Archives of
Microbiology
Arch. Microbiol. 109, 315-317 (1976)
9 by Springer-Verlag 1976
Short Communications The Use of Stable Sulfur Isotope Labelling to Elucidate Sulfur Metabolism by Clostridium pasteurianum RONALD G. L. McCREADY 1*, EDWARD J. LAISHLEY 1**, and H. ROY KROUSE 2 1 Department of Biology and 2 Department of Physics, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Abstract. An unique stable isotope labelling experi-
ment was conducted whereby mixtures of sulfate,and sulfite of different isotopic compositions were metabolized by Clostridiumpasteurianum. The results showed during reduction of 1 mM SO~ plus 1 mM SO2, essentially all evolved H2S arose from the sulfite whereas in the case of cellular sulfur, 85 ~ was derived from sulfite and the remainder from sulfate. Key words: Sulfur isotopes num - Sulfur metabolism.
Clostridium pasteuria-
McCready et al. (1975 b)reported large inverse isotope effects during the reduction of sulfite by Clostridium pasteurianum. On the basis of these unusual isotopic patterns, both assimilatory and dissimilatory sulfite reductive pathways were postulated for this organism. Further work showed growth enhancement when sulfate ion was added to cultures reducing sulfite (McCready et al., 1975a). It was therefore tempting to conclude that sulfate was metabolized by the assimilatory pathway while sulfite was primarily metabolized by the dissimilatory reductase system. There was also concern that some sulfite might be oxidized to sulfate during the experiments. This concern may be directed to many reported sulfite reduction experiments. To test the concept of two metabolic pathways and to further evaluate the oxidation possibility, experiments were devised in which compounds of differing 348/328 abundance ratios were metabolized. Clostridium p a s t e u r i a n u m strain W5 was grown in 10 1 batches of mineral salts-sucrose medium as previously described (McCready Present address: Elliot Lake Laboratory, CANMET, E.M.R., P.O. Box 100, Elliot Lake, Ontario, P5A 2J6, Canada ** To whom offprint requests should be sent *
et al., 1975b). In one experiment, cells were grown on a mixture of i m M SO~- (~34S = 0~ and 1 m M SO 2 (63, = + 3.1u/oo) while in the second experiment, cells were grown in a mixture of 1 m M SO~ (634S = 0~ and i m M SO 4 (634S = + 50.2~ where:
634Sin ~ =
[34S/32S]sample ] [3~S/3zS]referencesulfite - I x t000.
The cultures were incubated at 37~ and anaerobic conditions were maintained by bubbling high purity nitrogen gas through the medium. This also displaced the H2S gas produced which was fractionally collected and prepared for isotopic analyses by mass spectrometry. Intracellular sulfur was extracted for isotopic analysis as described by McCready et al. (1974).
The results of these two experiments are presented in Figure 1. The growths (upper curves) are slightly different in the two experiments. This may reflect the use of Na2SO4 in experiment A and a natural gypsum (CaSO4 92 H20) in experiment B. Unfortunately, sulfate so highly enriched in 34S was not available as the Na salt for experiment B. The x-dimensions of the horizontal lines in the bottom of the figure represent the fractions of H2S successively collected while the y coordinate represents the isotope composition of each fraction. The curves constructed through these lines represent the isotopic composition of the H2S produced at any instant during the conversion. The gross features of the isotopic patterns for both experiments are very similar and display the previously reported effects. Initially, the evolved H2S differs little isotopically from the starting sulfite. Up to about 20 reaction a normal kinetic isotope effect exists whereby the product H2S becomes increasingly enriched in the lighter 32S compared to the sulfite. Then the H2S starts to become isotopically heavier. At about 30 reaction, it is more enriched in 34S than the culture medium, i.e. an inverse kinetic isotope effect exists. This inverse isotope effect maximizes with 6-value of
316
Arch. Microbiol., Vol. 109 (1976)
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Fig.]. The growth curves and isotopic pattern observed during the reduction of 1 m M SO a in the presence of 1 mM isotopically heavy and light SO 4 by Clostridium pasteurianum. A is the "light" SO[ experiment (634S = + 3.1% 0). B is the "heavy" SO2 experiment (634S = + 50.2o/oo). The percent HaS produced is calculated on the basis of the original SO;- since negligible H2S comes from the SO 2
the HzS near + 25~ Thereafter, the a-value of the HzS decreases until activity ceases. In the H2S minimum troughs, there is "fine structure" in the form of positive peaks. One is duplicated convincingly in both experiments in the range J 5 - 1 7 ~ conversion. This corresponds to early logarithmic growth in both experiments. We have not explained these smaller peaks but we feel that they are related to recycling of sulfur compounds in the complex dissimilatory pathway previously described (McCready et al., 1975b). For this communication, the similarity of the curves is important rather than explanations of details. The similarity means that the H2S arises almost solely from sulfite. Any H2S arising from sulfate should have a-values in excess of + 50%0 and this was not found.
