JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1377-1382

Vol. 133, No. 3

0021-9193/78/0133-1377$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Nutritional Studies with Pseudomonas aeruginosa Grown on Inorganic Sulfur Sources LAWRENCE B. SCHOOK AND RICHARD S. BERK* and Microbiology, Wayne State University School of Medicine, Detroit, Immunology Department of

Michigan 48201 Received for publication 8 November 1977

Pseudomonas aeruginosa was grown on a succinate-basal salts medium supplemented with various inorganic sulfur compounds as its sole source of sulfur. The organism was able to grow on the sodium salts of sulfide, thiosulfate, tetrathionate, dithionite, metabisulfite, sulfite, or sulfate, but not on those of dithionate. Analyses of the culture media after 24 h of growth indicated accumulation of sulfate from each inorganic sulfur source except sulfate. Manometric studies with resting cells obtained by growth on each of these sulfur sources yielded net oxygen uptake for all substrates except sulfite and dithionate. Similar results were obtained with extracts from these cells by spectrophotometric techniques. Thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, with little or no activity observed when cells were grown on inorganic sulfur sources of higher oxidative states. Metabisulfite oxidase appeared to be associated with growth on all inorganic sulfur compounds. Rhodanese activity appeared to be constitutively present, and its activity, observed only in soluble fraction, seemed independent of the growth medium employed. Thiosulfate and tetrathionate oxidase activities were studied in greater detail than some of the other sulfur oxidases, and both were found to be distributed between particulate and soluble fractions.

Although extensive work has been done on thiobacilli for utilization of inorganic sulfur autotrophic utilization of reduced inorganic sul- sources (10). fur compounds by the thiobacilli, the utilization MATERIALS AND METHODS of these compounds by heterotrophs has received little attention (10). Particularly imporOrganism. The organism used throughout these tant is the possibility that soil heterotrophs may studies was P. aeruginosa ATCC 17934. The original play a dominant role in the oxidation of reduced culture was stored in 2x skim milk (Difco Laboratosoil sulfur (18). ries, Detroit, Mich.) at -700C in a Revco freezer. Trudinger (14) isolated two heterotrophic bac- Working cultures were grown on tryptose agar (Difco) teria from soil which oxidize thiosulfate to tet- slants and kept at room temperature. Media. The organism was grown in a 1% (wt/vol) rathionate. The enzyme system in one of the sulfur-free sodium succinate-basal salts medium that isolates was constitutive, whereas in the other was singly supplemented with various inorganic sulfur isolate it was induced by either thiosulfate or The basal salts medium contained (per liter): tetrathionate. Work in our laboratory (5) sources. K2HPO4, 0.75 g; KH2PO4, 0.25 g; NH4Cl, 5 g (J. T. showed that a thiosulfate oxidase could be in- Baker Chemical Co.); MgCl2-6H20, 0.5 g; NaCl, 0.5 g; duced by growth of a soil isolate of Alcaligenes FeCl3*6H20, 10 ,ug; CaCl2 * 2H20, 2 Ag. Fisher Scientific sp. on mercaptosuccinate. Resting cells and pur- Co., Fairlawn, N.J., produced all chemicals unless othified thiosulfate oxidase prepared from them erwise noted. After autoclaving, medium was adjusted converted thiosulfate (labeled in the inner sulfur aseptically with either 0.1 N HCl or NaOH to 7.0. The sodium succinate and sulfur sources were atom) to tetrathionate, the only detectable prod- pH filter sterilized by using a 0.22-,um membrane filter uct. (Millipore Corp., Bedford, Mass.) and then added to Because of the paucity of reports on auto- the autoclaved basal salts solution. trophic sulfur metabolism by heterotrophs, the Growth conditions. The organism was inoculated present investigation was initiated to further into the succinate-basal salts solution and incubated characterize the nutritional and metabolic prop- for 18 h at 350C on a New Brunswick shaker. The erties of these organisms. Our results suggest resulting cultures were used either to study initial that Pseudomonas aeruginosa possesses an ex- growth factor requirements or to reinoculate media tensive biochemical system similar to that of the under similar conditions. 1377

