Download Chromatium tepidum sp. nov. a Thermophilic Photosynthetic

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JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 222-227
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Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 2
Chromatium tepidum sp. nov. a Thermophilic Photosynthetic
Bacterium of the Family Chromatiaceae?
MICHAEL T. MADIGAN
Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901
A new species belonging to the photosynthetic bacterial genus Chromatium is described. This new organism
differs from all other Chromatium species in its thermophilic character and hot-spring habitat. In addition, the
combination of its carotenoid pigments, physiological peculiarities, and deoxyribonucleic acid base composition
clearly define this isolate as a new species of photosynthetic purple bacteria. The organism is a rod-shaped,
gram-negative bacterium which produces bacteriochlorophylla,, and grows photoautotrophically with sulfide
as an electron donor at an optimum temperature of 48 to 50°C. No growth is observed below 34°C or above
57OC. Globules of elemental sulfur are produced from the oxidation of sulfide and are stored intracellularly.
Acetate and pyruvate are the only organic compounds that are photoassimilated. The major carotenoids of the
new organism are rhodovibrin and spirilloxanthin, and the deoxyribonucleic acid base composition is 61 mol%
guanine plus cytosine. Based on these characteristics, I propose a new species, Chromatium tepidum; the specific
epithet refers to the moderately thermophilic nature of this hot-spring photosynthetic bacterium.
Purple sulfur bacteria were observed in warm thermal
springs as early as 1897 (13). From recent surveys of
photosynthetic procaryotes that inhabit thermal springs it is
clear that purple bacteria which resemble Chromatium are
present at temperatures between 40 and 60"C, especially in
springs containing significant levels of hydrogen sulfide
(1-3). The first pure culture of a purple bacterium capable of
growth at temperatures above 50°C was obtained by me and
was identified as a Chromatium species by its rod-shaped
morphology, assemblage of pigments, and ability to oxidize
sulfide and store elemental sulfur intracellularly (8). Because
the characteristics of this organism are unique among published descriptions of purple sulfur bacteria (family
Chromatiaceae), the new organism is more thoroughly characterized in this paper and is described as a new species of
the genus Chromatium, Chromatium tepidum.
trace element solution containing deionized water (1,OOO ml),
ethylenediaminetetraacetate (5.2 g), FeC12 4H20 (1.5 g),
ZnC12 (70 mg), MnC12 - 4H20 (100 mg), H3B03 (6 mg),
CoC12 * 6H20 (190 mg), CuC12 * 2H20 (17 mg), NiC12 - 6H20
(25 mg), Na2Mo04 - 2H20 (188 mg), V0S04 * 2H20 (30 mg),
and Na2W04 - 2H20 (2 mg). The modified enrichment medium lacked yeast extract and vitamin BI2.
Growth conditions. Cultures were grown photosynthetically in completely filled 17-ml to 1-liter tubes or in bottles
that were incubated in a water bath or in a light cabinet at
48°C. Freshly inoculated vessels were always placed in
darkness for 2 to 4 h before they were incubated photosynthetically at 1,500 to 2,000 lx of incandescent illumination.
Absorption spectra. Spectra of intact cells were recorded
by suspending sulfur-free cells in 30% bovine serum albumin
(10) and measuring absorption spectra with a Perkin-Elmer
model 552A ultraviolet-visible double-beam spectrophotometer against 30% bovine serum albumin. Pigment extracts
were obtained by extracting pellets for 30 min with acetonemethanol (7:2) in darkness at -20°C. Following centrifugation, the absorption spectra of the extracts were measured
against acetone-methanol blanks.
Electron microscopy. Cells were fixed by the method of
Ryter et al. (21), dehydrated in an ethanol series, and
embedded in Spurr low-viscosity embedding medium. Sections were stained with uranyl acetate and lead citrate and
examined with a Philips model 1300 electron microscope at
60 kV.
