JOItRNAL o(F BACTERIOI.OGY, (0et. 19719. p). 30;-308 (0)21-9193/9/'/ 10O-0300f/03$02.00,5 )
\ol. 140,
No. 1
Partial Purification and Characterization of Thiosulfate Oxidase from Pseudomonas aeruginosa LAWRENCE B. SCHOOKt ANI) RICHARI) S. BERK* Department of Immunology and Mlicrobiology, W'ayne State UnitlersitY, Detroit, Mlichigan 48201
Received for publication 25 Jtune 1979
Soluble thiosulfate oxidase from Pseudomonas aeruginosa was purified 85-fold and converted thiosulfate to tetrathionate by using either ferricvanide or cytochrome c as an electron acceptor. Extensive literature has accumulated regarding the metabolism of inorganic sulfur com-
pounds by autotrophic organisms (1, 2, 6, 9, 10,
13, 17). However, the relative distribution of sinilar enzymes in heterotrophs has not been extensively studied (3, 4, 19). Recent studies in our laboratorv indicate that Pseudomoi as aeruginosa can grow on a succinate-basal salts medium supplemented with the sodium salts of sulfide, thiosulfate, tetrathionate, sulfite, or sulfate (15). In addition, P. aeruginosa can also use elemental sulfur as its sole sulfur source (unpublished data). Analvses of the culture media after 24 h of growth indicated an accumulation of sulfate from each of the inorganic sulfur sources (other than sulfate). Two enzymes associated with the metabolisnm of thiosulfate were detected in cell extracts and consisted of rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1) and thiosulfate oxidase. Rhodanese activity was found to be constitutively present regardless of the growth medium. However, thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, but not by sulfite or sulfate (15). Because little is known about the functional role and metabolic pathways of inorganic sulfur compounds in heterotrophs, the purpose of this study was to isolate and characterize an inducible thiosulfate oxidase from P. aeruginosa. The thiosulfate oxidase was partially purified by the five-step process described in Table 1. Examination of crude extracts after ultracentrifugation indicated the presence of thiosulfate oxidase and tetrathionate oxidase activity in both the soluble (S8145) and particulate fractions (Pi45). A similar distribution of thiosulfate oxidase between soluble and particulate fractions has been described for Thiobacillus ferr oaxiclans by Tuovinen et al. (19). Attempts to solubilize the particulate activity by techniques employing 2%4 Triton X-100 (8) or I mg of sodium t Present address: Ilst it oe of Berne, Beroe', Sawit /wll
versit v
o,f ( li iIoi t(lld.
ItoIoIIIIologI O .
i
deoxycholate per nmg of protein (12) were unsuccessful. Consequently, all further purification and enzyme characterization studies were performed only on the S145 fractions. Ultrafiltration studies revealed that 7064 of the S.1,-, enzymatic activitv was associated with the molecular weight fraction greater than 300,000, 1864 was associated with the 100,000 to 300,000 range, and 12"- was associated with the 100,000 and below. P'ortions of the ultrafiltration fraction of molecular weight greater than 300,00 were used for the subsequent chromatographic steps. This distribution of enzymatic activity may represent various degrees of aggregation of the enzyme. Stepwise elution of thiosulfate oxidase from I)EAE-A-25 resulted in a series of enzyme peaks near the solvent front and after the initiation of stepwise elution with 0.2 and 0.5 M NaCl. In addition, four tetrathionate oxidase (15) peaks were also detected close to the three thiosulfate oxidase peaks. The pooled peak of thiosulfate oxidase obtained after elution with 0.2 M NaCl was chromatographed on Sephacrvl S-200, resulting in an enzyme peak free of tetrathionate oxidase and rhodanese activity (16). The incubation of post-Sephacryl S-200 thiosulfate oxidase fraction with thiosulfate at 30°C resulted in tetrathionate as the only detectable product when chromatographed by the ascending techniques of Rov and Trudinger (14). Paper chromatography revealed that the relative amount of tetrathionate increased with time. The S-200 fraction exhibited activity from pH 4.5 to 9.0 with optimal activity between pH 5.5 and 6.5 (determined by using citrate-phosphate, potassium phosphate, and Tris-hydrochloride [final ionic strength, 0.05]). Enzymatic activity declined rapidly when stored at 4°C with only 5064 activity after 3 days and 15%7 remaining after 14 days. The optimal temperature for activity was determined to be 30 to 42°C (MicroThunberg tubes were preincubated for 5 min before addition of substrate and then further incubated during the 5-min reaction period). Kinetic experiments, using ferricyanide as elec-
307
NOTES
VOL. 140, 1979
TABLE 1. Summary of partial purification of thiosulfate oxidase from P. aeruginosa" Protein (mg/ Fold purificaTotal U' Yield M Step Vol (ml) Sp act (U/mg)
Mno
tion
1.0 9,750 18.5 100 Crude CFE 75 7.0 Ultracentrifugation 1.1 8,250 20.0 85 S145 75 5.5 0.6 756 10.8 8 10 7.0 P145 Ultrafiltration 2.3 5,775 42.0 59 XM300 retentate 13.75 10.0 0.6 1,460 11.4 15 XM100A retentate 15 8.5 0.4 950 7.5 10 23 5.5 XM1OOA filtrate 4,385 227.0 45 12.3 DEAE-Sephadex A-25 25 0.8 950 1,575.0 10 85.2 Sephacryl S-200 5 0.12 a P. aeruginosa were grown as previously described (15) by using 0.1% sodium thiosulfate as the sole sulfur source. All fractionation steps were performed at 4°C with reduction in volume of fractions accomplished using Aquacide II. CFE were prepared by passage of cell suspensions through a French pressure cell at 16,000 lb/in2 (15), centrifuged at 35,000 rpm (145,000 x g) for 1 h, and designated S145 or P14r,. The S145 fraction was enzymatically treated for 60 min at 4°C for removal of nucleic acids using 5 jg of DNase I and RNase (both bovine pancreas) per ml. The S145 fraction was added to a Diaflo ultrafiltration cell fitted with either an XM1OOA or XM-300 membrane and washed with 5 volumes of 50 mM potassium phosphate buffer (pH 7.0) under 10 to 15 lb/in2 of nitrogen. The concentrated preparation was adsorbed to a column (2.5 by 25 cm) of DEAE-Sephadex A-25 equilibrated with 100 mM Tris-hydrochloride buffer (pH 7.6). After 30 min of equilibration, the column was washed with Tris-hydrochloride buffer until no more unadsorbed protein could be eluted. Subsequent elutions were performed by using a stepwise elution with NaCl of 0.2, 0.5, and 0.8 M in Tris-hydrochloride buffer. Peak enzymatic fractions were pooled, concentrated, and dialyzed overnight against 100 mM phosphate buffer (pH 6.2). The concentrated post-DEAE fraction (0.2 M NaCl elution peak) was then chromatographed on a column (2.5 by 90 cm) of Sephacryl S-200 equilibrated with 100 mM phosphate buffer (pH 6.2). b Determined by the microbiuret method of Koch and Putnam (5). 'Enzyme activity (20 to 30 jig of protein) was assayed in micro-Thunberg tubes by following the reduction of ferricyanide at 420 nm as described by Trudinger (17) and Schook and Berk (15).
