1 Department of Microbiology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118-2526, USA
2 The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA
Correspondence
Eva R. Kashket
ekashket{at}bu.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The main mechanism by which butanol exerts its toxic effects on C. beijerinckii, C. acetobutylicum and other solvent-producing clostridia has generally been taken to be its chaotropic effect on the integrity of the cell membrane. Butanol addition leads to leakage of ions and other solutes from clostridia (Hutkins & Kashket, 1986); with excessive ion leakage the cell cannot maintain its transmembrane ion gradients by ion pumping (Terracciano & Kashket, 1986
). However, butanol toxicity is not necessarily a limiting factor in solvent production by these organisms. Mutants that produce high concentrations of solvent (>13 g butanol l1, levels that previously were thought to be limiting) have been isolated without direct selection for butanol tolerance. The mutants include the hyperamylolytic C. beijerinckii NCIMB 8052 mutant, BA101 (Formanek et al., 1997
) and a mutant of C. acetobutylicum ATCC 824 with an inactivated solR gene (Harris et al., 2001
). The existence of these strains suggests that butanol at the concentrations that accumulate in cultures may not massively damage the cell membrane, and raises the possibility that the most sensitive target of butanol toxicity is not the phospholipid bilayer per se, but one or several specific cellular functions. For example, a target of butanol appears to be a function(s) involved in septation/cell separation, as suggested by the formation of filamentous forms when low concentrations of butanol were added to growing C. beijerinckii cells (E. R. Kashket, unpublished data).
The isolation of C. beijerinckii mutants that are more tolerant of butanol than the wild-type offered the opportunity to investigate whether specific functional components of the cell are especially sensitive targets of butanol toxicity. In the present studies we focused on the activities of the cell membrane which are required for maintaining transmembrane ion gradients. We report that a butanol-tolerant mutant, strain BR54 (Liyanage et al., 2000), undergoes less dissipation of the H+ gradient than the wild-type at a given concentration of butanol. We also provide evidence that the dissipation of
pH by Na+/H+ antiporter activity can be specifically blocked by low concentrations of butanol under conditions of limited cellular Mg2+ concentration. Finally, observations of differences in the content of arginyl residues between mutant BR54 and wild-type membrane proteins suggest that the proteins of the former are more extensively glycated by cellular methylglyoxal, which is consistent with the decreased ability of the mutant to detoxify metabolically generated methylglyoxal (Liyanage et al., 2001
). The mutant's apparent greater Na+/H+ antiporter activity may be a result of an increased degree of protein glycation compared to the wild-type.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
pH measurements.
The incubations were carried out at pH 5·0, since at this external pH the protonmotive force of the clostridia consists essentially of the pH (Terracciano & Kashket, 1986
), and it is therefore not necessary to measure
, the transmembrane electrical potential. The
pH was measured by the accumulation of low concentrations of a radioactively labelled, nonmetabolizable organic acid, [14C]benzoic acid (Baronofsky et al., 1984
; Terracciano & Kashket, 1986
). Weak organic acids such as benzoic acid or acetic acid distribute themselves across the membrane according to their pKa' values and the difference in pH (
pH) on the two sides (reviewed by Kashket, 1985
). The cells were harvested in late exponential phase (OD600 0·9), washed and suspended in the buffer. The buffers used were citric acid (12·5 mM) adjusted to pH 5·0 with either sodium phosphate (NaCP buffer) or potassium phosphate (KCP buffer), and supplemented with 1 % (w/v) glucose. Clostridial Basal Medium (CBM) (O'Brien & Morris, 1971
) containing 1 % (w/v) Casamino acids (Difco) was added to the
pH assay mixture where indicated. The
pH values were determined in triplicate and are reported as the means and standard deviations of at least three cultures per growth condition. P values were calculated by Student's t test.
Measurement of arginine residues in membrane proteins.