DNA composition. The deoxyribonucleic acid (DNA) base
ratio of C. tepidum was kindly determined by Henry Burr,
Wayne State University, Detroit, Mich., by using Escherichia coli, Micrococcus luteus, and chicken DNAs as standards. DNA from C. tepidum that was purified by the
method of Marmur and Doty (12) was subjected to thermal
denaturation, and the guanine-plus-cytosine content was
calculated from the melting profile.
MATERIALS AND METHODS
Source of the organism. Enrichment cultures were
established from reddish mat material that was attached to the
calcareous sinter of a small hot spring in the Upper Terrace
area of Mammoth Hot Springs, Yellowstone National Park.
The series of springs sampled were the so-called Stygian
Springs, which lie on top of a ridge in the southwest corner
of the Upper Terraces. The springs in this area have been
mapped by Castenholz (2), and the Stygian Springs are
indicated by this author.
Media. The new organism was isolated in a modified
version of the standard liquid enrichment medium used for
photosynthetic purple bacteria described by Pfennig (14).
This medium contained (per liter of deionized water) 200 mg
of MgS04 - 7H20, 50 mg of CaC12 * 2H20, 400 mg of NH4C1,
400 mg of NaCl, 0.5 g of KH2P04,1g of sodium acetate, 1ml
of a trace elements solution (15), 1 g of Na2S 9H20, 2 g of
NaHC03, 100 mg of yeast extract, and 20 pg of vitamin B12.
Pure cultures were grown in the enrichment medium in early
experiments, but in most of the work cultures were grown in
enrichment medium modified so that it contained 500 mg of
ammonium acetate in place of sodium acetate and 1 ml of a
-
RESULTS
Isolation and culture. During a survey of alkaline hot
springs for photosynthetic purple bacteria, thin reddish mats
were observed in certain sulfide-rich springs in the Upper
Terrace region of Mammoth Hot Springs, Yellowstone National Park. These springs are rich in calcium carbonate and
t This paper is dedicated to Thomas D. Brock, who first introduced me to the fascinating world of hot-spring microbiology.
222
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VOL. 36, 1986
THERMOPHILIC CHROMA TZACEAE
223
FIG. 1. Photomicrographs of C. tepidum. (A) Cells from mid-logarithmicgrowth filled with elemental sulfur (arrow). (B) Sulfur-free cells
from stationary-phase cultures. The cells were grown photoheterotrophically with sulfide, acetate, and C 0 2 at 48°C. Bar = 3 pm.
contain sulfide of geochemical origin at concentrations of
about 2 mg/liter (2). The thin mats were firmly embedded in
carbonaceous sinter from the springs and were composed of
rod-shaped bacteria, most of which contained bright
refractile globules that strongly resembled the sulfur globules of purple sulfur bacteria. Material collected from one
inoculated into a sulfide-acetate enrichsuch spring (44”C),
ment medium, and incubated photosynthetically at 52°C
yielded a bright red-pigmented culture within 72 h. The
spring which was sampled smelled of sulfide and was devoid
of cyanobacteria. Other rod-shaped bacteria that were free
of refractile globules also were present in the mat sample.
The primary enrichment culture was observed microscopically, and a 5% inoculum was transferred to fresh medium.
The predominant organism was a highly motile rod-shaped
bacterium containing sulfur globules, which displayed a
distinct phobophototactic response (24) when the microscope field was darkened. The major contaminant was a long
thin rod-shaped organism that was devoid of sulfur globules.
The transferred enrichment grew within 24 h and was kept at
4°C for 2 weeks before pure culture isolation was performed.
A pure culture of the new organism was eventually obtained following successive applications of the agar shake
culture technique (17). Bacto-Agar (Difco Laboratories) was
the solidifying agent used. By the third shake culture series
the only remaining contaminant was the long thin rod-shaped
organism mentioned above. In shake cultures prepared
thereafter the long thin rod-shaped organism was present in
low numbers until shake tubes were prepared by using agar
that had been washed with deionized water, followed by
washes in ethanol and then acetone. The washes converted
the slightly yellow agar preparation into a much whiter
crystalline powder. Media prepared from this “purified”
agar readily yielded pure cultures of the thermophilic
phototroph following two shake tube applications. Appar-
ently, crude agar preparations were supplying the contaminant with needed nutrients. Liquid cultures of the new
isolate, previously referred to as Chromatium sp. strain MCT
(T = type strain) (8), were grown in the modified enrichment
medium described in Materials and Methods, and samples of
log-phase cells were frozen at -80°C in growth medium
containing 10% glycerol; frozen cultures remained viable for
more than 1 year.