TABLE 2. Effect of various inhibitors on thiosulfate a Km value for thiooxidase actiVitya sulfate and ferricyanide of 6.7 x 10-4 and 1.1 x 10-3 M, respectively. Tetrathionate, dithionate, Final Activity (% of concn Inhibitor sulfite, and sulfate did not yield a reduction of control) (mM) ferricyanide with the S-200 enzyme fraction. In addition to potassium ferricyanide (420 nm), Dithionate .1.0 82 0.1 85 horse heart cytochrome c (550 nm) could couple 0.01 85 the thiosulfate oxidase reaction. Substitution of 15 1.0 2,6-dichlorophenol-indophenol (600 nm), meth- Sulfite 52 0.1 ylene blue (688 nm), flavin adenine dinucleotide 67 0.01 (450 nm), NAD (340 nm), and NADP (340 nm) Sulfate 67 1.0 as the electron acceptor did not result in the 75 0.1 specific reduction of these compounds during 0.01 93 thiosulfate oxidation. 0 1.0 Mercuric chloride 63 0.1 The effect of various inhibitors on enzyme 87 0.01 activity is shown in Table 2. Inhibitors affecting 53 1.0 sulfhydryl groups, such as mercuric chloride, p- p-Chloromercuribenzoate 93 0.1 chloromercuribenzoate, and N-ethylmaleimide, 0.01 100 significantly lowered activity. Chelators of met60 1.0 als had no effects on thiosulfate oxidase activity N-Ethylmaleimide 75 0.1 and, conversely, the addition of cations such as 0.01 100 Fe3+, Mg2+, Mn2+, or Ca2+ at concentrations be- EDTA 87 1.0 tween 10-3 and 10' M had no effect on enzyme 97 0.1 100 0.01 activity. 100 The properties of the enzyme with regards to Control substrate specificity and susceptibility to inhibNo inhibition of activity was detected with a final itors are quite similar to the thiosulfate oxidase concentration of 0.01, 0.1, and 1 mM of tetrathionate, of both Alcaligenes species (4) and the auto- potassium cyanide, sodium azide, hydroxylamine, iotrophs Thiobacillus novellus and T. thioparus doacetate, 2,2'-dipyridyl and diethyldithiocarbamate, (2, 6). In addition, the ability of our enzyme from respectively. tron acceptor, established
a
308
J. BACTERIOL.
NOTES
P. aeruginosa to couple with mammalian cytochrome c suggests a relationship of this enzyme to the membrane electron transport chain. This hypothesis is supported in part by Trudinger's findings that endogenous cytochromes of the ctype were reduced when thiosulfate was added to extracts of induced but not uninduced heterotrophic bacteria (18). Recent studies in our laboratory indicate that P. aeruginosa can convert sulfide, thiosulfate, or tetrathionate to sulfate (15) and suggest an inorganic sulfur pathway from sulfide to sulfate via polythionates as previously described in certain thiobacilli (7). On the basis of the present studies, it would appear then that thiosulfate is converted to sulfate via tetrathionate as an intermediate. Though little is known about inorganic sulfur metabolism in heterotrophs, previous results (15) suggest that sulfate may be the preferred sulfur compound for assimilation into organic cellular components. In addition, Pardee et al. have studied binding and transport of sulfate, and their results suggest that certain bacteria possess highly specific binding sites for sulfate on the cell surface (11). LITERATURE CITED 1. Charles, A. M., and I. Suzuki. 1965. Sulfite oxidase of a facultative autotroph, Thiobacillus nouellus. Biochem. Biophys. Res. Commun. 19:686-690. 2. Charles, A. M., and I. Suzuki. 1966. Mechanisnm of
thiosulfate oxidation by Thiobacillus nouellus. Biochim. Biophys. Acta 123:510-521. 3. Hall, M. R., and R. S. Berk. 1968. Microbial growth on mercaptosuccinic acid. Can. J. Microbiol. 14:515-523. 4. Hall, M. R., and R. S. Berk. 1972. Thiosulfate oxidase from an Alcaligenes grown on mercaptosuccinate. Can. J. Microbiol. 18:235-245. 5. Koch, A. L., and S. L. Putnam. 1971. Sensitive biuret method for determination of protein in an impure system such as whole bacteria. Anal. Biochem. 44:239-245.