This verifies that oxidation of sulfite to sulfate was negligible and did not give rise to the unusual isotopic pattern. It further suggests that sulfite reduction experiments previously described in the literature are valid. Since the addition of sulfate had a stimulatory effect on growth, it is informative to examine the isotopic composition of the cells. The intracellular sulfur for experiment A lxad a a-value of - 0 . 7 % 0 while that of experiment B was + 6.2O/oo. Two equations of isotopic balance can be used to estimate the percentages of cellular sulfur derived from the SO~ and SO~. Let a and b be the percentages of cellular sulfur derived from SO4 and SO 3 respectively. Assume negligible isotopic selectivity during SO~ assimilation. This contributes little error on the basis of previous SO~ reduction experiments with this (McCready et al., 1975b) and other organisms (Kaplan and Rittenberg, 1964). Further, since little SO~ is utilized and the isotopic fractionation is small, there is a negligible "reservoir" effect. In contrast, the isotopic composition of the reservoir SO~ changes drastically because copious H2S is liberated with large isotope effects. This unknown is designated 6SO 3 and is nearly the same for both experiments since SO~ of the same isotopic composition was used and the isotopic patterns for the released H2S were similar. The equations of isotopic balance are: Experiment B a(50.2) + b(aSO;-) = (a + b) 6.2; Experiment A a (3.1) + b(aSO3) = (a + b) ( - 0.7). Subtraction eliminates the term b (aSO~) and since (a + b ) corresponds to the total or 100~ cellular sulfur, we find a = 14.7~o and b = 85.3~. Substitution of a and b into either equation gives SO2 = - 1.3~ This value falls in the range of cellular sulfur derived from SO~ alone ( - 0 . 5 _ 2.5O/oo std. dev., 21 experiments with a variety of carbon sources). The value of + 6.2~ for cellular sulfur of experiment B falls well outside of this range attesting to some SO 2 incorporation in the cells. However, if the intracellular sulfur had been derived solely from SO2 then a a-value near + 50%o should have been found in experiment B. It was initially a surprise that despite the stimulatory effect of SO47 on growth (e.g. generation times; l m M SO~, 85min; l m M S O ~ + 0 . 1 m M SO2, 50min; l m M S O ~ + 10raM SO2, 55min), the labelling experiments show that sulfite provide the majority of intracellular sulfur. This is consistent with some related experiments where the production of amylopectin storage granules was studied. These are normally produced during growth on SO~7 but not SO~ (McCready et al., 1976). In mixtures of SO2
R. G. L. McCready et al. : 348/32S Labelling of Sulfur Metabolism by Clostridium pasteurianum
317
and SO 3, they are absent suggesting the SO~ is inhibiting their synthesis even in the presence of SO4-. All evidence attests to the preferential metabolism of SO2 and we also conclude that growth is stimulated markedly with very little SO2. Using labelling experiments with various isotope compositions for the nutrients, one could derive more equations and the isotopic selectivity during SO4 and SOl incorporation can be treated as unknowns rather than assumed negligible. Experiments can also be carried out with differing sulfate to sulfite ratios. Whereas radioactive 35S would have provided some of the conclusions of this study, there are certain advantages to using stable isotope labelling in terms of storage and quantitative calculations. Compounds of different 3~S/32S abundances can be made from natural samples or by utilizing chemical kinetic isotope effects or exchanges in laboratory experiments. Recent announcements predict that sulfur highly enriched in 34S can be produced by laser separation at low cost 1. This being the case,, biological labelling experiments with stable sulfur isotopes might achieve the prom!nence currently identified with ~SN.