Morphology. C . tepidum consists of rod-shaped cells
which measure 1.2 by 2.8 to 3.2 p,m and occasionally form
short chains of two to four cells (Fig. 1). Cells in young
cultures (Fig. 1A) contain two or more sulfur globules, while
cells in stationary phase (Fig. 1B) are generally sulfur free.
Although motile cells are occasionally observed, pure cultures of the new organism have not been observed to be as
highly motile as the original enrichment cultures. Electron
microscopy of thin sections of C . tepidum (Fig. 2 ) revealed
features that are typical of Chromatium species. The intracellular membranes of C. tepidum are of the vesicular
(chromatophore) type (Fig. 2). Large holes in the cell periphery are frequently observed, and these presumably represent
the locations of elemental sulfur or poly-P-hydroxybutyrate
granules before the embedding and dehydration process;
large amounts of poly-P-hydroxybutyrate are produced by
cells of C. tepidum that are grown photoheterotrophically on
acetate. A fibrous material arranged in clumps (Fig. 2) was
observed in the cytoplasm of thin sections of C. tepidurn,
and these structures appeared to be loosely attached via
membrane connections to the cytoplasmic membrane; their
function, if any, is unknown.
The cell wall of C. tepidum is morphologically distinct.
Outside what appears to be a typical lipopolysaccharide
layer, a dense, darkly staining area of unknown chemical
composition is present (Fig. 2). Although chemical assays of
the cell wall of C. tepidurn have not been performed, the
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INT. J. SYST.BACTERIOL.
MADIGAN
aoa
FIG. 2. Electron micrograph of thin sections of C. tepidum cells.
The arrow indicates the intracellular membrane of the
chromatophore type. Note the darkly staining outer cell wall layer.
Bar = 0.5 pm.
organism is highly susceptible to penicillin and therefore
presumably contains peptidoglycan. However, the thickness
of this outer layer is not typical of peptidoglycan in gramnegative bacteria and the layer may consist of other polysaccharide material. In addition to penicillin, which inhibits
the growth of C. tepidum at a concentration of 10 pg/ml, the
cell wall inhibitors vancomycin and cycloserine also inhibit
the growth of C. tepidum at the same concentration. The
protein synthesis inhibitors chloramphenicol and oxytetracycline are growth inhibitory at a concentration of 10 yg/ml
as well.
Pigments. C . tepidurn produces bacteriochlorophyll a as
its sole bacteriochlorophyll. Spectra of intact cells suspended in 30% bovine serum albumin are shown in Fig. 3.
Prominent peaks are observed at 858, 808, and 599 nm.
Acetone-methanol extracts (Fig. 4)produce peaks at 770 and
596 nm, which are typical of bacteriochlorophyll a (4). The
esterifying alcohol of the bacteriochlorophyll a of C .
tepidum is phytol, based on high-pressure liquid chromatography-mass spectrometer analyses kindly performed by
Hans Brockmann, Universitat Bielefeld.
The carotenoids of C . tepidum are mostly carotenoids of
the normal spirilloxanthin series (22) ; however, the proportions of the various components are not typical of any known
Chromatium species. The major carotenoid, as kindly determined by Karin Schmidt, Universitat Gottingen, is
rhodovibrin (or a rhodovibrinlike compound), and this pigment makes up nearly 50% of the total carotenoid pool.
Other major carotenoids include spirilloxanthin (20%),
rhodopin (15%), anhydrorhodovibrin (12%), lycopene (3%),
and demethylated spirilloxanthin (3 %). In addition, a small
amount (-2%) of glycosidic carotenoids of unknown chemical structure is produced by C. tepidum.
