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Journal of General Microbiology (1972)~ 70,555-566 Printed in Great Britain 555 Physiology of a New Facultatively Autotrophic Thermophilic Thiobacillus By R. A . D. W I L L I A M S * A N D T H E L A T E D. s. H O A R E Department of Microbiology, The University of Texas, Austin, Texas 78712 , U.S.A. (Acceptedfor publication 2I December I 971) SUMMARY A new thermophilic thiobacillus (G+ C = 66.2 mol%) has been isolated in pure culture. The temperature optimum for growth was 50'. Heterotrophic growth occurred on nutrient broth, but not on single organic compounds. No a-ketoglutarate dehydrogenase was present but unrestricted acetate incorporation took place via the glyoxylate cycle. INTRODUCTION Thiobacilli should be abundant in areas where reduced inorganic sulphur compounds are found. Such areas have been called ' Sulphataras'. Hot springs are frequently associated with sulphataras and might be expected to support the growth of thermophilic thiobacilli (Sokolova & Karavaiko, I 968). A unique spore-forming thiobacillus was isolated by Soviet investigators and was named Thiobacillus thermophilica (Egorova & Deryugina, I 963). Although there have been some other investigations of thiobacilli from hot springs (Zavarzin & Zhilina, 1964), there have been no other studies of pure cultures of thermophilic thiobacilli. Brierley (1966) studied the thiobacilli in some of the Hot Springs of Yellowstone National Park and isolated another spore-forming thiobacillus but made no physiological studies and the organism is no longer available. We therefore set out to enrich and to isolate thermophilic thiobacilli from hot spring waters in Yellowstone National Park. This paper reports the isolation and study of a new thermophilic non-spore-forming strain. METHODS Media. Autotrophic medium contained (% w/v) : Na2S20,.5H20, 0.5 ; NH,CI, 0.1; MgS04.7Hz0, 0.08 ; KH2P04,0.4; NaOH, 0.1; bromothymol blue, 0.002 and I ml/Ioo ml of a trace metal solution (Vishniac & Santer, 1957) at pH 7.0. The MgS0,.7Hz0 and trace metals were sterilized separately. Elemental sulphur, sterilized by intermittent steaming, was also used as an energy source in place of thiosulphate. In some experiments the medium, containing an appropriate indicator in place of bromothymol blue, was adjusted to different pH values with hydrochloric acid. Plates of thiosulphate medium contained 1.5 yo agar in addition to the above components. Both plates and liquid media were supplemented, when necessary, with 0.1yo (w/v) yeast agar or various organic compounds. Autotrophic medium supplemented with vitamins and bases contained (mg/Ioo ml) adenine, guanine, thymine, cytosine and uracil, I ; Ca pantothenate, I ;pyridoxamine, 0.4; thiamine, riboflavine and nicotinamide, 0.2 ; and biotin, 0.001. Ability to grow anaerobically with nitrate was tested in completely filled tubes containing * Present address : Department of Biochemistry, The London Hospital Medical College, Turner Street, London E I 2 A D . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 556 R. A. D. W I L L I A M S A N D D. S. H O A R E (yow/v) : Na2S203.5H20, 0.5 ; NH4Cl, 0.1; MgS04.7H20, 0.08; K2HP04, 0.2 ; KN03, FeSO,. 7H20, 0.002; NaHCO,, 092; and I ml/Ioo ml of a trace metal solution (Vishniac & Santer, 1957) at pH 7.0. Isolation of the organism. Water specimens from Yellowstone National Park were kindly provided by T. D. Brock in August, 1968. The samples were well shaken and 5 ml of each was inoculated into 25 ml sulphur medium, pH 7.0 and incubated at 50' for 7 days. Of five enrichments, three showed distinct growth of similar Gram-negative non-motile, non-sporeforming rods. The organism described here was isolated from the enrichment culture of a water sample obtained from just above the experimental channel at Nymph Creek (field temperature 38.2", field pH 2.7). Enrichment cultures incubated at 60" did not grow in 7 days. All six subsequent transfers were carried out in thiosulphate medium in which the growth of the organism was more readily observed. Subcultures were made every 3 to 4 days, the final pH being 2-7 to 3-2 and the last liquid culture was streaked out on nutrient agar and thiosulphate agar, Small translucent colonies grew on the autotrophic growth medium, and more yellowish colonies of similar appearance grew on the nutrient agar plates. Single colonies from either medium grew on the other. Well-isolated