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

Abstract

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.

Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum

“…2) a set of 50 genes (see below) from the new butanol-stress data [from the wild-type (WT) C. acetobutylicum strain] to the data of our earlier studies (Alsaker et al, 2004;Tomas et al, 2004), whereby we used partial-genome microarrays (Tomas et al, 2003a) to analyze gene expression among four different C. acetobutylicum strains in response to 0.25-0.75% (v/v) butanol, 10 min to 24 h after stress. Those strains were: the plasmid-control strain 824(pIMP1) (Mermelstein et al, 1992) for a study involving strain 824(pMSPOA), which overexpresses the spo0A gene (Harris et al, 2002); strain 824(pGROE1), which overexpresses the groESL operon (Tomas et al, 2003b), and its corresponding plasmid-control strain 824(pSOS95del) (Tomas et al, 2003b; we note that no butanol-stress data have been previously reported for the WT strain). While there were significant differences in the butanol response among those experiments (as there should be considering the variability in strains and butanol concentrations used), we identified a set of universally regulated butanol-response genes (regardless of butanol concentration and genotype).…”

Simultaneous saccharification and fermentation of hemicellulose to butanol by a non-sporulating Clostridium species

“…S-3AÀC), suggesting that strain MF28 is asporogenic and solventogenic, unlike many other wellknown Gram-positive Clostridium species, including C. acetobutylicum (Harris et al, 2002), C. beijerinckii (Ravagnani et al, 2000), and Clostridium sp. strain BOH3 (Li et al, 2014) that initiated sporulation at day 5 of fermentation ( Fig.…”

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

“…These findings reveal that butanol stress results in increased expression of solvent formation genes and proteins. Regulation of the solvent formation genes has been strongly linked to expression and subsequent activation (by phosphorylation) of spo0A (21,40). The expression pattern of spo0A (Fig.…”

“…While an exactly analogous phosphorelay system has not been identified in C. acetobutylicum, Spo0A has been shown to play a major role in differentiation. An activated Spo0A has been recently shown to induce all key solvent formation genes (21,40), thereby mediating many, if not all, possible triggers of solventogenesis.…”