Arch. Mikrobiol.

84, 54--68 (1972)

9 by Springer-Verlag 1972

Sulfolobus: A New Genus of Sulfur-Oxidizing

Bacteria Living at Low pH and High Temperature
THOMAS D. :BRocK, KATHERINE M. :BRocK,
ROBERT T. BELLY, a n d I ~ I C I ~ D L. W ~ I s s
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706,
U.S.A.,
Department of Microbiology, Indiana University, Bloomington, Indiana 47401,
U.S.A.

Received January 6, 1972

Summary. Sul/olobus is a new genus of bacteria characterized as follows:

1. generally spherical cells producing frequent lobes; 2. facultative autotrophy
with growth on sulfur or on a variety of simple organic compounds; 3. unusual cell
wall structure devoid of peptidoglycan; 4. acidophilic, pH optimum of 2--3 and
range from 0.9--5.8; 5. thermophilic with temperature optimum of 70--75~ and
range from 55--80~ (one strain grew at 85~ The DNA base composition of five
strains was determined by cesimn chloride density gradient centrifugation and
found to be 60--680/0 guanine plus cytosine. SulJolobus apparently has no close
relationship with any previously described bacteria, either he~erotrophic or autro-
trophic. Techniques are presented for distinguishing SulJolobus from Thermoplasma,
another genus of acidophilic thermophilic spherically shaped organisms. Sul/olobus
has been isolated from a variety of natural acidic thermal habitats, both terrestrial
and aquatic. Most isolations have been from habitats in Yellowstone National Park,
hut strains were also isolated from Italy, Dominica and E1 SaIvador. I t is suggested
that SulJolobus may be an important geochemical agent in the production of sulfuric
acid from sulfur in high temperature hydrothermal systems.

W e r e p o r t here t h e isolation a n d c h a r a c t e r i z a t i o n of a new genus of

sulfur-oxidizing b a c t e r i a able to grow a t high t e m p e r a t u r e a n d low p g .
This genus, which we h a v e d e s i g n a t e d Sul/olobus gem nov., is characteriz-
ed b y cells which are l o b e d spheres. So far, all strains i s o l a t e d are a b l e to
grow h e t e r o t r o p h i c M l y as well as a u t o t r o p h i e a l l y . O r g a n i s m s of this
genus are w i d e s p r e a d in s o l f a t a r a areas a n d can be r e a d i l y i s o l a t e d from b o t h
t h e r m a l a c i d soils a n d acid h o t springs. J . A. B r i e r l e y (1966) first r e p o r t e d
t h e isolation of a n o r g a n i s m r e s e m b l i n g SulJolobus, b u t d i d n o t charac-
terize i t in detail. C. L. B r i e r l e y (1971) has r e c e n t l y e x t e n d e d t h e w o r k of
J. A. Brierley.
I n a d d i t i o n to t h e i n h e r e n t i n t e r e s t in a new g r o u p of sulfur-oxidizing
b a c t e r i a , this genus is of i n t e r e s t b o t h e v o l u t i o n a r i l y a n d geochemieMly.
 Sul/oIobus: A New Genus 55

S o m e strains grow in c u l tu r e a t t e m p e r a t u r e s of 80~ an d h a v e been f o u n d

in n a t u r e a t similar t e m p e r a t u r e s . T h e i r a b i l i t y to grow a t these t e m p e r -
a t u r e s p r o b a b l y explains t h e origin of sulfuric acid in high t e m p e r a t u r e
t h e r m a l systems, a previous]y u n a n s w e r e d g e o ch em i cal q u e s t i o n (Schoen
an d Erlich, 1968).
Sul/olobus has been isolated f r o m a wide v a r i e t y of acid t h e r m a l areas,
b o t h t er r es t ri al a n d a q u a t i c . I n earlier studies ( D a r l a n d et al., 1970) we
described a n e w group of acidophilic a n d t h e r m o p h i l i c m y c o p l a s m a s
wh i ch we h a v e d e s i g n a t e d Thermoplasma. Because of t h e similar environ-
m e n t a l r e q u i r e m e n t s a n d generally spherical shape of b o t h fhermoplasma
a n d Sul/olobus, we m a d e especial effort in t he p r e s e n t w o r k to c o m p a r e
a n d c o n t r a s t these tw o groups of organisms. As we will show here, t h e
t w o genera are q u i te u n r e l a t e d a n d can be easily d i st i n g u i sh ed b o t h in
n a t u r e a n d in culture. Sulfolobus is also u n r e l a t e d to Bacillus acido-
caldarius, a spore-forming r o d i s o l a t e d f r o m acid t h e r m a l e n v i r o n m e n t s
b y D a r l a n d a n d B r o c k (1971).