Acknowledgements.This work was supported by National Research Council of Canada Grants A 5058 (E. J. L.), A 8176 (H. R. K.) and the University of Calgary Interdisciplinary Sulphur Research Group (UNISUL). The authors also acknowledge the technical assistance of Miss N. Enojo, Miss J. Pontoy, and Miss E. Bigornia.
1 Laser separation of isotopes: Big Step Science News 107, 284 (1975)
Received March 1, 1976
REFERENCES Kaplan, I. R., Rittenberg, S. C.: Microbiological fractionation of sulfur isotopes. J. gen. Microbiol. 34, 195-212 (1964) McCready, R. G. L., Costerton, J. W., Laishley, E. J. : Morphological modification of cells of Clostridium pasteurianum caused by growth on sulfite. Canad. J. Microbiol. 22, 269-275 (1976) McCready, R. G. L., Kaplan, I. R., Din, G. A.: Fractionation of sulfur isotopes by the yeast Saccharomyees cerevisiae. Geochim. cosmochiflq. Acta 38, 1239-1253 (1974) McCready, R. G. L., Krouse, H. R., Laishley, E. J. : Sulfur isotope effects during growth of Clostridium pasteurianum on mixtures ofsulfite and sulfate. Canad. Fed. bioI. Soc. Proc. 18, 525 (1975 a) McCready, R. G. L., Laishley, E. J., Krouse, H. R. : Stable isotope fractionation by Clostridiumpasteurianum. 1. 34S/32S: inverse isotope effects during SO]- and SO32- reduction. Canad. J. Microbiol. 21, 235-244 (1975b)
Microbiology
Arch. Microbiol. 109, 315-317 (1976)
9 by Springer-Verlag 1976
Short Communications The Use of Stable Sulfur Isotope Labelling to Elucidate Sulfur Metabolism by Clostridium pasteurianum RONALD G. L. McCREADY 1*, EDWARD J. LAISHLEY 1**, and H. ROY KROUSE 2 1 Department of Biology and 2 Department of Physics, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Abstract. An unique stable isotope labelling experi-
ment was conducted whereby mixtures of sulfate,and sulfite of different isotopic compositions were metabolized by Clostridiumpasteurianum. The results showed during reduction of 1 mM SO~ plus 1 mM SO2, essentially all evolved H2S arose from the sulfite whereas in the case of cellular sulfur, 85 ~ was derived from sulfite and the remainder from sulfate. Key words: Sulfur isotopes num - Sulfur metabolism.
Clostridium pasteuria-
McCready et al. (1975 b)reported large inverse isotope effects during the reduction of sulfite by Clostridium pasteurianum. On the basis of these unusual isotopic patterns, both assimilatory and dissimilatory sulfite reductive pathways were postulated for this organism. Further work showed growth enhancement when sulfate ion was added to cultures reducing sulfite (McCready et al., 1975a). It was therefore tempting to conclude that sulfate was metabolized by the assimilatory pathway while sulfite was primarily metabolized by the dissimilatory reductase system. There was also concern that some sulfite might be oxidized to sulfate during the experiments. This concern may be directed to many reported sulfite reduction experiments. To test the concept of two metabolic pathways and to further evaluate the oxidation possibility, experiments were devised in which compounds of differing 348/328 abundance ratios were metabolized. Clostridium p a s t e u r i a n u m strain W5 was grown in 10 1 batches of mineral salts-sucrose medium as previously described (McCready Present address: Elliot Lake Laboratory, CANMET, E.M.R., P.O. Box 100, Elliot Lake, Ontario, P5A 2J6, Canada ** To whom offprint requests should be sent *
et al., 1975b). In one experiment, cells were grown on a mixture of i m M SO~- (~34S = 0~ and 1 m M SO 2 (63, = + 3.1u/oo) while in the second experiment, cells were grown in a mixture of 1 m M SO~ (634S = 0~ and i m M SO 4 (634S = + 50.2~ where:
634Sin ~ =
[34S/32S]sample ] [3~S/3zS]referencesulfite - I x t000.
