Progress 08/15/01 to 08/31/04
Outputs
During the grant period our goal has been to establish the molecular mechanism(s) by which the fermentation end product, 1-butanol, and the toxic metabolic intermediate, methylglyoxal (MG), affect the growth and metabolism of the solvent-producing clostridium, Clostridium beijerinckii. The ultimate objective is gain an understanding of the factors that limit the production of butanol and acetone by these cells in order to increase the production of useful chemicals from renewable biomass. We have isolated butanol-tolerant transposon insertion mutants of this organism and showed that they contained higher cellular levels of MG than the wild type. The higher MG levels in the mutants were caused by the decreased expression of the gldA gene that encodes glycerol dehydrogenase, resulting in less detoxification of MG. We showed that the butanol-tolerant mutant is able to maintain a higher transmembrane gradient of H+ ions than the wild type when the cells are challenged by
increasing butanol concentrations. The H+ gradient was expressed as the Delta pH, the difference in pH between the interior and the exterior, and was determined from the distribution of radioactive benzoic acid between cells and the medium. Thus, our novel finding is that butanol specifically influences a membrane-linked function, the maintenance of a cellular ion gradient, in glycolyzing clostridial cells. We have also found that the Delta pH slowly decreased when glycolyzing clostridia were depleted of Mg++. Unexpectedly, the addition of low (0.8%) butanol concentrations prevented the decrease in the Delta pH. The sparing effect of butanol was seen in Na+-containing buffer, but not in K+ buffer, i.e., when the cells are carrying out Na+/H+ antiporter (exchange) activity, but not K+/H+ antiporter activity. These data were interpreted to mean that a putative Na+/H+ antiporter is inhibited by low levels of butanol. Since the Na+/H+ antiporter activity is higher in the butanol-tolerant
mutant than the wild type, this protein (as well as other proteins) may be more extensively glycated in mutant cells by the higher MG levels present; MG is known to modify arginine and lysine residues in proteins. To test the working hypothesis, that proteins are more extensively glycated when cellular MG levels are increased, we estimated the content of arginine residues of hydrolyzed membrane proteins. We measured the extent of glycation of arginine residues from the ornithine content of hydrolyzed membrane proteins treated with arginase, which removes unmodified, but not glycated guanidino groups from arginine. We found a 25-30% reduction in the arginines in membrane proteins of mutant cells compared to wild types. In addition, growth of the clostridia with MG addition to the medium reduced the arginine content of wild type membranes to those of the mutant. Thus, our data suggest that butanol can specifically affect the activity of at least one membrane protein, and are consistent
with the idea that the degree of protein modification by MG can affect cellular metabolism.
Impacts
It is now clear that the mechanisms of toxicity of the fermentation end product, butanol, on solvent-producing Clostridia are more complex than previously realized. Our new findings have shown that butanol specifically affects the activity of at least one membrane protein, and suggest that the degree of protein modification by methylglyoxal affects the butanol sensitivity of the cells. Indeed, our findings suggest the possibility that protein modification by MG might play a role in cellular metabolism. Our findings have added to our fundamental knowledge about the metabolism and cellular effects of the toxic metabolites, butanol and methylglyoxal, in solventogenic clostridia. An unexpected discovery during this grant period was that glycerol dehydrogenase performs an essential function in the detoxification of methylglyoxal in C. difficile as it does in C. beijerinckii, which may lead to the development of novel agents to cure infections by this prominent
antibiotic-resistant nosocomial pathogen.
Publications
- Liyanage, H., Young, M., and Kashket, E.R. 2000a. Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA. J. Molec. Microbiol. Biotechnol. 2: 87-93.
- Liyanage, H., Holcroft, P., Evans, V.J., Keis, S., Kashket, E.R., and M. Young. 2000b. A new insertion sequence, ISCb1, from Clostridium beijerinckii NCIMB 8052. J. Molec. Microbiol. Biotechnol., 2: 107-113.
- Liyanage, H., S. Kashket, M. Young, and E.R. Kashket. 2001. Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase. Appl.Environ. Microbiol. 67:2004-2010.
- Kashket, S., M.F.J. Maiden, A.D. Haffajee, and E.R. Kashket. 2003. Accumulation of methylglyoxal in the gingival crevicular fluid of chronic periodontitis patients. Journal of Clinical Periodontology, 30: 364-367.
- Wang, F., S. Kashket, and E.R. Kashket. 2004. Maintenance of Delta pH by a butanol-tolerant mutant of Clostridium beijerinckii. Submitted.