Materials and Methods

Cultures. All of the Sul/olobus cultures were isolated in the present work. The
Thermoplasma cultures were isolated by Darland et al. (1970) and by R. T. Belly
from self-heating coal refuse piles in southern Indiana.
Culture Media. For most studies a basal salts medium modified from Allen
(1959) was used. This medium contains (NH~)2SOd, 1.3 g; KH2POd, 0.28 g; MgSO,
9 7 H20, 0.25 g; CaC12 92 1~20, 0.07 g; FeC1a - 6 H20 , 0.02 g; MnCI2 - 4 I~20, 1.8 rag;
-Na2B~O~ - 10 H20, 4.5 rag; ZnSO~ 97 H~O, 0.22 rag; CuC12 - 2 H20, 0.05 rag;
NaMoOt 92 H20, 0.03 rag; VOSOr 92 H20, 0.03 rag; CoSQ, 0.01 rag; distilled or
deionized water, 1 1. The pI~ was adjusted with 10 1~ H~SOt.
Organic supplements were added to the basal medimn either as dry powder
before autoclaving or from 100-fold concentrated sterile stock solutions (for sugars)
after autoclaving. Elemental sulfur was sterilized by steaming in the dry state for
3 h on each of 3 successive days and added at a concentration of about 1 g/100 ml
to sterile media.
When agar plates or slants were made, a twice-concentrated basM salts solution
containing all necessary ingredients was autoclaved and mixed after cooling to
45~ with an autoclaved and twice-concentrated agar sol, and yeast extract to a
final concentration of 0.1 ~ was added aseptically. The final mixture was dispensed
into plates or tubes which were allowed to solidify. The final pH of the agar was
measured by inserting a glass electrode directly into the agar gel. If the agar was
mixed with the medimn at acid pH before autoclaving, solidification did not occur.
Colony formation occurred more reproducibly if Ionagax 4~ 2 (Oxoid) was used
instead of Difeo Agar. Ionagar was used at a final concentratio1~ of 1.2~
For most work, liquid cultures were prepared in screw-capped tubes or bottles.
When sulfur was used as energy source, tile bottles were gassed with carbon dioxide
by passing 1000/0 C02 into the gas phase for a few seconds immediately before
sealing.
Biochemical Methods. Cultures were grown in 1 1 volumes in unshaken 3 1
Fernbach flasks, and were harvested in the early stationary phase by eentrifugation
56 T. D. Brock, K. M. Brock, R. T. Belly, and R. L. Weiss:

