA)). While creating strain SKO1, a novel protocol was developed for generating targeted gene inactivations in C. acetobutylicum using a replicative plasmid capable of double crossover integration into the chromosome. Strains SKO1 and 824(pMSPOA) were characterized in pH controlled batch fermentations to determine the effects of altered spo0A expression on product formation. The morphology of these strains was examined using phase contrast microscopy. Northern Blot analysis was used to examine the expression patterns of several genes ( spo0A, thl, adc, aad-ctfA /B, ptb-buk, and sigE-sigG ) in response to spo0A inactivation and overexpression.; The spo0A knockout strain, SKO1, was severely deficient in solvent formation. Only 2 mM of acetone and 14 mM of butanol were produced by SKO1 compared to the 93 mM of acetone and 171 mM of butanol produced by the wildtype strain. The morphology of the SKO1 also differed from the wildtype. After 72 hours of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate properly. SKO1 cells never achieved the swollen clostridal form typical of wildtype and did not form endospores. Additionally, mRNA levels produced during SKO1 fermentations proved that the spo0A transcript was not made in SKO1 and showed limited transcription of two solvent formation operons (aad-ctfA/B and adc) in the absence of Spo0A.; Product formation patterns in strain 824(pMSPOA), the spo0A overexpression strain, were unaltered compared to the control strain. However, microscopy indicated that spore formation occurred earlier due to spo0A overexpression. Expression of four solvent formation genes (aad, ctfA, ctfB, and adc) and two sporulation specific genes (sigE and sigG) was observed earlier in 824(pMSPOA) than in the control strain.; This work provides support for the hypothesis that Spo0A is a transcriptional regulator that controls the onset of stationary phase phenomena including sporulation and solvent production. Spo0A positively regulates both solvent production and sporulation in C. acetobutylicum ATCC 824. |