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Progress 10/01/02 to 09/30/03
Outputs
We are continuing studies on the mechanisms of butanol toxicity in the solvent-producing clostridium, C.beijerinckii. Previously we had shown that a butanol-tolerant mutant of this organism has higher cellular levels of methylglyoxal (MG) than the wild type. The mutant maintains higher ion gradients across the cell membrane than the wild type when challenged by increasing butanol concentrations (>1.25 %). The ion gradient measured was the Delta pH (difference in pH between the interior and the exterior of the cell, which at pH 5 is equivalent to the transmembrane electrochemical gradient of H+). We also found that the Delta pH decreased when glycolyzing cells were depleted of Mg++, but the addition of low butanol concentrations (0.8%) prevented the decrease in Delta pH. The sparing effect of butanol was seen in Na+ containing buffer, but not in K+ buffer. The data were interpreted to mean that the Delta pH is dissipated in the absence of Mg++ by a Na+ or K+-linked
process and that the putative Na+/H+ antiporter is inhibited by low levels of butanol. These findings are consistent with the ability of butanol to selectively affect membrane-associated functions. It was proposed that in mutant cells an Na+/H+ antiporter protein (as well as other proteins) is more extensively glycated by the higher MG levels present in the cells, and is thus more susceptible to inhibition by butanol. Since quantitative assays of isolated Na+/H+ antiporter reconstituted into proteoliposomes are not feasible for technical reasons, we are first testing the working hypothesis, that proteins are more extensively glycated when cellular MG levels are increased and by comparing membrane proteins of wild type and mutant cells. MG is known to modify arginine and lysine residues in proteins. We measure the extent of glycation of arginine residues from the ornithine content of hydrolyzed membrane proteins treated with arginase, which removes unmodified but not glycated guanidino
groups from arginine. We have found a 25-30% reduction of unmodified arginine residues in the membrane proteins of MG-grown cells compared to untreated cultures. There was no detectable, statistically significant difference between the mutant and wild type membrane proteins. We are continuing to refine the methodology for assessing protein glycation by MG. The methods are based on modification of arginine and lysine residues by MG which alter the electrophoretic mobility of proteins (because of the decrease of positive charges), as well the pattern of peptides obtained by trypsin treatment (since trypsin does not hydrolyze peptide bonds of arginines with modified guanidine groups), to be followed by the identification by 2 dimensional gel electrophoresis of potentially glycated individual proteins.
Impacts
It is now clear that the mechanism of toxicity of the fermentation end product, butanol, on solvent-producing Clostridia is more complex than previously realized. Our new findings have shown that butanol specifically affects the activity of at least one membrane protein, and suggest that the degree of protein modification by methylglyoxal affects the butanol sensitivity of the cells. The impact of our studies will be demonstrated in the applicability of basic findings to long-range improvements in the microbiological conversion of waste agricultural biomass material to alcohol fuels and solvents.
Publications
- No publications reported. 2003
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Progress 10/01/01 to 09/30/02
Outputs
Studies were continued on the mechanisms of butanol toxicity in solvent-producing clostridia. We tested the working hypothesis that glycation of key protein targets by methylglyoxal (MG) can result in reduced butanol susceptibility of these bacteria; specifically, that protein glycation affects the functional integrity of cell membranes. We assessed Clostridium beijerinckii NCIMB 8052 and a butanol tolerant, transposon insertion mutant, strain BR54, whose decreased glycerol dehydrogenase activity is associated with increased cellular MG levels, for their ability to maintain transmembrane ion gradients upon butanol challenge. The magnitude of the Delta pH (difference in pH between the interior and the exterior of the cell) was measured with 14C-benzoate using late exponential phase cells suspended in citrate-phosphate buffer at pH 5 to maximize the Delta pH component of the protonmotive force (the transmembrane electrochemical gradient of hydrogen ions), and
supplemented with glucose and Mg2+. We found that the Delta pH of mutant cells was significantly more tolerant of added butanol than the wild type cells. Thus, when butanol was added to a concentration of 15 g/l (1.5%), the mutant cells maintained a Delta pH = 1.4, while the wild type Delta pH was dissipated. With 1.75% (w/v) butanol, the mutants' Delta pH was also dissipated. In the absence of added Mg2+ the Delta pH of both strains decreased slowly over time. However, the addition of low butanol concentrations (0.8%) inhibited this Delta pH decrease. The Delta pH-sparing effect was seen in cells suspended in citrate-sodium phosphate buffer, but not in citrate-potassium phosphate buffer. Sparing of the Delta pH was complete in mutant cells, and less effective in wild type cells, i.e., the Delta pH decreased, but less rapidly than without butanol addition. We interpret these data to mean that the Delta pH is dissipated in the absence of Mg2+ by a Na+ or K+-linked process, possibly by
a Na+/H+ or a K+/H+ antiporter, and that the former is inhibited by butanol. In the mutants, presumably, the Na+/H+ antiporter protein is more extensively glycated by the higher MG levels present in the cells, and is thus more susceptible to inhibition by butanol. These findings demonstrate that butanol, at sublethal concentrations, can selectively affect a membrane-associated function.
Impacts
The impact of our studies will be demonstrated in their applicability to long-range improvements in the microbiological conversion of waste agricultural biomass material to alcohol fuels and solvents. Specifically, increasing our basic understanding of the mechanisms of butanol action at the molecular level should lead to new approaches for strain improvement with regard to the capacity of Clostridia to produce solvents. Our new findings have already shown that the mechanism of toxicity of the end product, butanol, is more complex than previously realized.
Publications
- No publications reported this period
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