at about 10000X g. The cell pellets were frozen at --20~ until processing. The
frozen pellets were thawed and suspended in a small volume of water and the suspen-
sion was then dispensed into tubes for extraction and hydrolysis.
For hexosamine analyses, aliquots of suspension were hydrolyzed in sealed
tubes in 6 N HCI at 105~ for 11--16 h. The hydrolysates wore carefully neutralized
with 5 N NaOH using p H paper. AIiquots of neutralized extracts were then assayed
for hexosamine by the Tipper (1968) modification of the Morgan-Elson method,
using glueosamine as a standard. For ribonueleic acid analyses, aliquots of cell
suspensions were assayed directly using the orcinol method of Chargaff and David-
son (1955) with yeast ribonueleic acid as a standard. For protein assays, cell suspen-
sions were first extracted in boiling water with 1 N N a O t t and aliquots assayed using
the method of Lowry et al. (1951) with crystalline lysozyme as a standard. Total
carbohydrate analyses were performed directly on cell suspensions using the anthrone
method of Trevelyan and Harrison (1952) with glucose as a standard.
Sul/ate was assayed by the turbidimetric method with barium chloride (Stan-
dard Methods for the Examination of Water and Wastewater, 12th edition, Ameri-
can Public Health Assoc., New York, 1965).
Deoxyribonucleic Acid (DNA) Base Composition. DNA was isolated and purified
by the method of Marmur (1961) after the cell suspension was initially lysed by
sodium lauryl sulfate. Base compositions were calculated from the buoyant density
of the D~qA as determined in a Beckman model E ultracentrifuge in CsC1, with
bacteriophage T 2 DNA as a standard, following the method of Schildkraut et al.
(1962).
Electron Microscopy. Cultures were grown at 70~ in basal salts plus 0.1 D/0
yeast extract at p H 2 with trace elements deleted. For ultrathin sectioning, loga-
rithmically growing cells were cooled to room temperature and 10~ (V/V) glutar-
aldehyde in 0.1 M cacodylate-HC1 (pH 3.4) was added to obtain a final concentra-
tion of 20/0 (V/V) glutaraldehyde. After prefixation for 15 min at room temperature
the culture was cooled to 0--4~ for 2 h. The bacteria were harvested by centrifuga-
tion at 8 000 x g, washed in buffer, and fixed with 1 ~ in veronal buffer by the
method of Ryter and Kellenberger (1958). After fixation the cells were washed in
buffer and embedded in agar. The agar blocks were treated for 2 h in 0.5~ (W/V)
uranyl acetate. The cells were then dehydrated in a graded acetone series and embed-
ded in Epon 812. Ultrathin sections were cut on a Porter-Blum MT-2 ultramicro-
tome with a glass knife and post-stained with uranyl acetate and lead citrate
(Reynolds, 1963).
For negative staining a drop of the culture was placed on a carbon supported
parlodion-coated grid to allow organisms to attach to the grid. The organisms were
then stained with 1 ~ (W/V) aqueous uranyl acetate and dried immediately with
filter paper.
All samples were observed wi~h an Hitachi H U l l O electron microscope
operated at 50 kV. The magnification was calibrated with a diffraction grating
replica (2160 lines per mm). Several strains were studied, but the photographs
presented here were all derived from studies on the type strain, 98-3.
Environmental Methods. Temperatures of hot springs and other thermal habitats
were measured with a Yellow Springs Instrument Co. (Yellow Springs, Ohio)
thermistor and bridge. For pH, water samples were collected in plastic bottles and
the p H was measured after cooling using an Orion Model 401 p i t meter and Broadley-
James combination electrode. Care was taken to be certain that gases were not lost
from the water samples during cooling.
 Sul]olobus: A New Genus 57

Results
Habitats
Sul/olobus has been isolated from acid thermal soils and acid hot
springs in Yellowstone National Park, E1 Salvador, Dominiea, and Italy.
In many of the habitats from which the organism has been isolated, it
was present in such large numbers that it was visible by direct phase
microscopy of water or soil. Virtually all of the springs containing
Sul/olobus were of low p H (less than 3.0) and high temperature (65-- 90~
Over 80 ~ of the springs in which SulJolobus were found had p i t between
1.5--2.4 and temperature between 76 and 90~ Sul/olobus-type cells can
also be observed microscopically in aqueous suspensions of some acid
thermal soils, but a detailed microscopic survey of the presence of the
organism in soils has not been made.
Cultures have been obtained from a number of aquatic and terrestrial
habitats in geothermal areas and details of culture methods are presented
below.

Enrichment and Isolation

Originally enrichment cultures from acid thermal habitats were set
up both heterotrophieally (0.1~ yeast extract) and autotrophically (on
elemental sulfur). (The original isolations on sulfur were done by Flier-
mans and Davidson.) I t was then discovered that the heterotrophic
isolates would grow autotrophieally on sulfur and the autotrophie
isolates would grow heterotrophically on yeast, suggesting that Sul[o-
lobus is a facultative autotroph. I t is striking, however, that simple
heterotrophic enrichment with yeast extract should result in the selec-
tion of organisms able to grow autotrophically on sulfur. Presumably
the habitat of SulJolobus is so extreme that obligate heterotrophs, which
might normally be expected to be enriched with a yeast extract medium,
are not present in tile habitat so that the only organism able to grow in
hetcrotrophie enrichment culture is Sul]olobus.
The simplest procedure for the enrichment of Sul]olobws is to add
0.1 ~ of an acidified solution of yeast extract to a sample of spring
water and incubate at 70~ After 3--7 days heavy turbidity may be
seen, often with a surface pelliele, and microscopic examination may
reveal large numbers of typical Sul[olobus cells. A culture can then~be
established by transferring to salts medium containing 0.10/0 yeast
extract. For culturing from soil, about 0.1--0.5 g of soil can be added'to
0.1 ~ yeast extract medium. The best p H for heterotrophic enrichment
of Suljolobus is 2.0 although p H 1 was used when enriching at temperatu-
res of 55~ to prevent the growth of Bacillus acidocaldarius. When auto-
trophie enrichments arc desired, an aliquot of spring water or soil is
58 T.D. Brock, K. M. Brock, 1~. T. Belly, and 1~. L. Weiss:

added to salts medium containing about 1 ~ elemental sulfur. For auto-

trophic enrichments, pII 3.0 was used, the oxidation of sulfur reducing
the p t I to 2.0 or less.
I n general, cultures were maintained b y successive transfer in the
liquid state. Although colonies could usually be obtained by s~realdng on
0.1 ~ yeast extract agar, reproducible growth on agar was not obtained
so t h a t maintenance of cultures in the liquid state was preferable. When
colonies were obtained on agar, they were small, medium, or large, and
had a smooth surface and a glistening texture. Only a single colony type
was obtained on each plate. The individual isolates are considered dis-
tinct because they were independent isolates from geographically sepa-
rate springs. The colonies of Sulfolobus are quite different from those of
Thermoplasma acidophiIura. The latter forms typical "fried-egg" myco-
plasma-like colonies, rather t h a n the smooth glistening colonies formed
by Sul/olobus.

Morphological Characteristics
Two kinds of spherical organisms have been isolated from acidic
thermal habitats, Thermoplasma and Sul/olobus. They can be distinguish-
ed under the phase microscope b y careful examination. Thermoplasma
cells are completely round whereas Sul]olobus cells are more irregular, and
when ceils are tumbling through the field, distinct lobes can be seen. The
diameter of Sul/olobus cells is about 0.8--1 ~m and little size variation is
seen. Thermoplasma cells are quite variable in diameter, ranging from
about the limit of resolution at 0.1--0.2 ~m to large cells 5--10 ~m in
diameter. Thermoplasma cells seem to reproduce b y budding or b y narrow
hyphae whereas SuIjolobus cells apparently reproduce by septation of
lobes, although details of this process have not been studied. Sul/olobus
cells are more refractile t h a n Thermoplasma, presumably because of the
presence of a cell wall (see later). I n Thermoplasma cultures large e m p t y
cells 5 - - 10 ~m in diameter are often seen, within which denser structures
can be seen; when e m p t y cells are seen in Sul/olobus cultures they are no
larger than normal cells and lack the denser interior structures seen in
Thermoplasma cultures. Probably the best characteristic for distinguish-
ing the two organisms is sphericity, Thermoplasma always being evenly
spherical and Nul/olobus being lobed spheres. I n nature, SulJolobus cells
have a morphology similar to those cultured in the laboratory, and the
criteria listed above can be used to recognize them. Photomicrographs of
Sul/olobus are shown in Fig. 1.
When growing on sulfur, both in nature and in the laboratory, Sul/o-
lobus cells are often seen attached to sulfur crystals, either singly or in
large numbers. They can be readily observed upon sulfur crystals b y
treating with acridine orange and examining with a fluorescence micro-
 Sul/olobus: A New Genus 59

Fig. 1. Phase contrast photomicrographs of Sul[olobus strain 98-3. Magnification,

2300 x

scope equipped with a vertical illuminator. Even b y ordinary phase micros-

copy, a t t a c h m e n t to sulfur can often be observed when the edge of a
crystal is examined.

Electron Microscopy
A characteristic feature of Sul[olobus is the presenee of irregular
lobed structures of the cells shown in ultrathin sections (Fig.2).
The various shapes of Sul]olobus can be seen much better in the
eleetron microscope than in the light microscope. A typical multilobed
cell is presented in Fig. 2. Measurements from lobe to lobe at the widest
p a r t of this cell yield a distance of about 1 ~m. The narrowest distance
measured across a single lobe was about 0.25 ~m, while the distance
measured across the widest lobe was 0.5 ~m. The highly irregular
appearance of other cells (Fig. 3) is sometimes due to the presence of a
straight area of the wail observed between two rather large lobes.
An unusual feature of the cell envelope of this bacterium is the cell
wall (Figs.3 and 4) which appears to be different from both gram-
positive and gram-negative cell walls. An enlarged portion of the cell
60 T. D. Brock, K. M. Brock, R. T. Belly, and R. L. Weiss:

wall (Fig. 4) shows t h e presence of a diffuse electron t r a n s p a r e n t l a y e r of

s u b u n i t s a n d t h e absence of a t y p i c a l p e p t i d o g l y c a n layer. F u r t h e r details
of t h e wall s t r u c t u r e are p r e s e n t e d in n e g a t i v e l y s t a i n e d p r e p a r a t i o n s
of Sul/olobus (Fig.5). T h e s u b u n i t s t r u c t u r e , which is p a r t i c u l a r l y
e v i d e n t along t h e m a r g i n of t h e celt, a p p e a r s to be c o n t i n u o u s over the
entire cell surface as a r e g u l a r a r r a y of electron t r a n s p a r e n t units. The
cell wall s t r u c t u r e a n d l o b e d n a t u r e of n e g a t i v e l y s t a i n e d Sul/olobus
(Fig. 5) can be c o m p a r e d w i t h t h a t of Thermoplasma. T h e s m o o t h surface
of n e g a t i v e l y s t a i n e d Thermoplasma (Fig.6) lacks t h e electron t r a n s -