1378

SCHOOK AND BERK

Resting cells. For experiments requiring resting cells, the culture was centrifuged at 10,000 x g for 15 min at 4°C. The resulting cell pellet was suspended in the basal salts solution without a carbon and sulfur source and recentrifuged. This cell pellet was suspended in the basal salts solution before use in assay procedures. Chemicals. The following inorganic sulfur sources supplemented the succinate-basal salts medium: Na2S4O6, sodium tetrathionate (K & K Laboratories, Inc., Plainview, N.J.); Na2S 9H20, sodium sulfide (Mallinckrodt Chemical, St. Louis); Na2.%Os, sodium bisulfite (General Chemical, New York, N.Y.); Na2SO3, sodium sulfite; Na2S20s- 2H20, sodium dithionate; Na2S204, sodium hydrosulfite (dithionite); Na2S203 5H20, sodium thiosulfate; and Na2SO4, sodium sufate (Fisher Scientific Co.). Compounds were of the highest available purity. Preparation of cell-free extracts. Cell-free extracts (CFE) were prepared by passage of cell suspensions in 0.1 M phosphate buffer (pH 7.5) through a French pressure cell at 16,000 lb/in2. All cell suspensions were treated twice, and whole cells and debris were removed by centrifugation at 10,000 x g for 10 min before assay. Manometric techniques. Oxygen uptake was measured in a Warburg apparatus (Gilson Medical Electronics, Middleton, Wis.), using standard methods described by Umbreit (17). The manometry flasks contained: 0.2 ml of 20% KOH, 0.2 ml of substrate, 1.0 ml of cells or CFE, and 1.8 ml of 0.1 M phosphate buffer (pH 7.5). Substrate (autoxidation) and endogenous controls were routinely employed in each experiment. Protein determination. The protein concentration of CFE was determined by the microbiuret method of Koch and Putnam (8), using Armour bovine serum albumin as the standard. Sulfate assay. The amount of soluble sulfate concentration during growth of the organism with the various inorganic sulfur compounds, with resting cells or with CFE, was determined by the method of Berglund and Sorbo (3). Standard curves were used to determine the extent of interference these compounds had on the determination of free sulfate. Sulfide assay. Quantitative determinations of free sulfide were made by the method of Siegel (11). The extent of interference by media constituents on the detection of sulfide was also determined. Assay of thiosulfate-oxidizing enzyme. Enzyme activity was assayed in micro-Thunberg tubes by following the reduction of ferricyanide as described by Trudinger (15). The standard assay mixture contained in a total volume 3.0 ml: 3.0,umol of K3Fe(CN)6 (Fisher Scientific Co.), 20 .mol of Na2S2O3, 300 ltmol of potassium phosphate buffer (pH 6.2), and 0.2 ml of CFE. The assay system was mixed, and the reaction was initiated by tipping in the substrate from the upper reservoir of the Thunberg tube. Initial attempts to use this assay system in an aerobic environment were difficult to interpret because of nonenzymatic oxidation of thiosulfate; therefore, before the reaction was initiated, the sealed tubes were gassed with nitrogen (10 alternate cycles of evacuation and gassing using a three-port manifold and an aspirator pump). Ferricyanide reduction was followed at 420 nm in a Beckman