6. Lyric, R. M., and I. Suzuki. 1970. Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. III. Properties of thiosulfate-oxidizing enzyme and proposed pathway of thiosulfate oxidation. Can. J. Biochem. 48:355-363. 7. London, J., and S. C. Rittenberg. 1964. Path of sulfur in sulfide and thiosulfate oxidation bv thiobacilli. Proc. Natl. Acad. Sci. U.S.A. 52:1183-1190. 8. MacGregor, C. J. 1975. Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease. J. Bacteriol. 121:1102-1110. 9. Oh, J. K., and I. Suzuki. 1977. Isolation and characterization of a membrane-associated thiosulfate oxidizing system of Thiobacillus notellus. J. Gen. Microbiol. 99: 397-412. 1(). Oh, J. K., and I. Suzuki. 1977. Resolution of a membrane-associated thiosulfate-oxidizing complex of Thiobacillus notellus. J. Gen. Microbiol. 99:413-423. 11. Pardee, A. B., L. S. Prestidge, M. B. Whipple, and J. Dreyfuss. 1966. A binding site for sulfate and its relation to sulfate transport into Salmonella typhimurium. J. Biol. Chem. 241:3962-3969. 12. Radcliffe, B. C., and D. J. D. Nicholas. 1970. Some properties of a nitrate reductase from Pseudomonas dentrificans. Biochim. Biophys. Acta 205:273-287. 13. Ross, A. J., R. L. Schoenhoff, and M. I. H. Aleem. 1968. Electron transport and coupled phosphorylation in the chemoautotroph, Thiobacillus neopolitanus. Biochem. Biophys. Res. Commun. 32:301-306. 14. Roy, A. B., and P. A. Trudinger. 1970. The biochemistry of inorganic compounds of sulfur. Cambridge University Press, London. 15. Schook, L. B., and R. S. Berk. 1978. Nutritional studies with Pseudomonas aeruginosa grown on inorganic sulfur sources. J. Bacteriol. 133:1377-1382. 16. Sorbo, B. H. 1960. Rhodanese. Methods Enzymol. 11: 334-337. 17. Trudinger, P. A. 1961. Thiosulfate oxidation and cytochromes in Thiobacillus X. 2. Thiosulfate oxidizing enzyme. Biochem. J. 68:680-686. 18. Trudinger, P. A. 1967. Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria in soil. J. Bacteriol. 93:550-559. 19. Tuovinen, 0. H., B. C. Kelley, and D. J. D. Nicholas. 1976. Enzymatic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobac/llus ferroxidans. Can. J. Microbiol. 22:109-113.
\ol. 140,
No. 1
Partial Purification and Characterization of Thiosulfate Oxidase from Pseudomonas aeruginosa LAWRENCE B. SCHOOKt ANI) RICHARI) S. BERK* Department of Immunology and Mlicrobiology, W'ayne State UnitlersitY, Detroit, Mlichigan 48201
Received for publication 25 Jtune 1979
Soluble thiosulfate oxidase from Pseudomonas aeruginosa was purified 85-fold and converted thiosulfate to tetrathionate by using either ferricvanide or cytochrome c as an electron acceptor. Extensive literature has accumulated regarding the metabolism of inorganic sulfur com-
pounds by autotrophic organisms (1, 2, 6, 9, 10,
13, 17). However, the relative distribution of sinilar enzymes in heterotrophs has not been extensively studied (3, 4, 19). Recent studies in our laboratorv indicate that Pseudomoi as aeruginosa can grow on a succinate-basal salts medium supplemented with the sodium salts of sulfide, thiosulfate, tetrathionate, sulfite, or sulfate (15). In addition, P. aeruginosa can also use elemental sulfur as its sole sulfur source (unpublished data). Analvses of the culture media after 24 h of growth indicated an accumulation of sulfate from each of the inorganic sulfur sources (other than sulfate). Two enzymes associated with the metabolisnm of thiosulfate were detected in cell extracts and consisted of rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1) and thiosulfate oxidase. Rhodanese activity was found to be constitutively present regardless of the growth medium. However, thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, but not by sulfite or sulfate (15). Because little is known about the functional role and metabolic pathways of inorganic sulfur compounds in heterotrophs, the purpose of this study was to isolate and characterize an inducible thiosulfate oxidase from P. aeruginosa. The thiosulfate oxidase was partially purified by the five-step process described in Table 1. Examination of crude extracts after ultracentrifugation indicated the presence of thiosulfate oxidase and tetrathionate oxidase activity in both the soluble (S8145) and particulate fractions (Pi45). A similar distribution of thiosulfate oxidase between soluble and particulate fractions has been described for Thiobacillus ferr oaxiclans by Tuovinen et al. (19). Attempts to solubilize the particulate activity by techniques employing 2%4 Triton X-100 (8) or I mg of sodium t Present address: Ilst it oe of Berne, Beroe', Sawit /wll
versit v
o,f ( li iIoi t(lld.