Fig.2. Multilobed cell. • 34000; bar represents 0.5 ~m (for reproduction reduced
to 9/10)
Fig. 3. Cell showing a straight wall area and two large lobes. • 41000; bar represents
0.5 ~zm (for reproduction reduced to 9/10)
Fig.4. Enlargement of the cell envelope of Fig.4 showing the plasma membrane
(pm) and the cell wall (cw). • 173000; bar represents 0.1 ~m (for reproduction
reduced to 9/10)
Fig.5. Negatively stained Sul/olobus cell showing the lobed nature of the cell and
the subunit structure of the cell wall. • 61000; bar represents 0.5 9m (for reproduc-
tion reduced to 9/10)
Fig.6. Negatively stained Thermoplasma cell showing the spherical shape and ab-
sence of cell wall • 29 000; bar represents 1 ~zm (for reproduction reduced to 9/10)
Sul/olobus: A New Genus 61

Fig.5

Fig.6
62 T.D. Brock, K. M. Brock, R. T. Belly, and R. L. Weiss:

parent structures which constitute the cell wall of Sul/olobus (Fig.5).

Darland et al. (1970) reported that ThermopIasma is a free-living myco-
plasma bounded by a single well-defined unit membrane. When the spheri-
cally shaped cells of Thermoplasma are compared with the lobed Sul/o-
lobus cells, the unusual morphology of Sul/olobus is emphasized.

Hexosamine Content
The absence of a peptidoglycan structure in the cell wall of Sul]o-
lobus recognizable with the electron microscope prompted a study of
hexosamine content of these organisms and a comparison with Thermo-
plasma. As a control, assays were also done on Bacillus acidocaldarius, a
rod-shaped organism with normal wall able to live at high temperature
and low pH.
As shown in Table 1, the Sul/olobus strains have low but detectable
amounts of hexosamine, and rather high amounts of carbohydrate. All
Thermoplasma strains are devoid of hexosamine, confirming and extend-
ing the results of Darland et al. (1970). AssaysofSul/olobushydrolysatesby
Dr. R. Wheat, Duke University, using the amino acid analyzer, failed
to detect muramic acid and showed that all of the hexosamine was
glucosamine. These data confirm the electron microscopic data indicating
the absence of peptidoglycan in Sul/olobus.

Effect of Antibiotics which Inhibit Cell Wall Synthesis

As a further indication of the unusual nature of the cell wall of
Sul/olobus, the effects of two antibiotics which inhibit cell wall synthesis,

Table 1. Biochemical composition o/acido-thermophilic bacteria

Protein I~NA Hexosamine Carbohydrate

Sul/olobus
98-3 1 0.18 0.054 0.33
106-3 1 0.16 0.047 0.30
115-2 1 0.16 0.055 0.38
129-1 1 0.20 0.029 0.34
140-5 1 0.26 0.030 0.21
Bacillus acidocaldarius 1 0.10 0.i26 0.i1
Thermoplasma
3-24 1 0.24 0 0.09
22-7 1 0.14 0 0.04
122-1B3 1 0.18 0 0.06
R8C 1 0.32 0 0.09
Relative proportions by weight of various components normalized to the
protein contents of the cell suspensions.
 Sul/olobus: A New Genus 63

r i s t o c e t i n a n d v a n e o m y c i n , were studied. (Penicillin, which is labile a t

low p t I , could n o t be used.) As a control on t h e a c t i v i t y of these anti-
biotics a t high t e m p e r a t u r e a n d low p H , t h e s p o r e - f o r m i n g r o d Bacillus
acidocaldarius was used. B o t h Thermoplasma a n d Sul]olobus were h i g h l y
r e s i s t a n t to r i s t o c e t i n a n d v a n c o m y c i n , a l t h o u g h t h e y were sensitive to
novobiocin, a n a n t i b i o t i c which does n o t i n h i b i t cell wall synthesis. These
d a t a , t a k e n t o g e t h e r w i t h t h e d a t a in t h e previous two sections, p r o v i d e
strong evidence t h a t Sul/olobus does n o t c o n t a i n a n o r m a l p e p t i d o g l y c a n -
c o n t a i n i n g cell wall.