colonies were picked from the autotrophic plates under a binocular microscope, streaked on fresh thiosulphate plates and incubated at 50° for 3 days. This procedure was repeated five times and single colonies from the final plate were transferred into liquid thiosulphate medium. After 4 days of incubation the culture was slightly turbid, contained strands of aggregated bacteria, and was acid (pH 2.0). This culture contained short rods in which some dark areas were visible under phase contrast. Most had slightly curved long sides and rounded ends, and were sometimes in pairs like diplococci, but a few were long filaments. No growth occurred anaerobically in nitratethiosulphate medium and no nitrite was produced. Colonies grew on nutrient agar or on thiosulphate agar in 3 days at 50". After some preliminary experiments the culture was streaked on thiosulphate agar and single colonies transferred on the autotrophic medium four more times. The organism was maintained on plates of thiosulphate agar (pH 6.0) at 50' and transferred every 3 to 5 days. Growth experiments. Growth was studied in 125 ml Erlenmeyer flasks with side arms and containing 25 ml medium. The inoculum was usually I % (v/v). Cultures were incubated at 50" in a rotary shaking incubator (Lab-line Instruments Inc., Melrose Park, Illinois, U S A . ) and growth was followed turbidimetrically using a Klett-Summerson colorimeter with a red filter (transmission 640 to 700 nm). This method of estimating growth is prone to errors due to variation in cell size and shape and the organism described here tended to grow in strands, which complicated the determination of turbidity. To reduce such errors, duplicate and, wherever possible, triplicate flasks were used. The results quoted are the means of such multiple determinations. Temperature range experiments were conducted using unstirred flasks in water baths at temperatures between 30" and 60". In pellets harvested from different media to determine the yield and in suspensions used for Warburg experiments, protein was determined by the method of Lowry, Rosebrough, Farr & Randall (1951) after hydrolysis for I h with 0.14wsodium hydroxide at 100". Spent medium was examined for thiosulphate and polythionates by chromatography (Trudinger, 1965) and the assay method of Sorbo (1957). The method of Lu (1939) was used to detect ketoacids. For experiments with bacterial suspensions and preparation of extracts 10x 2 1flasks each containing 500 ml of thiosulphate medium (pH 6.0) were shaken at 50" for 3 to 4 days. In the case of mixotrophic cultures two days' growth was used. The bacteria were harvested by 0.2; Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 Thermophilic thiobacillus 557 centrifugation, washed three times in 0-01M-phosphate buffer, pH 7.0, and finally resuspended in the same buffer and used immediately, or stored at - 20’ for preparation of extracts. Preparation ofcell-freeextracts. Bacteria(o.7 to I mogwet wt) suspended ino-orgmpotassium phosphate buffer pH 7.0 (5 ml) were disrupted, in a beaker surrounded with crushed ice, by 5 min treatment with a sonic disintegrator (M.S.E. Ltd, London). Whole bacteria and debris were removed by 10 min centrifugation at 10000 g and the ‘particulate’ (130000g pellet) fraction was resuspended by homogenizing in 5 ml of 0-01M-phosphate buffer, pH 7.0. Spectrophotometric methods. Spectrophotometric assays were carried out using a Gilford 2000 Multiple Absorbance Recorder with a Beckman DU- I spectrophotometer. Succinic dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) oxidase were assayed in the particulate fraction. NADH-cytochrome c reductase was assayed in both soluble and particulate fractions. All other enzymes were assayed in the soluble fraction. A model 14 Cary spectrophotometer was used to obtain difference spectra of the soluble ‘c’ cytochrome. Sulphite oxidase and rhodanese were assayed by the spectrophotometric methods described by Taylor & Hoare (1969) using both potassium ferricyanide and cytochrome c as electron acceptors. Thiosulphate oxidase was measured by replacing the sodium sulphite of the sulphite oxidase assay with sodium thiosulphate. Rhodanese was also assayed by the colorimetric method of Bowen, Butler & Happold (1965) for determining the temperature optimum. For the assay of formic dehydrogenase, cuvettes contained in I ml (pmol): sodium phosphate, pH 7.0, 50; nicotinamide adenine