Protoplasts were prepared by treating washed cell pellets from 100 ml cultures suspended in 1·0 ml of a mixture containing 2·5 g sucrose l1, 50 mM Tris buffer, pH 8·0, and 55·5 mM EDTA, pH 8. Lysozyme (Sigma-Aldrich) was added to a final concentration of 2·5 mg ml1, and the cell suspension was incubated at 35 °C for 30 min, at which time conversion to round forms was found to be complete, as monitored by phase-contrast microscopy. The protoplasts were collected by centrifugation at 4000 r.p.m. (1900 g) for 10 min in Servall centrifuge, SS34 rotor, at 4 °C. The resulting pellet was suspended in 30 ml 10 mM sodium phosphate buffer, pH 7·0, and approximately 1 mg crystalline DNase (Sigma-Aldrich) was added at room temperature. The suspension clarified within a few minutes and became more yellow in colour. The lysed protoplasts were centrifuged at 9500 r.p.m. (10 800 g) for 20 min in a Servall SS34 rotor at 4 °C to remove any remaining intact protoplasts and debris. The supernatant fluid was centrifuged at 30 000 r.p.m. (166 000 g) for 1·5 h in a Servall Ultracentrifuge 4 °C with a Surespin 630 rotor. The resulting membrane pellet was suspended in 10 mM sodium phosphate buffer, pH 7·0, to a final volume of 400 µl. Part of the suspension (10 µl) was assayed for protein by the Folin-phenol (Lowry) method. A 250 µl aliquot of the membrane suspension was acid-hydrolysed under N2 with an equal volume of concentrated HCl at 105 °C, for 24 h. The hydrolysate was dried at 70 °C under N2, dissolved in 500 µl water, dried again, and dissolved in 250 µl 100 mM CAPS (Sigma-Aldrich) buffer, pH 9·0. The pH was readjusted to 9 with 1 M NaOH. Aliquots of 1030 µl of the hydrolysates were assayed for ornithine (Chinard, 1952), using CAPS buffer, pH 9·0, with and without treatment with beef liver arginase (EC 3.5.3.1; MP Biomedicals) for 60 min at room temperature, followed by heating at 100 °C for 10 min. The increase in the ornithine content of samples after arginase treatment reflected unmodified arginine residues, since arginine modified by MG at the guanidino group is not subject to hydrolysis by the enzyme (Greenberg, 1960
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The differences in butanol tolerance seen in wild-type and mutant cells were more pronounced in KCP buffer than in NaCP buffer (Fig. 1b). The
pH of the wild-type was lower with increasing butanol concentrations, and was completely dissipated at 1·5 % butanol. In the mutant the
pH decreased more gradually than in the wild-type, from 1·77 at 0 % butanol to 1·37 at 1·5 % butanol, and to 0·27 at 1·75 % butanol. The difference in butanol sensitivity between the strains was significant when 0·51·5 % butanol was added (P<0·001). Thus, the butanol-tolerant mutant, compared to the wild-type, was able to maintain a higher electrochemical gradient of H+ ions when challenged with butanol.
Effect of Mg2+ deprivation on pH
Wild-type C. beijerinckii and butanol-tolerant mutant BR54 cells incubated in MES buffer, pH 5·2, and supplied with glucose as fermentable energy source, maintained a pH of 1·63±0·02 (n=3) and 1·67±0·13 (n=4), respectively, for at least 20 min. However, when the cells were incubated in citrate/phosphate buffer, either NaCP or KCP, the
pH gradually dissipated. Thus, in mutant BR54 cells incubated in KCP buffer supplemented with glucose the
pH decreased significantly from 1·75 after 3 min to 1·27 at 30 min, a decrease of 0·48 pH units (Fig. 2
). When Mg2+ was included in the incubation mixture, however, the
pH was maintained over at least 30 min. In the experiment shown in Fig. 2
, Mg2+ was supplied by substituting 25 % of the KCP buffer with growth medium CBM (equivalent to 0·8 mM Mg2+). The
pH decreased to 1·69, a decrease of only 0·06 pH units. The components of CBM medium (potassium phosphate, cysteine, p-aminobenzoic acid, biotin, thiamine, MnSO4, FeSO4 and MgSO4), as well as casamino acids, were tested separately for ability to prevent or lessen the decrease in the proton electrochemical gradient in glucose-supplemented citrate/phosphate buffer. Only Mg2+ was found to lessen the dissipation of the
pH (data not shown). The same effect of Mg 2+ deprivation on
pH was seen in NaCP buffer and with wild-type cells (data not shown). We attribute the decrease in
pH in the absence of added Mg2+ to the chelating effects of citrate and phosphate and the resulting depletion of cellular Mg2+. Without adequate Mg2+, nucleotide-dependent reactions of energy metabolism, such as ATP-powered H+ extrusion, are apparently insufficient to replenish the proton electrochemical gradient in these anaerobes.