J. BACTERIOL. DB spectrophotometer (10-mm light path), and the activity was recorded as absorbancy change over a 5min period. Controls consisted of a complete assay system minus either the CFE, potassium ferricyanide, or thiosulfate. One unit of enzyme was defined as the amount reducing 1 ,umol of ferricyanide per min under standard assay conditions. Assay of AMP-independent sulfite oxidase. Sulfite oxidase (sulfite:oxygen oxidoreductase, EC 1.8.3.1) was detected by a modification of the method of Charles and Suzuki (4). The enzyme activity was determined by following the rate of reduction of ferricyanide at 420 nm with a Beckman DB spectrophotometer. Standard assay mixtures contained, in a 3-mil volume, 3 ,umol of K3Fe(CN)6, 20 Mmol of Na2SO3, 150 ,umol of tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.6), and 0.2 ml of the CFE preparation. Assays were performed in micro-Thunberg tubes under an atmosphere of nitrogen. One unit of enzyme was defined as that amount reducing 1 ,umol of ferricyanide per min under standard assay conditions. Sulfite oxidase activity was also measured by the method of Johnson and Rajagopalan (7). The enzymatic activity was determined by following the rate of reduction of cytochrome c (horse heart, type VI, Sigma Chemical Co.) at 550 nm using a Beckman DB spectrophotometer. Micro-Thunberg tubes contained 0.25 ml of CFE preparation, 0.04 mM ferricytochrome c, 0.4 mM sodium sulfite, and 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.5) to a final volume of 2.5 ml. One unit of sulfite oxidase activity was defined as the amount of enzyme necessary to produce an absorbance change of 0.13/min, which corresponds to 1 gmmol of cytochrome c reduced per min. Adenosine 5'-phosphosulfate reductase assay. Adenosine 5'-phosphosulfate reductase (EC 1.8.99.2) activity was determined with ferricyanide or cytochrome c as the electron acceptor as described by Adachi and Suzuki (1). These assays were performed in Thunberg tubes under a nitrogen atmosphere to reduce nonspecific oxidation. Rhodanese assay. Rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1.) assays were performed on the CFE from cells grown with the various inorganic and organic sulfur sources by the method of Sorbo (12). One rhodanese unit is defined as the amount of enzyme that forns 10 yeq of thiocyanate (one microequivalent equals 0.104 optical density unit at 460 nm) after 5 mi. Assay for sulfide oxidase, tetrathionate oxidase, dithionite oxidase, metabisultfte oxidase, and sulfate oxidase activity. Enzyme activity was measured as described for the thiosulfate-oxidizing enzyme by following the reduction of ferricyanide at 420 nm under a nitrogen atmosphere with the respective sulfur source. Preparation of cell fractions. The crude CFE (see above) was fractionated at 4°C by differential centrifugation as outlined by Tuovinen et al. (16).

RESULTS Growth studies. Initially, studies were done to determine which inorganic sulfur compounds

GROWTH ON INORGANIC SULFUR SOURCES

VOL. 133, 1978

could be utilized as sources of sulfur for P. aeruginosa. Various inorganic sulfur sources were used to supplement a sulfur-free basal salts solution containing sodium succinate as the sole source of carbon. The sulfur compounds tested were sodium salts of sulfide, thiosulfate, tetrathionate, dithionite, metabisulfite, dithionate, sulfite, and sulfate. Primary cultures first grown with 0.5% sodium sulfate as the sulfur source were harvested, washed twice, and then used to inoculate nephelometer flasks containing the succinate-basal salts medium supplemented with different concentrations of the above inorganic sulfur compounds. Essentially similar results (Fig. 1 and 2) were achieved with each of the aforementioned compounds. Most of these compounds demonstrated an 18-h lag before significant growth occurred at each of the concentrations tested (0.001% to 1.0%). There were two exceptions to this observation: (i) no lag in growth was detected when either tetrathionate or metabisulfite was used as the sulfur source (although metabisulfite was inhibitory at higher concentrations, whereas with tetrathionate, growth was proportional to the amount present) and (ii) dithionate was not able to support growth at any of the tested concentrations even after addition of trace amounts of various vita10

1.0 0.9t 0.8 07

NO2S

0,9

*10%

N02S203

0.8 07

0 001%

i

D 00001 % 06. 0.6 05050.4 -04-

Ec 0

03

v

01

1.00.9

02 0.1

6 12

z

03

2

02

230 36

6

I0 09

N02S406

2 824 30 36

No2S204

(I)~~~~~~~~~~08 0A81 xe~ 08

6

12

8

24 30 36

6

12 824

30 36

HOURS 1. Response of sulfate-grown cells of P. aeruginosa subcultured on different concentrations of sulfide (Na2S), thiosulfate (Na2S203), tetrathionate (Na2S40), or dithionite (Na2S2O). FIG.