Physiology and biochemistry. C . tepidum is an obligately
phototrophic bacterium. This organism requires substrate
levels of sulfide for growth under any nutritional conditions
and grows photoautotrophically in mineral media containing
HC03- and C 0 2 as sole carbon sources. No vitamins,
400
500
600
700
800
900
Wavelength (nm)
FIG. 3. Absorption spectra of intact cells of C. tepidurn. The
cells were suspended in 30% bovine serum albumin.
including vitamin B12, are required for growth. The sulfide
tolerance of strain MCT is fairly high; cultures routinely
grow in the presence of 2 to 3 mM sulfide, and the upper limit
is near 4 mM sulfide. Acetate and pyruvate are photoassimilated (in the presence of sulfide) and substantially increase
cell yields. No other organic compounds tested (various
organic and amino acids, fatty acids, sugars, and complex
~
770
477
1
400
500
1
600
700
WAVELENGTH (nm)
800
FIG. 4. Absorption spectra of acetone-methanol (7:2) extracts of
C . tepidurn cells.
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VOL. 36, 1986
I
THERMOPHILIC CHROMA TZACEAE
I
I
I
I
1
57O
0
I
10
I
20
I
30
I
40
1
4
1
50
Time (h)
FIG. 5. Growth rate of C. tepidum as a function of temperature.
Cells were grown photoheterotrophically (acetate, COz, and sulfide)
at 2,000 lx at different temperatures and counted as described
previously (8, 9).
substrates, such as yeast extract and Casamino Acids)
stimulate the growth of C. tepidum. The only nitrogen
sources utilized by C. tepidum are ammonia, urea, and
glutamine; N2 does not support growth as a sole nitrogen
source when it is tested at either 37 or 48°C.
The sulfide requirement of C. tepidum does not simply
reflect a need for a biosynthetic sulfur source. No growth of
C. tepidum is obtained in growth tests when sulfide is
replaced with either cysteine (2.5 to 5 mM), thiosulfate (2 to
8 mM), or sulfite (0.5 to 2 mM) in a medium containing
acetate as the carbon source. Hence, sulfide probably serves
a dual role in the metabolism of C. tepidum (as a photosynthetic electron donor and as a biosynthetic sulfur source).
Hydrogen and thiosulfate do not serve as electron donors in
place of sulfide, nor do they stimulate the growth of C .
tepidum above the level achieved on sulfide alone. When the
gradient technique of Kampf and Pfennig (7) was used, no
dark growth of C. tepidum was obtained in a medium
containing sulfide and acetate as energy sources.
A major difference between recognized species of
Chromatium and C. tepidum concerns the temperature requirements for growth. Reflecting its hot spring origin, C.
tepidum grows optimally at temperatures of 48 to 50"C, but
it also grows at reasonable rates at temperatures as low as
37°C and as high as 55°C (Fig. 5). The minimum generation
time at the optimum growth temperature is about 3.5 h (8).
The mass doubling times at 55 and 38°C are 25 and 10 h,
respectively. Slow growth of C. tepidum occurs at 56 to
57°C; however, cells grown at this temperature are considerably smaller than cells grown at 48°C and produce long
chains, suggesting that the organism is experiencing problems with cell division. The minimum growth temperature
for C. tepidum has not been determined precisely, although
cultures incubated photosynthetically at 33 to 34°C did not
develop, while cultures at 37°C did. Fully grown cultures of
C. tepidum remain viable for days at room temperature if
they are left in an unopened, anaerobic state; cultures
exposed to oxygen lose viability more quickly. Cells of C.
225
tepidum withstand freezing at -80°C if they are suspended in
fresh growth medium containing 10% glycerol.
DNA base composition. The DNA of C. tepidum strain
MCT contains 61 mol% guanine plus cytosine, as determined
by thermal denaturation.