ItoIoIIIIologI O .
i
deoxycholate per nmg of protein (12) were unsuccessful. Consequently, all further purification and enzyme characterization studies were performed only on the S145 fractions. Ultrafiltration studies revealed that 7064 of the S.1,-, enzymatic activitv was associated with the molecular weight fraction greater than 300,000, 1864 was associated with the 100,000 to 300,000 range, and 12"- was associated with the 100,000 and below. P'ortions of the ultrafiltration fraction of molecular weight greater than 300,00 were used for the subsequent chromatographic steps. This distribution of enzymatic activity may represent various degrees of aggregation of the enzyme. Stepwise elution of thiosulfate oxidase from I)EAE-A-25 resulted in a series of enzyme peaks near the solvent front and after the initiation of stepwise elution with 0.2 and 0.5 M NaCl. In addition, four tetrathionate oxidase (15) peaks were also detected close to the three thiosulfate oxidase peaks. The pooled peak of thiosulfate oxidase obtained after elution with 0.2 M NaCl was chromatographed on Sephacrvl S-200, resulting in an enzyme peak free of tetrathionate oxidase and rhodanese activity (16). The incubation of post-Sephacryl S-200 thiosulfate oxidase fraction with thiosulfate at 30°C resulted in tetrathionate as the only detectable product when chromatographed by the ascending techniques of Rov and Trudinger (14). Paper chromatography revealed that the relative amount of tetrathionate increased with time. The S-200 fraction exhibited activity from pH 4.5 to 9.0 with optimal activity between pH 5.5 and 6.5 (determined by using citrate-phosphate, potassium phosphate, and Tris-hydrochloride [final ionic strength, 0.05]). Enzymatic activity declined rapidly when stored at 4°C with only 5064 activity after 3 days and 15%7 remaining after 14 days. The optimal temperature for activity was determined to be 30 to 42°C (MicroThunberg tubes were preincubated for 5 min before addition of substrate and then further incubated during the 5-min reaction period). Kinetic experiments, using ferricyanide as elec-
307
NOTES
VOL. 140, 1979
TABLE 1. Summary of partial purification of thiosulfate oxidase from P. aeruginosa" Protein (mg/ Fold purificaTotal U' Yield M Step Vol (ml) Sp act (U/mg)
Mno
tion
1.0 9,750 18.5 100 Crude CFE 75 7.0 Ultracentrifugation 1.1 8,250 20.0 85 S145 75 5.5 0.6 756 10.8 8 10 7.0 P145 Ultrafiltration 2.3 5,775 42.0 59 XM300 retentate 13.75 10.0 0.6 1,460 11.4 15 XM100A retentate 15 8.5 0.4 950 7.5 10 23 5.5 XM1OOA filtrate 4,385 227.0 45 12.3 DEAE-Sephadex A-25 25 0.8 950 1,575.0 10 85.2 Sephacryl S-200 5 0.12 a P. aeruginosa were grown as previously described (15) by using 0.1% sodium thiosulfate as the sole sulfur source. All fractionation steps were performed at 4°C with reduction in volume of fractions accomplished using Aquacide II. CFE were prepared by passage of cell suspensions through a French pressure cell at 16,000 lb/in2 (15), centrifuged at 35,000 rpm (145,000 x g) for 1 h, and designated S145 or P14r,. The S145 fraction was enzymatically treated for 60 min at 4°C for removal of nucleic acids using 5 jg of DNase I and RNase (both bovine pancreas) per ml. The S145 fraction was added to a Diaflo ultrafiltration cell fitted with either an XM1OOA or XM-300 membrane and washed with 5 volumes of 50 mM potassium phosphate buffer (pH 7.0) under 10 to 15 lb/in2 of nitrogen. The concentrated preparation was adsorbed to a column (2.5 by 25 cm) of DEAE-Sephadex A-25 equilibrated with 100 mM Tris-hydrochloride buffer (pH 7.6). After 30 min of equilibration, the column was washed with Tris-hydrochloride buffer until no more unadsorbed protein could be eluted. Subsequent elutions were performed by using a stepwise elution with NaCl of 0.2, 0.5, and 0.8 M in Tris-hydrochloride buffer. Peak enzymatic fractions were pooled, concentrated, and dialyzed overnight against 100 mM phosphate buffer (pH 6.2). The concentrated post-DEAE fraction (0.2 M NaCl elution peak) was then chromatographed on a column (2.5 by 90 cm) of Sephacryl S-200 equilibrated with 100 mM phosphate buffer (pH 6.2). b Determined by the microbiuret method of Koch and Putnam (5). 'Enzyme activity (20 to 30 jig of protein) was assayed in micro-Thunberg tubes by following the reduction of ferricyanide at 420 nm as described by Trudinger (17) and Schook and Berk (15).
