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

Zhao, Yinsuo; Hindorff, Lucia A.; Chuang, Amy; Monroe-Augustus, Melanie; Lyristis, Michael; Harrison, Mary L.; Rudolph, Frederick B.; Bennett, George N.

doi:10.1128/aem.69.5.2831-2841.2003

Cited by 92 publications

(82 citation statements)

References 55 publications

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“…However, the increase in cyclopropane fatty acid production during [C 2 mim]Cl exposure demonstrates the distinct effect of ionic liquid on the bacterium. A number of studies using E. coli and lactobacilli have demonstrated that methylation of the cis-acyl chain double bond to form a cyclopropane ring plays a significant role in tolerance to acid, salt, butanol, and other stressors by reducing membrane fluidity and decreasing permeability (50)(51)(52)(53)(54). It is probable that cyclopropane fatty acid production plays a similar role in [C 2 mim]Cl tolerance by stabilizing the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium,Enterobacter lignolyticus

Khudyakov

D’haeseleer

Borglin

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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To process plant-based renewable biofuels, pretreatment of plant feedstock with ionic liquids has significant advantages over current methods for deconstruction of lignocellulosic feedstocks. However, ionic liquids are often toxic to the microorganisms used subsequently for biomass saccharification and fermentation. We previously isolated Enterobacter lignolyticus strain SCF1, a lignocellulolytic bacterium from tropical rain forest soil, and report here that it can grow in the presence of 0.5 M 1-ethyl-3-methylimidazolium chloride, a commonly used ionic liquid. We investigated molecular mechanisms of SCF1 ionic liquid tolerance using a combination of phenotypic growth assays, phospholipid fatty acid analysis, and RNA sequencing technologies. Potential modes of resistance to 1-ethyl-3-methylimidazolium chloride include an increase in cyclopropane fatty acids in the cell membrane, scavenging of compatible solutes, up-regulation of osmoprotectant transporters and drug efflux pumps, and down-regulation of membrane porins. These findings represent an important first step in understanding mechanisms of ionic liquid resistance in bacteria and provide a basis for engineering microbial tolerance.osmotic stress | osmolytes | membrane lipids | differential gene expression | whole genome metabolic reconstruction

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Section: Discussionmentioning

confidence: 99%

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium,Enterobacter lignolyticus

Khudyakov

D’haeseleer

Borglin

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…However, expression of the putative kinA is higher in the butanolstressed 824(pSOS95del) culture. Alterations in cell membrane composition are the most established mechanism which cells utilize to adapt to high levels of solvents (58). The ability of cells to incorporate a higher percentage of transmembrane fatty acids (long-chain C 30 fatty acids) in the cell membrane has been shown to increase alcohol tolerance in Thermoanaerobacter ethanolicus (7) from 1.5 to 8% (vol/vol) ethanol.…”

Section: Discussionmentioning

confidence: 99%

“…The highly differentially regulated gene expression patterns for the entire fatty acid biosynthesis operon and several related genes provide clear evidence that butanol stress alters the regulation of the fatty acid biosynthesis machinery. Increased expression of the cyclopropane fatty acid synthase gene (cfa [CAC0877]) in C. acetobutylicum has been shown to result in butanol resistance (58). Elevated expression of cfa was observed early in the high-dose 824(pSOS95del) culture, while cfa was Ͻ1.7 times higher in the high-dose 824 (pGROE1) culture (Fig.…”

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confidence: 99%

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

2004

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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...

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“…Prior to transformation into C. acetobutylicum, plasmid DNA must be methylated by the ⌽3TI methyltransferase to prevent restriction by the clostridial endonuclease Cac824I (24). Methylation was achieved by transformation of the required plasmid into E. coli DH10␤ harboring vector pDHKM (43), which carries an active copy of the ⌽3TI methyltransferase gene. Electrotransformation of wild-type C. acetobutylicum or strain SKO1 was carried out according to the protocol developed by Mermelstein et al (23).…”

Section: Methodsmentioning

confidence: 99%

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

Scotcher

Bennett

2005

J Bacteriol

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Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation.The gram-positive, obligate anaerobe Clostridium acetobutylicum was used for the industrial production of the solvents acetone and butanol for over 60 years in the 20th century. With chemical synthesis of acetone and butanol proving significantly more economic, there are no reported industrial fermentation plants using C. acetobutylicum operational in the world today (11). However, over the last 20 years, the genetics and biochemistry of C. acetobutylicum have been investigated in detail, as we try to understand and improve upon the processes that control the production of solvents.The genes aad, ctfA, ctfB, and adc are required for solvent production and are located on a 192-kb megaplasmid designated pSOL1 (8,27). ctfA and ctfB code for a multifunctional coenzyme A (CoA) transferase, which transfers the CoA moiety from acetoacetyl-CoA to acetate or butyrate (40). Subsequently, acetoacetate is decarboxylated to form acetone, and acetyl-CoA and butyryl-CoA are converted to ethanol and butanol, respectively, via an aldehyde intermediary (26). For a detailed description of clostridial biochemistry, see the paper by Mitchell (26). It has been shown that C. acetobutylicum can lose pSOL1, rendering the cells unable to produce solvents. Cells and strains that have lost pSOL1 are termed "degenerate" (8, 28).Whereas much is known about the biochemistry of C. acetobutylicum metabolism and the genes and proteins that catalyze these processes, relatively little is known about the genetic control of the expression of these genes. Recently, it was shown that a homologue to Bacillus subtilis stage 0 sporulation protein A (Spo0A) controls both the onset of solventogen...

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

Cited by 92 publications

References 55 publications

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium,Enterobacter lignolyticus

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium,Enterobacter lignolyticus

Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum

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

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