Ecology. It is likely that the natural habitat of C. tepidum
is warm to moderately hot (up to -60°C) neutral to alkaline
hot springs containing sulfide. Although strain MCT is the
only strain to be studied in any detail, a second strain of an
organism that strongly resembles C. tepidum was isolated
from a New Mexico hot spring (8). The New Mexico isolate
differs from strain MCT in that the cells are slightly more
narrow and cultures are considerably less sulfide tolerant. Of
possible significance in the latter regard is the fact that the
New Mexico strain originated from filamentous cyanobacterial mat material and therefore may represent a less sulfidetolerant variant of C. tepidum. Like C. tepidum, however,
the New Mexico strain grows at 50"C, oxidizes sulfide, and
deposits elemental sulfur intracellularly .
DISCUSSION
Most of the properties of the thermophilic photosynthetic
bacterium described in this paper readily identify it as a
member of the genus Chromatiurn, including (i) the rodshaped cells, (ii) bacteriochlorophyll up as the sole bacteriochlorophyll, (iii) carotenoids of the normal spirilloxanthin
series, (iv) intracytoplasmic membranes of the vesicular
type, and (v) photoautotrophic growth with sulfide as the
electron donor and accumulation of elemental sulfur
intracellularly. When the properties of C.tepidum are compared with those of other small-celled species of
Chromatiurn, however, there are several differences (Table
1). The most obvious difference relates to the optimum
temperature for growth. C. tepidum grows optimally at 48"C,
whereas all other Chromatium species grow optimally at
temperatures between 25 and 35°C (16). Therefore, the
thermophilic character alone makes C. tepidum unique
among species of the Chromatiaceae. It should be pointed
out that some of the extremely halophilic Ectothiorhodospira species isolated from African saline lakes by Imhoff
and Triiper also show a rather high optimum temperature.
For example, Ectothiorhodospira halochloris grows optimally at 45 to 48°C (5). However, the optimum growth
temperature of E. halochloris is very close to its maximum
growth temperature (-50°C); C. tepidum is the only purple
sulfur bacterium that is capable of growth at temperatures
above 50°C. On the other hand, the thermophilic property of
C. tepidum is not extreme enough to suggest that the
organism is an archaebacterium. Indeed, the antibiotic susceptibility of C. tepidum indicates it is not an archaebacterium, and a preliminary analysis of the 16s ribosomal
ribonucleic acid sequences clearly identified C. tepidum as a
eubacterium (Carl Woese, personal communication).
The color of mass suspensions of C. tepidum resembles
the colors of suspensions of Chromatium vinosum and
Chromatium gracile (i.e., brownish red), but cultures of C.
tepidum are distinctly more red than cultures of the other
two species. This may be due to the predominance in C.
tepidum of rhodovibrin, a carotenoid that is essentially
absent from C. vinosum and C.gracile (22). Rhodovibrin is
not a common carotenoid in photosynthetic bacteria. It is
found in trace amounts in a variety of purple bacterial
species, but in substantial amounts only in the nonsulfur
purple bacterium Rhodospirillum photometricum and in certain strains of Rhodopseudomonas palustris and Rhodospirillum rubrum (22). C. tepidurn is morphologically similar
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INT. J. SYST.BACTERIOL.
MADIGAN
TABLE 1. Characteristics of C. tepidum and other small-celled Chromatium species“
Cell size (pm)
Species
C. tepidum
C . vinosum
C. minus
C. purpuratum
C . violascens
C. gracile
Width
Length
1.2
2
2
1.2-1.7
2
1.2
2.8-3.2
2.5-6
2.5-6
3
2.5-6
2-6
COlOP
Red
Brownish red
Purplish red
Purplish red
Purplish violet
Brownish red
Rhodovibrin, spirilloxanthin
Spirilloxanthin, lycopene, rhodopin
Okenone
Okenone
Rhodopinal
Spirilloxanthin, lycopene, rhodopin
61.5
64.3
52
68.4
62.2
69.9
-
+
+
+
+
Yes
No
Yes
No
No
No
-
+
+
+
+
-
Comparative data obtained from Imhoff and Truper (6) and Truper and Pfennig (26).
Photosynthetically grown cell suspensions.
Type strains (11, 16).