Temperature Range
The t e m p e r a t u r e r a n g e for g r o w t h of a n u m b e r of Sul/olobus strains
was d e t e r m i n e d . T h e criterion for g r o w t h was visible t u r b i d i t y a n d t u r b i d
cultures were checked microscopically to confirm t h a t t u r b i d i t y was i n d e e d
due to t y p i c a l Sul/olobus.
As can be seen in T a b l e 2, all Sul/olobus cultures g e n e r a l l y fell in t h e
s a m e t e m p e r a t u r e range, w i t h a m i n i m u m of 55--60~ a n d a m a x i m u m of
75--85~ A f t e r p r o l o n g e d i n c u b a t i o n (2 weeks), m o s t strains showed
slight g r o w t h a t 40--45~ T h e g r o w t h of s t r a i n 140-5 a t 85~ was confirmed
on r e p e a t e d tests. The o p t i m u m t e m p e r a t u r e for all cultures a p p e a r e d to
be in t h e range of 70--75~ T h e t e m p e r a t u r e range of Thermoplasma is
c o n s i d e r a b l y lower, 37--65~ so t h a t only a r o u n d 6 0 - - 6 5 ~ is t h e r e a n y
o v e r l a p p i n g of t h e two genera.

pH Range
Also shown in T a b l e 2, Sul/olobus strains are all acidophilic. The p H
v a l u e s listed on t h e T a b l e are initial p H values. I n all cases, t h e final p H

Table 2. Temperature and pH range/or growtho/ Sulfolobus

Strain Temperature (~ pH
Minimum Maximum Minimum Maximum
98-3 55 80 1 5.9
106-3 55 80 1 5.9
111-3 55 75 1 4.5
115-2 55 80 1 5.9
129-1 60 80 1.5 5.8
132-1 55 80 1 5.3
136-1 55 80 1 4.6
140-5 55 85 1 5.8
140-6 60 80 1 5.8

Tested in basal salts with O.l~ yeast extract added. For temperature serms,
all media were adjusted to pH 2. For pH studies, incubation was at 70~ Growth
was recorded after 4--6 days incubation.
64 T.D. Brock, K. M. Brock, 1%.T. Belly, and 1%.L. Weiss:

of the culture was also measured. I n general, at low initial p H the p H

remained constant during incubation whereas at higher initial p H the p H
rose during incubation. The optimum p i t for growth of Sul]olobus, as
determined by observing the p H at which m a x i m u m turbidity was obtain-
ed, was between 2 and 3, and the range was from about 1 to 5.9. For
most of the strains, the p H limits were determined two or three times.
There were slight differences in the limits obtained in the several experi-
ments and the data presented in Table 2 are the lowest and highest p H
values at which growth was obtained. The optimum p H for Thermo-
plasma is about 2 but the range of p H is somewhat lower, from 0.5 to
4.3.
DNA Base Composition
The DNA base compositions of five Sul[olobus strains were: strain
98-3, 61~ GC; 106-3, 65o/0 GC; 115-2, 680/0 GC; 129-1,600/0 GC; 140-5,
66 ~ GC. Thus, all Sul]olobus strains studied had fairly high guanine plus
cytosine content of the DNA, whereas the Thermoplasma strains have
fairly low G q- C content, 24--290/0 (Darland et al., 1970; Belly and
Brock, unpublished). These data further emphasize the differences be-
tween the two genera. The strains of Sul[olobus isolated from I t a l y and
E1 Salvador had DNA base compositions in the same range as the Yellow-
stone strains.
Autotrophie Nutrition
All Sul]olobus strains isolated grow on elemental sulfur as a sole
energy source. Even those strains which were enriched, isolated, and
maintained for m a n y generations on yeast extract grew on sulfur. Like-
wise, strains isolated on sulfur all grew on yeast extract. Growth did not
diminish after m a n y transfers on elemental sulfur, and growth on sulfur
was stimulated b y adding C02 to the gas phase. Examination by fluores-
cence microscopy of acridine orange-stained cultures revealed numerous
cells of Sul/olobus attached to sulfur crystals. Oxidation of elemental
sulfur occurred as indicated b y sulfate production and a drop in p H of
the culture. I f an initial p i t of 3 was used, the final p H of sulfur-grown
cultures was usually 2 or less. Oxidation of asS-labelled elemental sulfur
has been demonstrated b y Douglas Shivvers in this laboratory and
details of these experiments will be reported elsewhere.
Growth was always better on sulfur plus yeast extract than on sulfur
alone. Further, the addition of sulfur to yeast extract medium led to
increased growth over yeast extract alone. Sulfur oxidation was not
inhibited b y yeast extract, since the amount of sulfate produced was the
same in media with and without yeast extract, although the drop in p i t
did not occur, probably due to the buffering action of yeast extract.
Thiosulfate and hydrogen sulfide have not been tested as inorganic energy
 Sul[olobus: A New Genus 65