1379

E C

0

02

L z 0

0.9

No2SA

Q8

O.r

06

05 04 03 02 0.

1% 6

0.01% % 2e0A 0.1o

6 2 8 2430 36

HOURS FIG. 2. Response of sulfate-grown cells of P. aeruginosa subcultured on different concentrations of metabisulfite (Na2S2Or), sulfite (Na62Oa), or dithionate (Na2S206). The response of tryptone-grown cells subcultured on different concentrations of sulfate (Na2SO4) is also shown.

mins. In addition, supplements of dithionate (final concentrations of 0.001, 0.01, 0.1 and 1%, respectively) to a succinate-thiosulfate-basal salts medium did not inhibit growth. These results suggest that dithionate is not toxic to the cells.- In addition, the inability of resting cells and of CFE to metabolize dithionate also lends support to hypothesis that there is not a specific oxidase for this substrate in P. aeruginosa. Experiments were then carried out on organisms induced to grow on the succinate-basal salts medium by using each compound as a sole sulfur source. Because maximal growth was usually observed at concentrations of the sulfur source between 0.01 and 0.1%, the organism was grown for 18 h with each sulfur source (0.05%), harvested, washed twice, and used to inoculate a medium containing the same sulfur source at 0.05%. In these induced cultures, no lag period was seen. Maximal growth was usually seen with 10 to 12 h, except for sulfide-grown cells, for which maximal growth did not occur until 20 h. Analysis of the supernatant fractions from these induced cultures reveals that the pH of the medium increases from neutrality to pH 9.0

J. BACTERIOL.

SCHOOK AND BERK

1380

as a function of growth for all compounds tested. These supernatant fractions were also assayed for accumulation of extracellular sulfate (Table 1). Where growth occurred, all the inorganic sulfur compounds were oxidized by P. aeruginosa to sulfate in various amounts. Unlike the Alcaligenes sp. previously described (6), this organism was not capable of oxidizing mercaptosuccinate with the formation of sulfate. It seemed that the rate of appearance of extracellular sulfate was not directly related to growth TABLE 1. Accumulation of extracellular sulfate during growth of P. aeruginosa ATCC 17934 Source of sulfur for

Accumulation of extracellular sulfateb (jumol of S042-/ml of growth medium)

graowtha (4 ymol/ml)

O h 8 h 10 h 12 h 36 h 84 h 0 0 0 0 0 1.25 0 0 0 0 1.0 1.0 0 0 0.5 1.0 1.0 0 S20420 0.5 0.5 3.25 4.0 4.0 1.75 1.75 1.75 1.75 1.75 0 S20520 0.75 1.25 1.25 2.0 2.25 So32S0423.75 2.75 2.5 3.0 4.0 4.0 aThe organism was grown for 18 h with each of the sulfur sources, harvested, washed twice, and used to inoculate the same sulfur source. b Values given represent micromoles of S042- per milliliter of growth medium from induced cultures and have been corrected for spontaneous autoxidation.

S2S2032S4062-

rates but varied with the oxidative levels of the substrate. With sulfate-grown celLs, the medium sulfate decreased with cell growth but subse-

quently rose with declining cell growth, possibly indicating autolysis of the organism and liberation of intracellular sulfate. Also, there was no detectable accumulation of sulfide in any of the growth media, nor were any morphological differences seen with the organism, regardless of sulfur source. However, similar studies on resting cells from induced cultures grown on each sulfur source revealed no accumulation of extracellular sulfate during incubation with the individual sulfur sources during an 18-h incubation. Control experiments with resting cells showed that the lack of extracellular sulfate was not due to binding of the anion to the bacterial cell surface. CFE of these cells behaved, however, like growing cells and exhibited oxidation of the various inorganic sulfur compounds to sulfate (Table 2). Osidase activities. Manometric studies on resting cells that were induced to grow on each of the sulfur sources showed net oxygen uptake for each substrate except sulfite and dithionate (Table 3). CFE were prepared by using a French press. The ability of the organism to oxidize different inorganic sulfur sources when grown on individual sulfur compounds was then determined by following the reduction of ferricyanide spectro-