Heterotrophic or lithotrophic growth under microaerobic conditions (7).
to the small-celled Chromatium species Chromatium minus,
Chromatium purpuratum, and Chromatium violascens, but
clearly differs from these species in carotenoid content and
guanine-plus-cytosine base ratio (Table l), as well as in
thermal characteristics .
C . tepidum shares the following interesting physiological
characteristic with large-celled species of Chromatium
(Chromatium okenii, Chromatium weissei, and Chromatium
buderi) and with Thiospirillum jenense: the only organic
compounds photoassimilated are acetate and pyruvate. According to the classification of Triiper (25), C . tepidum
should therefore be included with the species listed above in
the nutritionally restricted Chromatiaceae I group. By contrast, the members of the Chromatiaceae I1 group ( C .
vinosum, C . violascens, C . gracile, C . purpuratum, and
others) photoassimilate, in addition to acetate and pyruvate,
C-4 citric acid cycle intermediates, fatty acids with longer
chains than acetate, and certain sugars (25) (Table 1).
Representatives of the Chromatiaceae I group also lack an
assimilatory sulfate reduction pathway and are unable to
grow under any nutritional conditions in darkness. These
properties are also observed in C. tepidum (Table 1) and
serve to further separate this species from the remaining
small-celled Chromatium species.
The observations of Castenholz (2) suggest that the
thermophilic chromatia are widely distributed in high-sulfide
neutral-pH springs in the Mammoth Hot Springs region, but
are generally absent from the springs dominated by stands of
Chloroflexus. Strains of Chloroflexus isolated from these
springs are dark green in color, reflecting their high bacteriochlorophyll contents, and are unable to grow by respiratory
means (R. W. Castenholz and S. J. Giovannoni, Abstr.
Annu. Meet. Am. SOC. Microbiol. 1981, 179, p. 100).
Anaerobic strains of Chloroflexus predominate in these
springs probably because the source temperature (-70°C)
excludes the development of C . tepidum-like organisms and
the sulfide content (1to 2 mglliter) excludes the development
of high-temperature species of cyanobacteria, such as
Synechococcus (2). Around 50 to 55°C thermophilic
chromatia begin to appear along with Spirulina, a major
cyanobacterial representative in sulfide-containing waters
with temperatures below 50°C (2). In high-sulfide springs at
temperatures of 45 to 50°C it is likely that Chromatium is the
favored photosynthetic bacterial species, since pure cultures
of C . tepidum grow optimally near 50°C and pH 7 and
Chloroflexus grows optimally at 55°C and at a more alkaline
pH (18, 19). In the spring from which C . tepidum was
isolated, for example, the temperature was 44”C, the pH was
7, and no cyanobacteria were present. C . tepidum probably
experiences little competition from Chloroflexus in such
springs, because both the temperature and the pH are
substantially below the optimum values for Chloroflexus.
A thermophilic filamentous photosynthetic bacterium that
resembles Chlorojlexus but lacks bacteriochlorophyll c, (or
any other type of chlorobium chlorophyll) and chlorosomes
has been described recently (20). Although not yet in pure
culture, this organism, which has been given the provisional
name “Heliothrix,” contains bacteriochlorophyll a and appears to be physiologically similar to the nonsulfur purple
photosynthetic bacteria and Chlorojlexus (18). “Heliothrix”
grows in cocultures with the filamentous aerobic heterotroph
Zsocystis pallida and photoassimilates organic compounds
under both aerobic and anaerobic conditions (20). In one
important respect “Heliothrix” resembles the recently described aerobic marine pigmented bacteria (23), because it
synthesizes bacteriochlorophyll under fully aerobic conditions (20). This is in contrast to what is observed in nonsulfur
purple bacteria and in Chloroflexus, in which oxygen serves
as a potent repressor of bacteriochlorophyll synthesis (4,
19). Additionally, “Heliothrix” requires unusually high light
intensities for growth and synthesizes carotene derivatives
(as does Chloroflexus [18]) as carotenoids (20). Hence,
“Heliothrix” shares properties with a variety of phototrophic bacteria, but shares relatively little with C . tepidum.