sources since at the temperatures and p H values used b o t h are rapidly

converted to elemental sulfur spontaneously.
Growth experiments performed with Thermoplasma indicated t h a t
neither autotrophic growth nor sulfur oxidation occurred, further
emphasizing the differences between Thermoplasma and Sul]olobus.

Heterotrophic Nutrition
Table 3 presents a s u m m a r y of d a t a on the heterotrophic nutrition of
five strains of Sullolobus. All strains grew on media containing yeast
extract, tryptone, peptone, or casein hydrolysate. Visible g r o w t h
occurred at concentrations of yeast extract as low as 0.01 ~ a n d best
g r o w t h occurred at 0.1~ A concentration of yeast extract of 0.25~
resulted in less g r o w t h t h a n 0.1~ suggesting toxicity at higher y e a s t
e x t r a c t concentrations. The doubling time measured on several strains
grown on 0.1~ yeast e x t r a c t varied from 6 . 5 - - 8 h.
All strains grew in synthetic m e d i u m with single compounds as sole
sources of carbon and energy, although the strains differed in the specific
c o m p o u n d s which t h e y used (Table 3). All strains used alanine and gluta-
mine a n d m o s t strains used g l u t a m a t e and aspartate. None of the strains
grew on phenylalanine, histidine, proline, or leueine. Of the sugars,
fructose was n o t used at all a n d glucose, galaetose, and lactose were
used only b y one strain. Sucrose, on the other hand, was used b y m o s t
strains, and ribose b y all. Strain 129-1 in particular seemed more adept

Table 3. Heterotrophic nutrition o] Sulfolobus strains

Nutrient Strain
98-3 106-3 115-2 129-1 140-5
Yeast extract + + + + +
Tryptone + + + + +
Peptone + -4- -4- -4- +
Casamino acids + -4- + + +
Casein hydrolysate + + + + +
Glutamate + -k -k :k +
Glutamine + i + =t= +
Alanine + + -[- • +
Aspartate + + + -- +
Fructose . . . . .
Glucose -- -- -- + --
G a l a e t o s e -- -- -- -}- --

Sucrose + + -- + +
Lactose -- -- -- + --

Ribose + + + + +
All tested at 0.1 ~ concentrations in basal salts at pH 2, incubation tempera-
ture 70~ -4- good growth; :L fair growth; -- no growth. No growth was obtained
with any strain with the amino acids phenylalanine, histidine, proline, or leucine.
5 Arch.B{ikrohioI.,Bd. 84
66 T. D. Brock, K. 1~I.Brock, R. T. Belly, and R. L. Weiss:

at using sugars and less adept at using amino acids than other strains.
Although glucose was not used by most strains as sole carbon source, it
stimulated the growth of all strains when present together with yeast
extract.
Phosphate at 0.1 ~ inhibited growth completely.
Thermoplasma, on the other hand, has complex nutritional require-
ments and good growth occurred only on a medium containing yeast
extract (Darland et al., 1970). Glucose did stimulate growth of all strains
in a yeast extract-containing medium (K. M. and T. D. Brock, unpublished
observations).
Species
I t seems preferable at the present to recognize only a single species of
Sul]olobus, which we propose to designate Sul]olobus acidocaldarius sp.
nov. The generic name is derived from the Latin noun sul]ur meaning
sulfur and the Latin noun lobus meaning lobe. The specific epithet is
derived from the Latin word acidus meaning acid and the Latin word
caldarius pertaining to warm or hot. Thus Sul]olobus acidocaldarius is a
lobed suffur-oxidizing organism which lives in acid thermal habitats.
Strain 98-3 is designated as the type. It was isolated from Locomotive
Spring in Yellowstone I~ational Park, the spring having a pH of 2.4 and a
temperature of 83~ It will be deposited with the American Type Culture
Collection.
Discussion
It seems clear from the data presented that we are dealing with an
entirely new group of organisms. The characteristics of Sul/olobus can be
summarized as follows: 1. generally spherical organisms producing
frequent lobes; 2. facultative autotrophs growing on sulfur or on a
variety of simple organic compounds; 3. unusual cell wall apparently
devoid of peptidoglycan; 4. acidophilie, with pH optimum of 2--3 and
range from 0.9--5.8; 5. thermophilic, with temperature optimum of
70--75~ and range from 55--85~ As far as we can tell, Sul]olobus has no
close relationships with any previously described bacteria, either hetero-
trophic or autotrophic. C. L. Brierley has recently characterized a sulfur-
oxidizing isolate which may be Sul]olobus, although it differs in some
respects (Brierley, 197i). Brierley's organism has a lower temperature
range (45--70~ a less well-defined cell wall although the DNA base
composition of this organism (60% G A- C, personal communication) is
in the range of the present Sul]olobus isolates.
In the initial stages of this work we were faced with the task of dis-
tinguishing Sul]olobus from ThermoTlasma, another organism of hot
acid environments. With experience, this distinction proved relatively
easy, even with the phase microscope, although when in doubt electron
 Sul/olobus: A New Genus 67