TABLE 2. Accumulation of SO2? from CFE grown on different inorganic sulfur sources S042/mlb Time-(hf Source of sulfur (h for growtha (4 mM) S2S2-' S2OS4062S2O SO:2' 5,;21 1.0 2.5 2.5 0 2.0 2.0 1.5 S21.25 2.0 0 1.5 1.5 2 2.75 2.5

Sime

S2032-

1 2

1.0 1.5

3.75 2.75

1.0 1.0

1.5

1.75

3.0 1.25

3.25 2.75

0 0

1 2

3.0 3.75

2.75 3.25

2.0 3.0

2.5

2.75

2.5 2.75

2.0 3.0

0 0

S2042-

1 2

1.5 2.75

1.75 2.75

1.25 1.75

1.0 1.0

2.5 1.25

2.5 2.0

0 0

S2 52-

1 2

1.0 1.0

0.25 1.5

0 1.0

0 0

0.5 0.5

0.5 1.25

0 0

S032-

1 2

1.0 1.75

2.25 2.0

1.5 1.75

0 0

2.0 1.25

2.0 2.0

0 0

S042-

1 2

1.5 2.5

2.25 2.75

0 0.75

0 0

2.0 1.25

2.0 1.75 (see text).

0 0

S406F-

CFE were prepared from cultures induced to grow on their respective sulfur sources reaction mixture contained 4 mM substrate, basal salts, and the corresponding CFE (2 mg/ml). Values given represent micromoles of S042- per milliliter of reaction mixture and have been corrected for spontaneous autoxidation. 'Substrate (4 mM). a

b The

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VOL. 133, 1978

photometrically. Table 4 shows that the CFE of P. aeruginosa was able to oxidize each substrate except S2062-. It appears that thiosulfate oxidase activity was induced by growth of the organism on sulfur sources that had low oxidative states (sulfide, thiosulfate, and tetrathionate), whereas little or no thiosulfate oxidase activity was observed with P. aerugunosa grown on compounds with higher oxidative states. Also, metabisulfite oxidase activity was associated with growth on all of the inorganic sulfur compounds listed in Table 4. Sulfite-grown cells appeared to have only small amounts of metabisulfite oxidase activity, whereas no sulfite oxidase activity was found when thiosulfate was used as the sulfur source. Other sulfite oxidase assays (see Materials and Methods) using cytochrome c were unable to detect activity in these CFE. The incorporation of molybdenum (Na2MoO4 2H20) into the medium at various concentrations (2.5, 25, and 250 fM) also failed to stimulate sulfite oxidase activity. Rhodanese, apparently involved in the intracellular turnover of reduced sulfur, appeared to be constitutively present, since equal amounts of activity, regardless of the source of sulfur for growth, were observed. Other studies (manuscript in preparation) show that rhodanese activity is also constitutive with regard to the carbon source of growth. We have also examined the oxidase activities

1381

of different cell fractions by differential ultracentrifugation of CFE and by analyzing them for thiosulfate and tetrathionate oxidase activities (Table 5). Thiosulfate oxidase activity was distributed in equal amounts in both the soluble (S108) and particulate fractions (P108), whether induced by growth on thiosulfate or tetrathionate. However, the specific activity of tetrathionate oxidase was higher in the particulate fraction (P108) when induced by thiosulfate or by tetrathionate. Detection of rhodanese activity only in the supernatant (S108) fraction was consistent with previous findings (13). DISCUSSION Although a great deal of work has been done on the metabolism of sulfur amino acids by heterotrophs, little is known about their nutritional requirements and metabolic activities regarding inorganic sulfur compounds. In addition, the control of sulfur oxidation in autotrophic thiobacilli is unknown. The ability of P. aeruginosa to utilize many inorganic sulfur compounds was surprising, yet more significant was the extensive oxidase system for these compounds. At present, it is not known why dithionate did not support the growth of this organism. However, since the CFE were also inactive, one must rule out impermeability as the reason. In regard to thiosulfate, our results are similar