TABLE 2. Effect of various inhibitors on thiosulfate a Km value for thiooxidase actiVitya sulfate and ferricyanide of 6.7 x 10-4 and 1.1 x 10-3 M, respectively. Tetrathionate, dithionate, Final Activity (% of concn Inhibitor sulfite, and sulfate did not yield a reduction of control) (mM) ferricyanide with the S-200 enzyme fraction. In addition to potassium ferricyanide (420 nm), Dithionate .1.0 82 0.1 85 horse heart cytochrome c (550 nm) could couple 0.01 85 the thiosulfate oxidase reaction. Substitution of 15 1.0 2,6-dichlorophenol-indophenol (600 nm), meth- Sulfite 52 0.1 ylene blue (688 nm), flavin adenine dinucleotide 67 0.01 (450 nm), NAD (340 nm), and NADP (340 nm) Sulfate 67 1.0 as the electron acceptor did not result in the 75 0.1 specific reduction of these compounds during 0.01 93 thiosulfate oxidation. 0 1.0 Mercuric chloride 63 0.1 The effect of various inhibitors on enzyme 87 0.01 activity is shown in Table 2. Inhibitors affecting 53 1.0 sulfhydryl groups, such as mercuric chloride, p- p-Chloromercuribenzoate 93 0.1 chloromercuribenzoate, and N-ethylmaleimide, 0.01 100 significantly lowered activity. Chelators of met60 1.0 als had no effects on thiosulfate oxidase activity N-Ethylmaleimide 75 0.1 and, conversely, the addition of cations such as 0.01 100 Fe3+, Mg2+, Mn2+, or Ca2+ at concentrations be- EDTA 87 1.0 tween 10-3 and 10' M had no effect on enzyme 97 0.1 100 0.01 activity. 100 The properties of the enzyme with regards to Control substrate specificity and susceptibility to inhibNo inhibition of activity was detected with a final itors are quite similar to the thiosulfate oxidase concentration of 0.01, 0.1, and 1 mM of tetrathionate, of both Alcaligenes species (4) and the auto- potassium cyanide, sodium azide, hydroxylamine, iotrophs Thiobacillus novellus and T. thioparus doacetate, 2,2'-dipyridyl and diethyldithiocarbamate, (2, 6). In addition, the ability of our enzyme from respectively. tron acceptor, established