microscopy permitted an unequivocal distinction. The presence of a wall

on Sul]olobus provides the best method for distinguishing Sul/olobus from
Thermoplasma, and simple negative staining with nranyl acetate is suffi-
cient (ef. Figs. 5 and 6) to determine whether a wall exists.
The wall of Sul/olobus is of interest because it appears to be different
from that of other bacteria, either gram-positive or gram-negative. The
lack of a peptidoglycan-containing wall, clearly shown here, is not
unprecedented, as this seems also to be the case in extreme halophiles
(Larsen, 1967). The wall, and perhaps the membrane, are the only cellular
structures which must be exposed to high hydrogen ion concentration of
the environment and conceivably the unusual wall of Sul/olobus may be a
factor permitting adaptation to this extreme condition.
The facultative autotrophy of Sul]oIobus is of interest in relation to
the enrichment procedures used in its isolation. Both heterotrophic and
autotrophie enrichments gave similar isolates. In most habitats it would
be very unlikely for heterotrophic enrichments to yield sulfur-oxidizing
organisms and the fact that Sul/olobus is routinely isolated in that way
suggests that in the hot acid environments in which it exists other
organisms are not present.
Detailed studies on the distribution of Sul]olobus have been completed
by Brock and l~liermans and will be reported elsewhere. It is clear from
the data presented here that it is an organism widely distributed in very
hot and very acid environments.
The probable geochemical significance of Sul]olobus should also be
noted. Until the present work, organisms able to oxidize sulfur at
temperatures above 55--60~ had not been isolated, and geoehemists had
difficulty in explaining the origin of sulfuric acid in high temperature
springs (Sehoen and l~ye, 1970). Although we have not shown directly
that Sul]olobus is responsible for sulfuric acid production in high temper-
ature environments, its ubiquity in such habitats suggests that it is of
major importance in acid production. Studies directly in solfatara areas
using radioisotopes are currently underway and should provide data
relevant to this point.
We should note that in moderately hot habitats (40--55~ thermo-
philie forms of Thiobacillus are very common (Kaplan, 1956; Sehoen and
Ehrlich, 1968; Schwartz and Schwartz, 1965), and are probably respon-
sible for sulfur oxidation. It is only in habitats above 60--65~ that
Sul]olobus is commonly found.
In addition to its ecological and geochemical interest, Sul]olobus may
prove to be a useful organism for studies on the biochemistry of sulfur
oxidization and autotrophic COs fixation. If it resembles other thermo-
philes, it should produce heat stable enzymes, making purification more
facile and thus permitting detailed biochemical study.
5*
68 T.D.Broek, K.M.Brock, R.T. Belly, and L.Weiss: Sul[olobus: A New Genus

Acknowledgements. This work was supported by research grants from the Natio-
nal Science Foundation (GB-7815, GB-19138 and GB-30075). R. Weiss received
support from a Microbiology Training Grant from the U.S. Public Health Service
(5 T1-GM-503). The technical assistance of Diane Smith, D. Simon, H. Pearce and
K. Boylen is gratefully acknowledged. R. T. Belly was a post-doctoral fellow of
the U.S. Public Health Service.

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Prof. Dr. T. D. Brock

Dept. of Bacteriology
University of Wisconsin
Madison, Wisconsin 53706, U.S.A.