|
|
Effect of MG on cell growth and pH
The difference between the wild-type and the butanol-tolerant mutant, BR54, is the degree of expression of the glycerol dehydrogenase gene, gldA, resulting in increased MG levels in the mutant cells (Liyanage et al., 2001). We postulated that the differences in butanol tolerance and rate of
pH dissipation in citrate/phosphate buffer seen in the mutant and the wild-type are due to differences in the extent of protein glycation by MG in the two strains. It is important to note that the concentrations of free MG measured in the cultures were in the micromolar range (Liyanage et al., 2001
). However, since MG is known to react with many constituents in the medium as well as in the cells, we tested the effects of micromolar (low) and millimolar (high) MG concentrations on
pH dissipation. When we added MG at the same time as [14C]benzoate (zero time) there was no effect on the rate of
pH decrease in either NaCP buffer or KCP buffer, in both mutant and wild-type cells (data not shown). When MG was added to high concentrations (millimolar) to growing cells for 12 h before harvest the culture continued to increase in optical density, albeit somewhat more slowly (not shown). The most striking effect on cell growth was the inhibition of cell separation. One hour of exposure of BR54 cultures to 3 mM MG resulted in a significant increase (P<0·0003) in the proportion of chains consisting of three or more cells that had not separated from each other, and a decrease in the proportion of single-cell lengths. Wild-type cells behaved in a similar way (not shown). When MG was added to cultures earlier in exponential phase (OD600<0·3), the chains were longer, consisting of four cell-lengths or more, and there were fewer in one or two cell-lengths (not shown).
The addition of MG to growing cultures 1 h before harvesting of both wild-type and mutant BR54 cells inhibited the dissipation of the pH of cells assayed in citrate/phosphate buffer in the absence of added Mg2+ (Fig. 4
). The MG effect on
pH was more pronounced at higher MG concentrations, reaching a maximum at 3 mM added MG. The inhibiting effect of MG was seen both in mutant BR54 cells and in the wild-type cells. Thus, in the absence of added Mg2+, MG-treated cells retained their
pH, particularly in KCP (Fig. 5
). The addition of 0·8 % butanol to MG-treated cells only had the usual small
pH-dissipating effect that is seen in Mg+-replete cells (see Fig. 1
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The second effect of added butanol manifested itself under conditions of Mg2+ deficiency. Under such conditions the cells cannot maintain their transmembrane proton electrochemical gradient by Mg2+-dependent H+-translocating ATPase, leading to a decrease in pH with time, catalysed by Na+/H+ or K+/H+ antiporter activity. However, in Na+-containing citrate/phosphate buffer the addition of 0·8 % butanol prevented
pH dissipation despite cellular Mg2+ deficiency. This
pH-sparing effect of 0·8 % butanol was not seen in Mg2+-deficient cells incubated in K+ buffer. In fact, only the
pH-dissipating activity of 0·8 % butanol was clearly seen in cells incubated in K+ buffer (Fig. 3b, d
). The simplest explanation for these observations is that butanol inhibits a Na+/H+ exchanger that ordinarily catalyses the extrusion of Na+ by exchange with H+, by analogy to antiporters identified in other bacteria (West & Mitchell, 1974
; Padan et al., 2001
; Terracciano et al., 1987
). Under Mg2+-deficient conditions, the
pH cannot be maintained by the H+-translocating ATPase and the proton gradient is dissipated in Na+ buffers. In K+ buffers,
pH dissipation would occur by exchange of K+ and H+ ions, and the protein catalysing this reaction is apparently not sensitive to butanol. These findings point to the ability of low concentrations of butanol to bring about a selective effect on a specific metabolic function.
An additional finding was that the dissipation of pH in mutant BR54 was faster than in wild-type cells in Na+-containing buffer under conditions of Mg2+ deficiency. The single transposon insertion in mutant BR54 results in decreased expression of the gldA gene and a 25 % reduction in glycerol dehydrogenase activity (Liyanage et al., 2000
). This essential enzyme is apparently involved in the detoxification of MG; indeed, the mutant is more sensitive to growth inhibition by MG addition than the wild-type and contains higher cellular levels of the toxic compound. The simplest explanation for the apparently higher Na+/H+ exchange activity in the mutant strain is that the mutant protein is more highly glycated than the antiporter in the wild-type cells. Since proteins, as well as nucleic acids, are glycated by MG (Thornalley, 1996
; Kalapos, 1999
; Oya et al., 1999
; Uchida et al., 1997
), one would expect that many cell proteins, in addition to the putative Na+/H+ antiporter, would be more extensively glycated in the mutant than in the wild-type. The present experiments showed that the concentration of arginine residues unmodified by glycation in membrane fractions was indeed significantly higher in the wild-type strain than in the mutant. The mechanism of butanol tolerance may be an indirect result of the elevated glycation of cell proteins in the mutant strain.
Growth of the cells with high levels of added MG resulted in a decrease of the concentration of unmodified arginine residues in wild-type membranes. Interestingly, the addition of MG at concentrations of 14 mM, which are considerably higher than the micromolar concentrations of unbound MG found in wild-type and mutant cultures, resulted in the inhibition of Na+/H+ and K+/H+ exchange activities in Mg2+-deficient buffers. Presumably cellular proteins, or other glycation targets, are more extensively glycated at the high concentrations of the added MG than when MG is derived only from cellullar metabolism.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chinard, F. P. (1952). Photometric estimation of proline and ornithine. J Biol Chem 199, 9195.