a
308
J. BACTERIOL.
NOTES
P. aeruginosa to couple with mammalian cytochrome c suggests a relationship of this enzyme to the membrane electron transport chain. This hypothesis is supported in part by Trudinger's findings that endogenous cytochromes of the ctype were reduced when thiosulfate was added to extracts of induced but not uninduced heterotrophic bacteria (18). Recent studies in our laboratory indicate that P. aeruginosa can convert sulfide, thiosulfate, or tetrathionate to sulfate (15) and suggest an inorganic sulfur pathway from sulfide to sulfate via polythionates as previously described in certain thiobacilli (7). On the basis of the present studies, it would appear then that thiosulfate is converted to sulfate via tetrathionate as an intermediate. Though little is known about inorganic sulfur metabolism in heterotrophs, previous results (15) suggest that sulfate may be the preferred sulfur compound for assimilation into organic cellular components. In addition, Pardee et al. have studied binding and transport of sulfate, and their results suggest that certain bacteria possess highly specific binding sites for sulfate on the cell surface (11). LITERATURE CITED 1. Charles, A. M., and I. Suzuki. 1965. Sulfite oxidase of a facultative autotroph, Thiobacillus nouellus. Biochem. Biophys. Res. Commun. 19:686-690. 2. Charles, A. M., and I. Suzuki. 1966. Mechanisnm of
thiosulfate oxidation by Thiobacillus nouellus. Biochim. Biophys. Acta 123:510-521. 3. Hall, M. R., and R. S. Berk. 1968. Microbial growth on mercaptosuccinic acid. Can. J. Microbiol. 14:515-523. 4. Hall, M. R., and R. S. Berk. 1972. Thiosulfate oxidase from an Alcaligenes grown on mercaptosuccinate. Can. J. Microbiol. 18:235-245. 5. Koch, A. L., and S. L. Putnam. 1971. Sensitive biuret method for determination of protein in an impure system such as whole bacteria. Anal. Biochem. 44:239-245.
6. Lyric, R. M., and I. Suzuki. 1970. Enzymes involved in the metabolism of thiosulfate by Thiobacillus thioparus. III. Properties of thiosulfate-oxidizing enzyme and proposed pathway of thiosulfate oxidation. Can. J. Biochem. 48:355-363. 7. London, J., and S. C. Rittenberg. 1964. Path of sulfur in sulfide and thiosulfate oxidation bv thiobacilli. Proc. Natl. Acad. Sci. U.S.A. 52:1183-1190. 8. MacGregor, C. J. 1975. Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease. J. Bacteriol. 121:1102-1110. 9. Oh, J. K., and I. Suzuki. 1977. Isolation and characterization of a membrane-associated thiosulfate oxidizing system of Thiobacillus notellus. J. Gen. Microbiol. 99: 397-412. 1(). Oh, J. K., and I. Suzuki. 1977. Resolution of a membrane-associated thiosulfate-oxidizing complex of Thiobacillus notellus. J. Gen. Microbiol. 99:413-423. 11. Pardee, A. B., L. S. Prestidge, M. B. Whipple, and J. Dreyfuss. 1966. A binding site for sulfate and its relation to sulfate transport into Salmonella typhimurium. J. Biol. Chem. 241:3962-3969. 12. Radcliffe, B. C., and D. J. D. Nicholas. 1970. Some properties of a nitrate reductase from Pseudomonas dentrificans. Biochim. Biophys. Acta 205:273-287. 13. Ross, A. J., R. L. Schoenhoff, and M. I. H. Aleem. 1968. Electron transport and coupled phosphorylation in the chemoautotroph, Thiobacillus neopolitanus. Biochem. Biophys. Res. Commun. 32:301-306. 14. Roy, A. B., and P. A. Trudinger. 1970. The biochemistry of inorganic compounds of sulfur. Cambridge University Press, London. 15. Schook, L. B., and R. S. Berk. 1978. Nutritional studies with Pseudomonas aeruginosa grown on inorganic sulfur sources. J. Bacteriol. 133:1377-1382. 16. Sorbo, B. H. 1960. Rhodanese. Methods Enzymol. 11: 334-337. 17. Trudinger, P. A. 1961. Thiosulfate oxidation and cytochromes in Thiobacillus X. 2. Thiosulfate oxidizing enzyme. Biochem. J. 68:680-686. 18. Trudinger, P. A. 1967. Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria in soil. J. Bacteriol. 93:550-559. 19. Tuovinen, 0. H., B. C. Kelley, and D. J. D. Nicholas. 1976. Enzymatic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobac/llus ferroxidans. Can. J. Microbiol. 22:109-113.
