JOURNAL OF BACTERIOLOGY, May 1971, p. 700-701 Vol. 106, No. 2 Copyright ( 1971 American Society for Microbiology Printed in U.S.A. Survey of the Photosynthetic Bacteria for Rhodanese (Thiosulfate: Cyanide Sulfur Transferase) Activity DUANE C. YOCH1 AND E. S. LINDSTROM Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 21 January 1971 Rhodanese activity was demonstrated in extracts from all three taxonomic fami-L: lies of photosynthetic bacteria, and this activity appeared to be uncorrelated witki-. thiosulfate metabolism. Rhodanese (thiosulfate: cyanide sulfur trans- TABLE 1. Rhodanese activity in photosynthetic ferase, EC 2.8.1.1) catalyzes the cleavage of bacteria thiosulfate (equation 1). Specific activity' CN- + S20 2- CNS- + S32- (I) Crude Solu- Chro- ble mato- This enzyme is widely distributed, having been Taxonomic Source of ex- ex- in liver and bacteria tract tract phores detected tissue, plant roots, Taxonomiy Sourac (super- (super- (pel- (4). In photosynthetic bacteria, rhodanese ac- natant natant let tivity was first demonstrated in Chromatium (5) 30,000 105,000105,000 and was later found in Rhodopseudomonas sphe- xg) xg) xg) rubrum Rho- roides and Rhodospirillum (6). Athio- Rhodospirtilum 88 171 9 danese may play a role in thiosulfate oxidation in rhodaceae rubrum Chromatium by cleaving the sulfur-sulfur bond Rhodopseudomonas 68 120 0 of thiosulfate to yield sulfur and sulfite (6). capsulata The rhodanese activity of the three taxonomic R. palustris 185 350 12 families of photosynthetic bacteria was surveyed Thio- Chromatium sp. 107 170 9 to determine if its occurrence is consistent with rhodaceae Ectothiorhodospira 116 117 8 its proposed role in thiosulfate oxidation. mobilis The organisms used in this were cultured study Chloro- Chlorobium 36 55 5 in the appropriate media as described by Bose bacteriaceae thiosulfatophilum (1) with only minor modifications. Extracts were Chloropseudomonas 209 121 187° prepared from a cell paste suspended in 25 mm ethylica tris(hydroxymethyl)aminomethane - hydrochloride a Nanomoles of DPIP reduced per minute per milligram of buffer [pH 7.8 (1:3 w/v)], disrupted by sonic os- protein. The complete mixture contained: Tris buffer (pH 8.7), cillation, and centrifuged as indicated in Ta- 300 jsmoles; sodium thiosulfate, 150 pmoles; half neutralized ble 1. Rhodanese activity was determined by the sodium cyanide (NaCN/HCI, 2.5/1, mole/mole), 80 pmoles; method of Smith and Lascelles (6), which is DPIP, 0.5 pmole; and PMS, 0.25 mg. The protein concentra- tion was 0.1 to 0.4 mg, and water was added to a final volume based on the N-methylphenazonium-methosul- of 3.0 ml. fate (PMS)-mediated reduction of 2, 6-dichloro- °Chromatophores were washed three times with 0.025 M phenol indophenol (DPIP) by sulfite (equation 2) Tris, pH 7.8. which results from the enzymatic cleavage of thiosulfate (equation 1). dopseudomonas palustris was linear with time SO2- + DPIP PMSy Q042 + DPIPH2 (2) for I min and was proportional to added protein up to about 0.4 mg of protein per 3 ml. Except Protein concentration was measured by the Folin for a variation in rates, extracts of the other pho- phenol method (2). tosynthetic bacteria used gave similar results. This reduction of DPIP by extracts of Rho- Table 1 compares the rhodanese activity in the 1 Present address: Department of Cell Physiology, Univer- crude extract with that of the supernatant and sity of California, Berkeley, Calif. 94720. chromatophore fractions from a number of pho- 700 VOL. 106, 1971 NOTES 701 tosynthetic bacteria. All photosynthetic bacteria Siskind, M. S. Thesis, The Pennsylvania State tested had rhodanese activity, suggesting that Univ., 1969), rhodanese may function in cyanide this enzyme is common to all species of this detoxification (4) by Athiorhodaceae. group. However, rhodanese does not appear to be associated with all photosynthetic tissue, as it We thank D. 1. Arnon for the cell material of several strains was not observed in extracts of the blue-green of bacteria and for the assay facilities. Authorized for publica- tion as paper no. 3803 in the journal series of the Pennsylvania alga Anabaena cylindrica or in spinach chloro- Agricultural Experiment Station. plasts (specific activities <1). With one excep- tion, rhodanese was found only in the superna- LITERATURE CITED tant fraction after centrifugation at 105,000 x g 1. Bose, S. K. 1963. Media for anaerobic growth of photosyn- for I hr (Table 1). In Chloropseudomonas ethy- thetic bacteria, p. 501-510. In H. Gest, A. San Pietro, lica extracts, activity was observed in both the and L. P. Vernon (ed.), Bacterial photosynthesis. The soluble and the chromatophore fractions. Antioch Press, Yellow Springs, Ohio. 2. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Washing the chromatophores three times with Randall. 1951. Protein measurement with the Folin 0.025 M Tris buffer did not diminish this activity. phenol reagent. J. Biol. Chem. 193:265-275. The reason for a chromatophore-bound rho- 3. Rolls, J. P., and E. S. Lindstrom. 1967. Induction of a thiosulfate-oxidizing enzyme in Rhodopseudomonas pal- danese in this particular organism is not clear. ustris. J. Bacteriol. 94:784-785. The occurrence of rhodanese activity in photo- 4. Roy, A. B., and R. A. Trudinger. 1970. The biochemistry of synthetic bacteria is apparently not correlated inorganic compounds of sulphur. Cambridge Univ. Press, either with the ability to metabolize thiosulfate Cambridge, England. 5. Smith, A. J. 1964. Sulfur metabolism of or with the route of thiosulfate metabolism. Chromatium strain D and rhodanese activity in extracts. J. Gen. Microbiol. Rhodanese may be involved in thiosulfate metab- 34:1X-X. olism in Chromatium (6), may not be in R. pal- 6. Smith, A. J., and J. Lascelles. 1966. Thiosulfate metabolism ustris (3, 7), and is certainly present in many and rhodanese in Chromatium sp. strain D. J. Gen. Mi- nonthiosulfate-oxidizing A thiorhodaceae crobiol. 42:357-370. (Table 7. Truper, H. G., and H. D. Peck, Jr. 1970. Formation of ad- 1). As growth in a cyanide-containing medium enylyl sulfate in phototrophic bacteria. Arch. Mikrobiol. doubles rhodanese activity in R. palustris (S. 73:125-142..