The cultures were incubated at 37~ and anaerobic conditions were maintained by bubbling high purity nitrogen gas through the medium. This also displaced the H2S gas produced which was fractionally collected and prepared for isotopic analyses by mass spectrometry. Intracellular sulfur was extracted for isotopic analysis as described by McCready et al. (1974).
The results of these two experiments are presented in Figure 1. The growths (upper curves) are slightly different in the two experiments. This may reflect the use of Na2SO4 in experiment A and a natural gypsum (CaSO4 92 H20) in experiment B. Unfortunately, sulfate so highly enriched in 34S was not available as the Na salt for experiment B. The x-dimensions of the horizontal lines in the bottom of the figure represent the fractions of H2S successively collected while the y coordinate represents the isotope composition of each fraction. The curves constructed through these lines represent the isotopic composition of the H2S produced at any instant during the conversion. The gross features of the isotopic patterns for both experiments are very similar and display the previously reported effects. Initially, the evolved H2S differs little isotopically from the starting sulfite. Up to about 20 reaction a normal kinetic isotope effect exists whereby the product H2S becomes increasingly enriched in the lighter 32S compared to the sulfite. Then the H2S starts to become isotopically heavier. At about 30 reaction, it is more enriched in 34S than the culture medium, i.e. an inverse kinetic isotope effect exists. This inverse isotope effect maximizes with 6-value of
316
Arch. Microbiol., Vol. 109 (1976)
0.80.4 E
/7"
c
o0.2
//
//z'/
r"i 0.4
d
,/// 1
I
2
4
I
I
I
I
6 8 40 TIME IN HOURS
/'\
,'
~2
~.
/! 4O
A La
o
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I
0
I
20 PERCENT
I
I
I
I
40 60 HzS PRODUCED
I
I
80
Fig.]. The growth curves and isotopic pattern observed during the reduction of 1 m M SO a in the presence of 1 mM isotopically heavy and light SO 4 by Clostridium pasteurianum. A is the "light" SO[ experiment (634S = + 3.1% 0). B is the "heavy" SO2 experiment (634S = + 50.2o/oo). The percent HaS produced is calculated on the basis of the original SO;- since negligible H2S comes from the SO 2
the HzS near + 25~ Thereafter, the a-value of the HzS decreases until activity ceases. In the H2S minimum troughs, there is "fine structure" in the form of positive peaks. One is duplicated convincingly in both experiments in the range J 5 - 1 7 ~ conversion. This corresponds to early logarithmic growth in both experiments. We have not explained these smaller peaks but we feel that they are related to recycling of sulfur compounds in the complex dissimilatory pathway previously described (McCready et al., 1975b). For this communication, the similarity of the curves is important rather than explanations of details. The similarity means that the H2S arises almost solely from sulfite. Any H2S arising from sulfate should have a-values in excess of + 50%0 and this was not found.