dinucleotide (NAD), 0.1 ; K,Fe(CN),, I ; sodium formate, 10. The following enzymes were also assayed spectrophotometrically using standard assay methods modified where so described: citrate synthase (Srere & Kosicki, 1961); aconitase and NADH oxidase (Smith & Hoare, 1968); isocitrate dehydrogenase (Kornberg, 1955); a-ketoglutarate dehydrogenase (Kaufman, I 955) (modified to include I pmol/ml of MgClz and 0.2 pmollml of thiamine pyrophosphate) ; pyruvate dehydrogenase was measured by the method for a-ketoglutarate dehydrogenase substituting sodium pyruvate as the substrate; succinate dehydrogenase (Jurtshuk, May, Pope & Aston, 1969) except that the reaction was started by adding both dyes simultaneously after 15 min incubation of the enzyme and substrate at room temperature; fumarase (Massey, 1955); malate dehydrogenase (Ochoa, 1955) modified by replacing pH 7.0 phosphate buffer for pH 7-4 glycyl-glycine buffer ; carboxydismutase (Hurlbert & Lascelles, I 963) ; phosphoenolpyruvate carboxylase, (Large, Peel & Quayle, 1962); isocitrate lyase and malate synthase (Dixon & Kornberg, 1959); NADH cytochrome c reductase (Nason & Vassington, I 955). Threonine deaminase was determined by the colorimetric method of Datta (1966) and protein by the method of Lowry et al. (1951). The oxidation of sulphur and inorganic sulphur compounds were studied in washed bacterial suspensions at 40”using standard manometric techniques. Incorporation of [114C]acetate, carbon dioxide production from this compound, and incorporation of carbon dioxide from NaH14C03 were also followed in the Warburg apparatus (Gilson Medical Electronics, Middleton, Wisconsin, U.S.A.). Suspensions in Warburg vessels were rapidly chilled in crushed ice and samples of 0.1 or 0.2 ml corresponding to 0.05 to 0-15mg protein were drawn through Millipore filters (average pore size 0.45 p). Where necessary the suspensions were homogenized gently with a hand homogenizer to break up clumps before filtration. The organisms retained on the filters were washed three times with 20 mM-NaHCO, in the case of C 0 2fixation experiments, or with 20 mM-sodium acetate in the case of acetate incorporation experiments, and then 36 MIC Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 70 R. A. D. W I L L I A M S A N D D. S. H O A R E 558 washed twice with water. The filters were then air-dried and glued to aluminium planchets for counting. To determine the production of 14C02from acetate, 0'I ml hyamine hydroxide (Nuclear Chicago Corp., Des Plaines, Illinois, U.S.A.) was placed in a glass vial in the centre well of the Warburg vessel. At the end of the experiment the vial and contents were placed in a screw cap tube containing 15 ml of Bray's solution (Bray, 1960) and stored in the refrigerator for scintillation counting. To study the distribution in the components of labelled carbon from [114C]acetatethe harvested bacteria were fractionated by the method of Roberts, Cowie, Anderson, Bolton & Britten (1955). The final residue was boiled under reflux with 20 ml of 6 N-HCI for 18 h to hydrolyse the protein. The hydrolysate was dried under a stream of air at 40" and dissolved in I ml water; this drying was repeated twice. The amino acids were partly resolved into four fractions; neutral and basic groups, glutamate and aspartate, by high voltage electrophoresis (Dixon, Kaufman & Neurath, 1958). These fractions were eluted and the neutral amino acids separated by two-dimensional paper chromatography (Benson et al. 1950). Radioactive areas on chromatography and electrophoresis papers were located by autoradiography, using NoScreen X-ray film (Eastman Kodak Co., Rochester, New York, U.S.A.), and the radioactive compounds eluted for counting. Poly-P-hydroxybutyrate was isolated from bacteria grown in thiosulphate medium containing I m ~ - [ ~ ~ ~ C ] s o acetate d i u m by the method of Williamson & Wilkinson (1958) in the presence of I 6 mg of pure carrier poly-P-hydroxybutyrate kindly provided by Dr P. Jurtshuk. The polymer was reprecipitated to constant specific activity and aliquot samples plated for counting. The radioactivity in the samples was determined with infinitely thin preparations on planchets, or on Millipore filters glued to planchets, using a model D 47 gas-flow planchet counter (Nuclear Chicago Corp., Des Plaines, Illinois, U.S.A.). Samples of hyamine in Bray's solution were counted using a series 3rqE Packard TriCarb liquid scintillation spectrometer (Packard Instrument Co. Inc., La Grange, Illinois, U.S.A.). RESULTS Growth The growth of the strain was sparse under all conditions. In autotrophic medium growth ceased when about 10 of the 18.7 pmollml of thiosulphate had been used. When 2 mMacetate, malate, aspartate or glutamate, or 0.1 % yeast extract was included in the medium, all the thiosulphate was oxidized, and the maximum turbidity increased from 9 to 2 6 k 2 Klett units. To obtain maximum growth, medium supplemented with 0-1yo yeast extract was used in experiments to determine the temperature optimum. The strain grew most rapidly at 50" (Fig. I) and less rapid growth was observed at 45", 40", 55" and 35" in descending order. No growth occurred at 60". We did not calculate mean generation times because of the slight growth and the tendency to grow in strands which appeared to contain elemental sulphur particles. These clumps tended to block pipettes and resisted dispersion in a hand homogenizer. Therefore we could not obtain representative samples for protein estimation as an estimate of growth. However, 25 to 30 Klett units approximately correspond to 40 to 50 pg proteinlml of culture. No increase in growth was obtained by lowering the phosphate or the thiosulphate concentration, nor by replacing the mineral medium with that described by Pfennig & Lippert (1966). No polythionates or ketoacids were detected in the spent medium. A precipitate was obtained with barium chloride, indicating extensive sulphate formation from thiosulphate. Continuous aeration of growing cultures with air Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 Thermophilic t hiobacillus 20 40 60 80 Hours 100 559 I20 Fig. I . Growth curve, thiosulphate utilization and acid production by thiobacillus at 50”. Thiowith a 3-day-old sulphate-mineral medium containing 0.1 ”/, yeast extract was inoculated ( I autotrophic culture. Initial thiosulphate concentration after inoculation was I 8.0 ,urnol/ml. -0-,Thiosulphate concentration; +, turbidity (Klett photometer units); -A-, pH. x) containing 5 yo(v/v) C 0 2 did not increase growth, and no significant increase was obtained by using a dialysis culture apparatus of the type described by Borichewski &Umbreit (1966). In autotrophic medium with one-tenth the normal phosphate adjusted to various pH values, growth at 50° occurred between pH 4.8 and 8.0 with an optimum at pH 5.6. No growth was detected at pH 3-8 in 160 h. The pH was maintained in each flask with the aid of appropriate indicators by intermittant addition of sterile sodium bicarbonate. Growth on various nitrogen sources decreased in the order ammonium chloride, sodium nitrate, sodium glutamate, sodium aspartate and urea. For this experiment ammonium ions were eliminated from the inocula by centrifuging an autotrophic culture in sterile bottles, and resuspending the pellet in sterile autotrophic medium without ammonium chloride. No growth was observed on ‘nitrogen free’ autotrophic medium. Substrate oxidation Oxidation of thiosulphate by bacterial suspensions occurred at all pH values tested, but was most rapid, and linear with time, at pH 6.0 to 8.0. Under more acid conditions ‘tailing off’ of oxygen consumption occurred, and at pH 4-0 and 4-8 oxidation was incomplete and proceeded very slowly after 90 min. At the optimum pH for autotrophic growth (pH 5-6) 36-2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 560 R. A. D. W I L L I A M S A N D D. S. H O A R E Table I . Efect of temperature on the speciJic activity of rhodanese and threonine deaminase in extracts of autotrophically grown thiobacilli Specific activity (%)* I Temperature 30" 35" 40" 45" 50" 55" Rhodanese Threonine deaminase 27 38 76 74 79 83 I00 100 87 73 9 41 * The maximum specific activities at 45" were: rhodanese, 455 nmol thiocyanate formed/mg protein/ min; threonine deaminase, 5.6 nmol a-ketobutyrate formedlmg proteinlmin. Threonine deaminase was 50 % inhibited by 4.5 x I O - ~M-isoleucine at 40". Table 2 . Carboxydismutase and enzymes of sulphur metabolism in extracts of thiobacillus Specific activity (nmol/mg proteinlmin) in extracts of bacteria grown on: Enzyme Sulphite oxidase Thiosulphateoxidase Rhodanese Carboxydismutase Thiosulphate-mineral medium Thiosulphate-mineral medium with 2 mM-acetate 16.0 0.9 I 7'65 25.2 5.89 0.30 4'17 22-9 the initial rate of oxidation was the most rapid, but slowing of the rate resulted in complete oxidation occurring in the same time interval as at higher pH values. The stoichiometry of thiosulphate oxidized indicated complete conversion to sulphate (2 pmol O,/pmol Na2S,0s). Tetrathionate was also oxidized extensively, the oxygen uptake being 78 % of that required for complete oxidation to sulphate. The rates of oxidation for thiosulphate and tetrathionate were identical. Sulphite and sulphur were oxidized very slowly and thiocyanate not at all. Temperature optima of enzymes Rhodanese and threonine deaminase were optimally active at 45" (Table I). Both enzymes exhibited a high proportion of their maximal activity at 40" and 50' but there was a marked decrease at 5 5 O , especially in the case of threonine deaminase. Enzymes of sulphur metabolism and carboxydismutase Cell-free extracts of autotrophically grown bacteria contained high activities of sulphite oxidase and rhodanese (Table 2), and also reduced ferricyanide in the presence of thiosulphate (thiosulphate oxidase), The sulphite oxidase activity was independent of adenosine monophosphate. The activities of all three enzymes were reduced in extracts of bacteria grown in the presence of 2 mwacetate. Carboxydismutase was, however, apparently only slightly repressed by acetate at this concentration. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 Thermophilic thiobacillus Table 3. Enzymes of the tricarboxylic acid and glyoxylate cycles, and related enzymes in extracts of thiobacillus Specific activity (mol/mg proteinlmin) in bacteria grown on: A f Enzyme Citrate synthase Aconi tase Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase Succinate dehydrogenase (particulate) Fumarase Malate dehydrogenase Isocitrate lyase Malate synthase Phosphoenol pyruvate carboxylase NADH oxidase (particulate) NADH cytochrome c reductase (a) Soluble (b) Particulate > Thiosulphate-mineral Thiosulphate-mineral medium with 2 mM-acetate medium 0.10 12.5 37'8 0 8.6 9-25 I08 60 0 I 1.8 1216 1050 77 176 86 2'7 10'1 2-76 19 4'9 I 2-10 Reduction of cytochrome c The soluble fraction of extracts contained a cytochrome with a Soret band at 410 nm, which was reduced by sodium dithionite to give absorption bands at 420, 522 and 552 nm typical of a c-type cytochrome. Thiosulphate or sulphite immediately produced reduced cytochrome peaks as intense as those caused by dithionite. Slight reduction was also obtained with NADH, and NADPH,, the peak heights being about 15% of those produced by thiosulphate. No attempt was made to exclude oxygen from the cuvettes in these experiments. No reduction of cytochrome c was detected with 10pm0l/3 ml cuvette of sodium tetrathionate, or with a small quantity of elemental sulphur. Enzymes of TCA and glyoxylate cycles and related enzymes Major features of the enzyme profiles are the absence of a-ketoglutarate dehydrogenase (Table 3) under all growth conditions and the marked increase in the other TCA cycle enzymes, together with the induction of isocitrate lyase, in bacteria grown on thiosulphatemineral medium containing 2 mwacetate. a-Ketoglutarate dehydrogenase was assayed in extracts of a facultatively autotrophic thiobacillus (strain A ~ Taylor ; & Hoare, 1969) and pyruvate dehydrogenase successfully detected in the thermophilic thiobacillus as controls. Malate synthase was constitutive, but its activity was higher in bacteria grown with acetate. NADH oxidase was readily demonstrated. In the particulate fraction it was 50 yoinhibited by 1 - 2x I O - ~M-potassium cyanide. No formate dehydrogenase was detected in extracts of bacteria grown in either medium. Incorporation of HI4CO3There was negligible incorporation of HC03- by suspensions in the presence of acetate, and very little in the presence of elemental sulphur (Table 4). No assimilation occurred with thiocyanate. Both thiosulphate and tetrathionate were efficient substrates for promoting carbon fixation. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 R. A. D. W I L L I A M S A N D D. S. H O A R E 562 Table 4. Fixation of N a H W 0 3 by thiobacillus suspensions with sulphur compounds Substrate fixed* c.p.m. x I O - ~ pmol NaHCO, fixed pmol substrate used 19.4 1.16 1.37 5 5 Thiosulphate Tetrathionate Thiocyanate Sulphur 23.1 0 0 0 n.d. 0.024 0.4 * Corrected for endogenous fixation of 2550 c.p.m./vessel. Warburg vessels contained bacteria grown autotrophically for 3 days, equiv. 