TABLE 3. Manometric studies on resting cells of P. aeruginosa and inorganic sulfur compounds Net nnol of 02 uptake with resting ceilsa 22S S,4 52o~ 2r623.5 0 0 S2_ 6.0 2.83 0 0 0 8.0 7.0 0 3.6 0 0 S2W32_ 0 7.0 10.0 S4062_ 7.65 16.2 0 0 0 0 7.65 5.4 0 0 0 S20420 0 16.2 0 0 S20520 0 so320 0 1.7 0 0 0 0 0 1.2 0.3 SO421.7 0 0 0 a Corrected for endogenous and autoxidation. Represents average of two separate experiments. b Substrate (10 ,umol). Source of sulfur for growth

S2b

S0420 2.0 0 0 2.0 0 2.6

TABLE 4. Oxidase activity of CFE for different inorganic sulfur compounds (P. aeruginosa ATCC 17934) a Source of Sp act (U/mg of protein) sulfur for growth S2(4 l*mol/ml) S40e2- S20( S2W3 &052- So32- S2062- So42- Rhodanese -b S2 12.8 4.6 59.2 46.4 9.6 0 7.2 0.10 19.6 34.6 0 11.7 36.4 0 0 0.09 S2032S4062-

-

11.7

7.0

11.3

21.0

5.8

0

4.9

0.11

0 0 11.3 16.9 18.3 0 0 0.06 S204221.2 23.8 3.2 0 0 2.3 21.0 S20520.09 0 0 1.5 0 5.8 0 0 0.08 S0320 4.9 0 15.6 16.9 0 0 S042 0.12 a One unit of enzyme was defined as the amount reducing 1 ,umol of ferricyanide per min at 420 nm. Enzyme activity was assayed in micro-Thunberg tubes under a nitrogen atmosphere for 5 min. One rhodanese unit was defined as the amount that forms 10 ueq of thiocyanate after 5 mi. b-, Not done.

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J. BACTERIOL.

SCHOOK AND BERK

TABLE 5. Enzyme activities in various ceU fractions of P. aeruginosa ATCC 17934a Enzyme

Cell fraction

Thiosulfate oxidase BC

Sio P1o S108 P108 Tetrathionate oxidase

BC S10

Pio S1os P1os

Sp act (U/mg of protein) Grown Grown on S20,0 l2 on S4 0,;2-

48.0 5.3 22.2 85.3 96.0

NDb 10.3 ND 113.5 120.0

10.66 42.7 18.5 37.3 86.4

6.0 44.6 4.4 43.6 211.2

0.01 0.03 0.0 P1O 0.05 s108 P108 0.0 a BC, Broken cells; 50, supernatant 10,000 x g, 10 min; P,,, pellet 10,000 x g, 10 min; S1o8, supernatant 108,000 x g, 90 min (prepared from Sio); P,(*, pellet 108,000 x g, 90 min. b ND, No activity detected.

Rhodanese

BC S10

0.01 0.04 0.0 0.02 0.0

to those of Trudinger (14), in which his heterotrophic soil isolate could be induced to produce thiosulfate oxidase by either thiosulfate or tetrathionate. He concluded that, because growth was unaffected by increasing amounts of thiosulfate, and because of the occurrence of an alkaline change in pH, the thiosulfate-oxidizing enzyme is probably not involved in detoxification or in the energy metabolism of the bacterium. Like Trudinger, we found that increasing amounts of thiosulfate in the growth medium do not stimulate growth. Trudinger did not study tetrathionate, but in our study it did appear to affect growth, since cell yield was proportional to the amount of tetrathionate present. Other studies (10) have shown that even with thiobacilli many variables can affect the outcome of thiosulfate oxidation. Also, the induction of an extensive inorganic sulfur enzyme system may serve other functions in the cell. Since thiosulfate oxidase activity was found in both particulate and soluble fractions, its role in the overall metabolism of the organism may be multifunctional. The particulate-associated enzyme may be involved in the transfer of electrons through the cytochromes to molecular oxygen and the generation of ATP, or may be source of lowpotential electrons (2, 9). The role of the soluble enzyme may be the same as that postulated for