Interestingly, however, the upper temperature limit for
photoassimilation of acetate (and, presumably, also for
growth) for “Heliothrix” is about 56°C (20), the same as the
upper limit of C. tepidum. Perhaps this is the upper temperature limit for photosynthetic purple bacteria in general.
Based on the novel assemblage of properties shown by
pure cultures of the thermophilic purple sulfur bacterium
described above, this organism, provisionally assigned to the
genus Chromatium and referred to previously ( 8 ) as
Chromatium sp. strain MCT, is proposed as a new species of
the genus Chromatium, Chromatium tepidum.
Description of Chromatium tepidum. Chromatium tepidum
(tepi.dum. L. neut adj. tepidum lukewarm) cells are rod
shaped and 1 to 2 pm wide by 2.8 to 3.2 pm long. Gram
negative, occasionally motile. Photosynthetic intracytoplasmic membrane system of the vesicular type.
Pigments. Absorption spectra of intact cells show peaks at
858, 808, and 599 nm, which are typical of bacteriochlorophyll a . The esterifying alcohol of bacteriochlorophyll a is phytol. Carotenoids of the normal spirilloxanthin series are present; rhodovibrin and spirilloxanthin
predominate.
Physiology. Obligately phototrophic. Growth occurs
photoautotrophically in mineral media supplemented with
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THERMOPHILIC CHROMATIACEAE
sulfide as electron donor. Sulfur is formed as an intermediate
product, which is further oxidized to sulfate. Hydrogen and
thiosulfate are not utilized as electron donors. Acetate and
pyruvate are photoassimilated in the presence of sulfide;
citric acid cycle intermediates, fatty acids other than acetate,
and sugars are not utilized. Ammonia, urea, and glutamine
serve as nitrogen sources. No growth factors are required.
Optimum pH, 7. Optimum growth temperature, 48 to 50°C;
no growth below 34°C or above 57°C. NaCl is not required
for growth and is growth inhibitory at concentrations above
1%(wthol). Catalase negative.
Storage material. Poly-P-hydroxybutyrate is a storage
material.
DNA base composition. The DNA contains 61.5 mol%
guanine plus cytosine, as determined by thermal denaturation.
Habitat. The habitat of C. tepidum: sulfide-containing hot
springs of neutral to alkaline pH at temperatures below 60°C.
Type strain. The type strain is strain MC, which was
isolated from Stygian Springs, located in the Upper Terraces
of Mammoth Hot Springs, Yellowstone National Park. This
strain has been deposited with the American Type Culture
Collection as strain ATCC 43061T.
ACKNOWLEDGMENTS
This work was supported by grant DMB-8505492from the National
Science Foundation and by the Coal Research Center of Southern
Illinois University.
I thank the National Park Service, U.S. Department of the
Interior, for permission to sample in Yellowstone National Park. I
especially thank Dave Ward for help in field sampling, Rudy Turner
for taking the electron micrographs, Joseph Mayers, Jr., for helpful
discussions, Sharon Cox for technical assistance, and Hans Truper
for suggesting the species epithet.
ADDENDUM IN PROOF
Near infra-red spectroscopy of membrane preparations
from cells of C. tepidum shows a distinct absorption peak at
910 nm due to an unusual antennae form of bacteriochlorophyl a (Andre VermCglio, personal communication). In
addition, “Heliothix’ ’ has been given formal taxonomic
standing with the description of Heliothrix oregonensis, gen.
nov. sp. nov. (B. K.Pierson, S. J. Giovannoni, D. A. Stahl,
and R. W. Castenholz. 1985. Arch. Microbiol. 142:164-167).
LITERATURE CITED
1. Brock, T. D. 1978. Thermophilic microorganisms and life at high
temperatures. Springer-Verlag, New York.
2. Castenholz, R. W. 1977. The effect of sulfide on the blue-green
algae of hot springs. 11. Yellowstone National Park. Microb.
Ecol. 3:79-105.
3. Castenholz, R. W. 1979. Evolution and ecology of thermophilic
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