This verifies that oxidation of sulfite to sulfate was negligible and did not give rise to the unusual isotopic pattern. It further suggests that sulfite reduction experiments previously described in the literature are valid. Since the addition of sulfate had a stimulatory effect on growth, it is informative to examine the isotopic composition of the cells. The intracellular sulfur for experiment A lxad a a-value of - 0 . 7 % 0 while that of experiment B was + 6.2O/oo. Two equations of isotopic balance can be used to estimate the percentages of cellular sulfur derived from the SO~ and SO~. Let a and b be the percentages of cellular sulfur derived from SO4 and SO 3 respectively. Assume negligible isotopic selectivity during SO~ assimilation. This contributes little error on the basis of previous SO~ reduction experiments with this (McCready et al., 1975b) and other organisms (Kaplan and Rittenberg, 1964). Further, since little SO~ is utilized and the isotopic fractionation is small, there is a negligible "reservoir" effect. In contrast, the isotopic composition of the reservoir SO~ changes drastically because copious H2S is liberated with large isotope effects. This unknown is designated 6SO 3 and is nearly the same for both experiments since SO~ of the same isotopic composition was used and the isotopic patterns for the released H2S were similar. The equations of isotopic balance are: Experiment B a(50.2) + b(aSO;-) = (a + b) 6.2; Experiment A a (3.1) + b(aSO3) = (a + b) ( - 0.7). Subtraction eliminates the term b (aSO~) and since (a + b ) corresponds to the total or 100~ cellular sulfur, we find a = 14.7~o and b = 85.3~. Substitution of a and b into either equation gives SO2 = - 1.3~ This value falls in the range of cellular sulfur derived from SO~ alone ( - 0 . 5 _ 2.5O/oo std. dev., 21 experiments with a variety of carbon sources). The value of + 6.2~ for cellular sulfur of experiment B falls well outside of this range attesting to some SO 2 incorporation in the cells. However, if the intracellular sulfur had been derived solely from SO2 then a a-value near + 50%o should have been found in experiment B. It was initially a surprise that despite the stimulatory effect of SO47 on growth (e.g. generation times; l m M SO~, 85min; l m M S O ~ + 0 . 1 m M SO2, 50min; l m M S O ~ + 10raM SO2, 55min), the labelling experiments show that sulfite provide the majority of intracellular sulfur. This is consistent with some related experiments where the production of amylopectin storage granules was studied. These are normally produced during growth on SO~7 but not SO~ (McCready et al., 1976). In mixtures of SO2
R. G. L. McCready et al. : 348/32S Labelling of Sulfur Metabolism by Clostridium pasteurianum
317
and SO 3, they are absent suggesting the SO~ is inhibiting their synthesis even in the presence of SO4-. All evidence attests to the preferential metabolism of SO2 and we also conclude that growth is stimulated markedly with very little SO2. Using labelling experiments with various isotope compositions for the nutrients, one could derive more equations and the isotopic selectivity during SO4 and SOl incorporation can be treated as unknowns rather than assumed negligible. Experiments can also be carried out with differing sulfate to sulfite ratios. Whereas radioactive 35S would have provided some of the conclusions of this study, there are certain advantages to using stable isotope labelling in terms of storage and quantitative calculations. Compounds of different 3~S/32S abundances can be made from natural samples or by utilizing chemical kinetic isotope effects or exchanges in laboratory experiments. Recent announcements predict that sulfur highly enriched in 34S can be produced by laser separation at low cost 1. This being the case,, biological labelling experiments with stable sulfur isotopes might achieve the prom!nence currently identified with ~SN.
Acknowledgements.This work was supported by National Research Council of Canada Grants A 5058 (E. J. L.), A 8176 (H. R. K.) and the University of Calgary Interdisciplinary Sulphur Research Group (UNISUL). The authors also acknowledge the technical assistance of Miss N. Enojo, Miss J. Pontoy, and Miss E. Bigornia.
1 Laser separation of isotopes: Big Step Science News 107, 284 (1975)
Received March 1, 1976
REFERENCES Kaplan, I. R., Rittenberg, S. C.: Microbiological fractionation of sulfur isotopes. J. gen. Microbiol. 34, 195-212 (1964) McCready, R. G. L., Costerton, J. W., Laishley, E. J. : Morphological modification of cells of Clostridium pasteurianum caused by growth on sulfite. Canad. J. Microbiol. 22, 269-275 (1976) McCready, R. G. L., Kaplan, I. R., Din, G. A.: Fractionation of sulfur isotopes by the yeast Saccharomyees cerevisiae. Geochim. cosmochiflq. Acta 38, 1239-1253 (1974) McCready, R. G. L., Krouse, H. R., Laishley, E. J. : Sulfur isotope effects during growth of Clostridium pasteurianum on mixtures ofsulfite and sulfate. Canad. Fed. bioI. Soc. Proc. 18, 525 (1975 a) McCready, R. G. L., Laishley, E. J., Krouse, H. R. : Stable isotope fractionation by Clostridiumpasteurianum. 1. 34S/32S: inverse isotope effects during SO]- and SO32- reduction. Canad. J. Microbiol. 21, 235-244 (1975b)