the particulate fraction or may play a role in the oxidation of sulfur-containing amino acids. Unlike Trudinger (14), we have been able to isolate two fractions containing thiosulfate- and tetrathionate-oxidiing activity. Work to further purify and characterize these enzymes is in progress, and we hope to evaluate their role in heterotrophic sulfur metabolism. ACKNOW LEDG&MENTIS This investigation was supported by Public Health Service general research support grant 5S01-RPR)5384 from the Division of Research Resources, National Institutes of Health.

LITERATURE CITED 1. Adachi, K., and I. Suzuki, 1977. A study on the reaction mechanism of adenosine 5'-phosphosulfate reductase from Thiobacillus thioparus, an iron-ulfur flavoprotein. Can. J. Biochem. 55:91-98. 2. Aminuddin, M., and D. J. D. Nicholas. 1974. An AMPindependent sulfite oxidase from ThiobaciUus denitrifican8: purification and properties. J. Gen. Microbiol. 82:103-113. 3. Berglund, F., and B. H.L Srbo. 1960. Turbidimetric analysis of inorganic sulfate in serum, plama, and urine. Scand. J. Clin. Lab. Invest. 12:147-150. 4. Charles, A. M., and L. Suzuki. 1965. Sulfite oxidase of a facultative autotroph, ThiobaciUus novelus. Biochem. Biophys. Res. Commun. 19:686-690. 5. Hall, ML R., and R. S. Berk. 1972. Thiosulfate oxidase from an Alcaligenes grown on mercaptosuccinate. Can. J. Microbiol. 18:235-245. 6. Hall, M. R., and R. S. Berk. 1968. Microbial growth on mercaptosuccinic acid. Can. J. Microbiol. 14:515-523. 7. Johnson, J. L, and K. V. Rajagopalan. 1976. Purification and properties of sulfite oxidase from human liver. J. Clin. Invest. 58:543-50. 8. 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. 9. Radcliffe, B. C., and D. J. D. Nicholas. 1970. Some properties of a nitrate reductase from Pseudomonas denitrificans. Biochim. Biophys. Acta 205:273-287. 10. Roy, A. B., and P. A. Trudinger. 1970. The biochemistry of inorganic compounds of sulfur. Cambridge University Press, London. 11. Siegel, L M. 1965. A direct microdetermination for sulfide. Anal. Biochem. 11:126-132. 12. S6rbo, B. H. 1960. Rhodanese. Methods Enzymol. 2:334-337. 13. Tabita, R., Silver, M., and D. G. Lundgren. 1969. The rhodanese enzyme of Ferrobacillus ferrooxidans (Thiobacillus ferroxidans). Can. J. Biochem. 47:1141-1145. 14. Trudinger, P. A. 1967. Metabolism of thiosulfate and tetrathionate by heterotrophic bacteria from soil. J.

Bacteriol. 93:550-559.

15. Trudinger, P. A. 1961. Thiosulfate oxidation and cytochromes in ThiobaciUus X. 2. Thiosulfate oxidizing enzyme. Biochem. J. 68:680-686. 16. Tuovinen, 0. H., Kelley, B. C., and D. J. D. Nicholas. 1976. Enzymatic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferroxidans. Can. J. Microbiol. 22:109-113. 17. Umbreit, W. W., R. H. Buris, and F. Stauffer. 1964. Manometric techniques, 4th ed. Burger Publishing Co.,

Minneapolis. 18. Vishniac, W., and M. Santer. 1957. The thiobacilli. Bacteriol. Rev. 21:195-213.