Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

Abstract

The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response. Strain 824(pGROE1) was generated by groESL overexpression. The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h. A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol). Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA. A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes. Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease. Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.Metabolically engineered solventogenic clostridia may potentially lead to industrial processes for the production of solvents (such as butanol and acetone), other oxychemicals, and enzymes (30,35,41) or for biotransformation (57). In addition, clostridia can degrade a number of toxic chemicals and have great potential for bioremediation applications (15,16,25,46,54). In all such applications, the ability of cells to withstand "stressful" conditions such as high concentrations of substrates and the accumulation of toxic products without loss of productivity is a most significant goal. The difficulty-but also the intellectual and biotechnological challenge-is that the desirable phenotypic trait may be determined by several genes or a complex regulatory network. Solvents are inherently toxic to bacteria. Solvent tolerance, particularly ethanol tolerance, has been extensively examined in gram-negative organisms (38) but rarely, if at all, in gram-positive organisms. Gram-negative bacteria are generally much more resistant to increasingly polar solvents that gram-positive prokaryotes (27,28,51). The initial effect is disruption of membrane fluidity, and cells may...

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

“…Third, our new microarray data show consistent patterns of expression for all predicted (Paredes et al, 2004) operons, and a select set of these data are shown and discussed below. Fourth, our data display consistent temporal patterns for genes belonging to the same operon or (Alsaker et al, 2004;Tomas et al, 2004). Gray indicates that the data were not reliable.…”

“…The validity of the microarray data used in this study was assessed based on five different approaches/metrics. First, the new microarray data of butanol stress were compared against the earlier, partial genomic data which were obtained with recombinant C. acetobutylicum strains (Alsaker et al, 2004;Tomas et al, 2004). Second, we expected that the acetate and butyrate stress would upregulate several general stress response genes, and also, based on earlier physiological studies (Huesemann and Papoutsakis, 1986;Papoutsakis, 1988, 1990), the solvent-formation genes.…”

“…First, we compared ( Fig. 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).…”

“…C, energy production and conversion; D, cell cycle control; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation; K, transcription; L, replication, recombination and repair; M, cell wall/membrane biogenesis; N, cell motility; O, post-translational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction; U, intracellular trafficking and secretion; V, defense mechanisms. genome microarray analysis of four different recombinant C. acetobutylicum strains (Alsaker et al, 2004;Tomas et al, 2004). Many of these genes are orthologous to Bacillus subtilis stress genes, specifically of the class I (HrcAregulated: hrcA-grpE-dnaK, groESL), class III (CtsR-regulated: ctsR-yacH-yacI-clpC), and class IV (htrA) genes, and correspond to proteins with chaperone and protease activities (Schumann et al, 2002).…”

“…There exists hardly any literature, in either Gram À or Gram þ organisms, as to how cells respond to stresses due to metabolites present simultaneously. We previously used partial (25% genome coverage) DNA microarrays to examine butanol stress in four recombinant C. acetobutylicum strains at varying stress levels to elucidate what genes were involved in the short-and long-term stress responses to butanol (Alsaker et al, 2004;Tomas et al, 2004). In this study, we used full-genome C. acetobutylicum microarrays to identify and compare the transcriptional programs associated with the cell response to the key and largely toxic primary metabolites acetate, butyrate, and butanol.…”