Clarke, D. J., Fuller, F. M. & Morris, J. G. (1979). The proton-translocating adenosine triphosphatase of the obligately anaerobic bacterium, Clostridium pasteurianum. Eur J Biochem 98, 597612.[Abstract]
Formanek, J., Mackie, R. & Blaschek, H. P. (1997). Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Appl Environ Microbiol 63, 23062310.[Abstract]
Greenberg, D. (1960). Arginase. In The Enzymes 4, pp. 257267. Edited by O. Boyer, H. Lardy & K. Myrback. New York: Academic Press.
Harold, F. M. (1972). Conservation and transformation of energy by bacterial membranes. Bacteriol Rev 36, 172230.[Medline]
Harold, F. M. (1986). The Vital Force: a Study of Bioenergetics. New York: W. H. Freeman.
Harris, L. M., Blank, L., Desai, R. P., Welker, N. E. & Papoutsakis, E. T. (2001). Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutyulicum with an inactivated solR gene. J Ind Microbiol Biotechnol 27, 322328.[CrossRef][Medline]
Hasan, S. M. & Rosen, B. P. (1979). Properties and function of the proton-translocating adenosine triphosphatase of Clostridium perfringens. J Bacteriol 140, 745747.[Medline]
Hutkins, R. W. & Kashket, E. R. (1986). Phosphotransferase activity in Clostridium acetobutylicum from acidogenic and solventogenic phases of growth. Appl Environ Microbiol 51, 11211123.
Kalapos, M. P. (1999). Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol Lett 110, 145175.[CrossRef][Medline]
Kashket, E. R. (1985). The proton motive force in bacteria: a critical assessment of methods. Annu Rev Microbiol 39, 219242.[CrossRef][Medline]
Liyanage, H., Young, M. & Kashket, E. R. (2000). Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA. J Mol Microbiol Biotechnol 2, 8793.[Medline]
Liyanage, H., Kashket, S., Young, M. & Kashket, E. R. (2001). Clostridium beijerinckii and Clostridium difficile detoxify methylglyoxal by a novel mechanism involving glycerol dehydrogenase. Appl Environ Microbiol 67, 20042010.
Mitchell, P. (1961). Coupling of phosphorylation to electron and hydrogen transport by a chemi-osmotic type of mechanism. Nature 191, 144148.
Mitchell, P. (1963). Molecule, group and electron translocation through natural membranes. Biochem Soc Symp 22, 142168.
Mitchell, P. (1966). Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol Rev 41, 445502.[Medline]
Nicholls, D. G. & Ferguson, S. J. (2002). Bioenergetics 3. London: Academic Press.
O'Brien, R. W. & Morris, J. G. (1971). Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol 68, 307318.[Medline]
Oya, T., Hattori, N., Mizuno, Y., Miyata, S., Maeda, S., Osawa, T. & Uchida, K. (1999). Methylglyoxal modification of proteins. J Biol Chem 274, 1849218502.
Padan, E., Venturi, M., Gerchman, Y. & Dover, N. (2001). Na+/H+ antiporters. Biochim Biophys Acta 1505, 144157.[Medline]
Terracciano, J. S. & Kashket, E. R. (1986). Intracellular conditions required for the initiation of solvent production by Clostridium acetobutylicum. Appl Environ Microbiol 52, 8691.
Terracciano, J. S., Schreurs, W. J. A. & Kashket, E. R. (1987). Membrane H+ conductance of Clostridium thermoaceticum and Clostridium acetobutylicum: evidence for electrogenic Na+/H+ antiport in Clostridium thermoaceticum. Appl Environ Microbiol 53, 782786.
Thornalley, P. J. (1996). Pharmacology of methylglyoxal: formation, modification of proteins and nucleic acids, and enzymatic detoxification a role in pathogenesis and antiproliferative chemotherapy. Gen Pharmacol 27, 565573.[CrossRef][Medline]
Uchida, K., Khor, O. T., Oya, T., Osawa, T., Yasuda, Y. & Miyata, T. (1997). Protein modification by a Maillard reaction intermediate, methylglyoxal. FEBS Lett 410, 313318.[CrossRef][Medline]
West, I. C. & Mitchell, P. (1974). Proton/sodium ion antiport in Escherichia coli. Biochem J 144, 8790.[Medline]
Received 25 August 2004;
revised 8 November 2004;
accepted 9 November 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |