Download Physiology of a New Facultatively Autotrophic

Journal of General Microbiology (1972)~
70,555-566
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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 .
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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;
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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
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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
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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
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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.
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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.
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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
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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).
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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.
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