3-56mg proteinlvessel, IOOpmol phosphate pH 6.5, 2 pCi NaH1*C03 at 2.6 pmollvessel, and 5 pmol of substrate. Table 5. Distribution of incorporated 1I4Cacetate in a growing culture of thiobacillus Thiosulphate-mineral medium at pH 6.0 containing 20 pCi sodium acetate at 2 mM. , 14Cincorporated c.p.m. x lop4 6.6 41.0 8.75 56.0 28.4 175.5 Fraction (Roberts et al. 1955) Cold TCA soluble Ethanol soluble Ethanol-ether soluble Hot TCA soluble Acid ethanol-ether soluble Residue Hydrolysed residue Glutamate Asparate Basic amino acids Neutral amino acids I 3-6 % 2.1 I 2-9 2.8 17'7 9-0 55'3 99'8 4'3 4'7 14.7 I 7.0 123 5'4 38-3 52'7 Table 6. The incorporation and oxidation of [ 114C]-and [214C]acetate by suspensions of thiobacillus Warburg vessels contained bacteria grown autotrophically for 3 days equivalent to 3.75 mg protein/ vessel, IOO pmol phosphate pH 6.5 and 0.5 pCi of acetate at 2-5 pmollvessel. Hyamine hydroxide (0.1ml) was placed in a glass vial in the centre well. Labelling position Thiosulphate (pmol) pmol acetate incorporated pmol acetate Total acetate used oxidized to COP oxidized to CO, (%) 0.85 0'2 I0 0.82 2.16 0.003 1.88 0.009 0.I 76 19.8 0.48 17.7 0.14 Utilization of [14C]acetate Bacteria grown on thiosulphate-mineral medium containing [I 14C]acetateincorporated radioactive carbon into the major macromolecules (Table 5). The largest amounts were found in the insoluble residue containing the protein, and the fraction soluble in hot trichloroacetic acid containing the nucleic acids. High voltage electrophoresis of the hydrolysed protein residue revealed unrestricted incorporation of acetate carbon into neutral, acidic and basic amino acids. Two dimensional chromatography and radioautography of the neutral amino acid fraction revealed eight distinct radioactive spots. Both [ 114C]-and [214C]acetate were readily assimilated in the absence of thiosulphate, by Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 Thermophilic thiobacillus 563 suspensions of autotrophically grown bacteria (Table 6). Oxidation of acetate to carbon dioxide accounted for 17 to 20 76 of the acetate used after 55 min.In the presence of 5 pmol thiosulphate the incorporation of acetate was enhanced by over twofold, while oxidation to carbon dioxide was reduced to less than 0.5 yo of the total acetate used. Poly-/?-hydroxybutyrate was synthesized by bacteria grown on thiosulphate-mineral medium containing I mwacetate, and represented 1-14o//o of the total acetate incorporated. Phospholipid composition The major component of autotrophically grown bacteria was phosphatidyl ethanolamine. Lesser amounts of phosphatidyl glycerol and diphosphatidyl glycerol were also detected, together with a fourth unidentified component (J. M. Shively, personal communication). DNA base cornposition The buoyant density of DNA isolated from bacteria grown on thiosulphate-mineral medium with or without acetate, and heterotrophically on yeast extract was 1.7248 (66.2 y o G + C ) (M. Mandel, personal communication). DISCUSSION The isolate described here has the structure and physiology of the genus Thiobacillus (Breed, Murray & Smith, 1957); it is a Gram-negative rod capable of obtaining its energy by oxidizing reduced sulphur compounds to sulphate and of obtaining carbon by carbon dioxide fixation. Extracts contained enzymes characteristic of sulphur metabolism in high activity, particularly an adenosine monophosphate-independent sulphite oxidase which has also been reported in Thiobacillus novellus (Charles & Suzuki, 1966). The status of the isolate as a thiobacillus is also confirmed by its DNA base composition, which falls within the range for the genus, close to that of T. novellus and T. denitrlficans (Jackson, Moriarty & Nicholas, 1968). The phospholipids positively identified are the same as those in all other thiobacilli (Barridge & Shively, 1968). However, methylated phosphatidyl ethanolamines, which were found in four of the five species tested by Barridge & Shively, were not detected in the present strain. One unidentified phospholipid component has not been reported in other thiobacilli, and in this respect phospholipid analysis of other thermophilic strains would be of interest in order to determine whether there is any relationship between temperature optimum and phospholipid composition. Growth was slight under all conditions tested. The reported yield of Thiobacillus interunedius, both in terms of turbidity and cell protein, was 3-8 times that of the present strain in autotrophic medium, and 3-6 times that in thiosulphate medium containing 0 - 1yo yeast extract (London, 1963; London & Rittenberg, 1966). The relationship between protein content of the culture and its turbidity was identical for T. intermedius and the thermophile in autotrophic medium. However, this relationship was not constant for either organism under different conditions of growth. This is probably a reflexion of differences in composition and form in diverse media. The incomplete utilization of thiosulphate in autotrophic medium remains unexplained. It is not a simple pH effect as intermittent neutralization did not permit complete utiIization. Furthermore, suspensions were able to oxidize thiosulphate completely. Utilization of thiosulphate by growing cultures of T. thermophilica is also reported to be incomplete (Hutchinson, Johnson & White, I 967). Autotrophic bacteria which grow scantily have also been isolated from sea water (S. Watson, personal communication). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 02:08:31 564 R. A. D. W I L L I A M S A N D D. S . H O A R E The acidic pH optimum is not surprising in an organism isolated from an acid hot spring, but failure to detect growth at pH 3.8 was unexpected as the pH of the water from which it was isolated was 2.7. However, slow growth may be possible at pH 2.7 at the temperature of the spring (38"), but not at 50'. While less heat-tolerant than the spore-forming Thiobacillus therrnophilica (Egorova & Deryugina, 1963) the present strain would nevertheless be regarded as a thermophile in view of its ability to grow at 55" (Farrell & Rose, 1967). As the optimum temperatures of the two enzymes examined were lower than the optimum for growth, it must be concluded that either the optimum in cell-free extracts is lower than in intact cells, or that the activities of the two enzymes are sufficient at 50° for them to be non-limiting on growth. The isolate was successfully maintained on yeast extract plates, and a sample grown in nutrient broth had the same DNA mean base composition as cells grown autotrophically. No satellite or contaminant bands of DNA were detected, and colonies grown on yeast extract plates could be transferred to thiosulphate medium. The strain was not, therefore, an obligate autotroph. Failure to grow the thermophile on single organic compounds might be due to inhibition due to metabolic imbalance. Such inhibitory effects have been produced in autotrophs by single amino acids (Kelly, 1971). The absence of a-ketoglutarate dehydrogenase has been cited as a possible metabolic basis for obligate autotrophy (Smith, London & Stanier, 1967). This metabolic block restricts incorporation of acetate carbon to leucine and the glutamate family of amino acids. Facultative autotrophy, and an unrestricted incorporation of acetate into amino acids, may be explained in the present strain by the ability of the glyoxylate cycle enzymes to bypass the block in the Krebs cycle. This metabolic combination has been reported in Chromatiurn (Truper, I 964). The inhibition of acetate oxidation and stimulation of acetate incorporation by thiosulphate resemble the effects of nitrite on acetate metabolism by Nitrobacter agilis (Smith & Hoare, 1968). Oxidation of both carbon atoms of acetate to carbon dioxide might take place by a dicarboxylic acid cycle (Kornberg & Sadler, 1961) operating in conjunction with the glyoxylate cycle. Energy might then be produced by oxidative phosphorylation involving cytochrome c, which can be reduced by thiosulphate or by reduced coenzymes produced by the metabolic scheme postulated. This work was supported by a grant from the Robert A. Welch Foundation. P. Jurtshuk kindly supplied pure poly-P-hydroxybutyrate prepared from Azotobacter vinelandii. D N A analyses were performed by M. Mandel of the University of Texas M. D. Anderson Hospital and Tumour Institute, Houston, and phospholipid analyses by J. M. Shively of the University of Nebraska, Lincoln, Nebraska. The helpful discussions and encouragement of Barrie F. Taylor are gratefully acknowledged. REFERENCES BARRIDGE, J. K. & SHIVELY, J. M. (1968). Phospholipids of the thiobacilli. Journal of Bacteriology 95,21822 I 85. BENSON,A. A., BASSHAM, J. A., CALVIN,M., GOODALE, T. C., HASS,V. A. & STEPKA,W. (1950). The path of carbon in photosynthesis. V. Paper chromatography and radio-autography of the products. 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