INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1986, p. 222-227 Vol. 36, No. 2 OO20-7713/86/020222-06$02.OO/O Copyright 0 1986, International Union of Microbiological Societies

Chromatium tepidum sp. nov. a Thermophilic Photosynthetic Bacterium of the Family ? MICHAEL T. MADIGAN Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901

A new species belonging to the photosynthetic bacterial 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 . The organism is a rod-shaped, gram-negative bacterium which produces bacteriochlorophyll a,, 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 trace element solution containing deionized water (1,OOO ml), springs as early as 1897 (13). From recent surveys of ethylenediaminetetraacetate (5.2 g), FeC12 4H20 (1.5 g), photosynthetic procaryotes that inhabit thermal springs it is ZnC12 (70 mg), MnC12 - 4H20 (100 mg), H3B03 (6 mg), clear that which resemble Chromatium are CoC12 * 6H20(190 mg), CuC12 * 2H20 (17 mg), NiC12 - 6H20 present at temperatures between 40 and 60"C, especially in (25 mg), Na2Mo04 - 2H20 (188 mg), V0S04 * 2H20 (30 mg), springs containing significant levels of hydrogen sulfide and Na2W04 - 2H20 (2 mg). The modified enrichment me- (1-3). The first pure culture of a purple bacterium capable of dium lacked yeast extract and vitamin BI2. growth at temperatures above 50°C was obtained by me and Growth conditions. Cultures were grown photosyntheti- was identified as a Chromatium species by its rod-shaped cally in completely filled 17-ml to 1-liter tubes or in bottles morphology, assemblage of pigments, and ability to oxidize that were incubated in a water bath or in a light cabinet at sulfide and store elemental sulfur intracellularly (8). Because 48°C. Freshly inoculated vessels were always placed in the characteristics of this organism are unique among pub- darkness for 2 to 4 h before they were incubated photosyn- lished descriptions of (family thetically at 1,500 to 2,000 lx of incandescent illumination. Chromatiaceae), the new organism is more thoroughly char- Absorption spectra. Spectra of intact cells were recorded acterized in this paper and is described as a new species of by suspending sulfur-free cells in 30% bovine serum albumin the genus Chromatium, Chromatium tepidum. (10) and measuring absorption spectra with a Perkin-Elmer model 552A ultraviolet-visible double-beam spectrophotom- MATERIALS AND METHODS eter against 30% bovine serum albumin. Pigment extracts Source of the organism. Enrichment cultures were were obtained by extracting pellets for 30 min with acetone- established from reddish mat material that was attached to the methanol (7:2) in darkness at -20°C. Following centrifuga- calcareous sinter of a small hot spring in the Upper Terrace tion, the absorption spectra of the extracts were measured area of Mammoth Hot Springs, Yellowstone National Park. against acetone-methanol blanks. The series of springs sampled were the so-called Stygian Electron microscopy. Cells were fixed by the method of Springs, which lie on top of a ridge in the southwest corner Ryter et al. (21), dehydrated in an ethanol series, and of the Upper Terraces. The springs in this area have been embedded in Spurr low-viscosity embedding medium. Sec- mapped by Castenholz (2), and the Stygian Springs are tions were stained with uranyl acetate and lead citrate and indicated by this author. examined with a Philips model 1300 electron microscope at Media. The new organism was isolated in a modified 60 kV. version of the standard liquid enrichment medium used for DNA composition. The deoxyribonucleic acid (DNA) base photosynthetic purple bacteria described by Pfennig (14). ratio of C. tepidum was kindly determined by Henry Burr, This medium contained (per liter of deionized water) 200 mg Wayne State University, Detroit, Mich., by using Esche- of MgS04 - 7H20, 50 mg of CaC12 * 2H20, 400 mg of NH4C1, richia coli, Micrococcus luteus, and chicken DNAs as stan- 400 mg of NaCl, 0.5 g of KH2P04,1 g of sodium acetate, 1ml dards. DNA from C. tepidum that was purified by the of a trace elements solution (15), 1 g of Na2S - 9H20, 2 g of method of Marmur and Doty (12) was subjected to thermal NaHC03, 100 mg of yeast extract, and 20 pg of vitamin B12. denaturation, and the guanine-plus-cytosine content was Pure cultures were grown in the enrichment medium in early calculated from the melting profile. experiments, but in most of the work cultures were grown in enrichment medium modified so that it contained 500 mg of RESULTS ammonium acetate in place of sodium acetate and 1 ml of a 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 t This paper is dedicated to Thomas D. Brock, who first intro- Terrace region of Mammoth Hot Springs, Yellowstone Na- duced me to the fascinating world of hot-spring microbiology. tional Park. These springs are rich in calcium carbonate and

222 VOL. 36, 1986 THERMOPHILIC CHROMA TZACEAE 223

FIG. 1. Photomicrographs of C. tepidum. (A) Cells from mid-logarithmic growth filled with elemental sulfur (arrow). (B) Sulfur-free cells from stationary-phase cultures. The cells were grown photoheterotrophically with sulfide, acetate, and C02at 48°C. Bar = 3 pm.

contain sulfide of geochemical origin at concentrations of ently, crude agar preparations were supplying the contami- about 2 mg/liter (2). The thin mats were firmly embedded in nant with needed nutrients. Liquid cultures of the new carbonaceous sinter from the springs and were composed of isolate, previously referred to as Chromatium sp. strain MCT rod-shaped bacteria, most of which contained bright (T = type strain) (8), were grown in the modified enrichment refractile globules that strongly resembled the sulfur glob- medium described in Materials and Methods, and samples of ules of purple sulfur bacteria. Material collected from one log-phase cells were frozen at -80°C in growth medium such spring (44”C),inoculated into a sulfide-acetate enrich- containing 10% glycerol; frozen cultures remained viable for ment medium, and incubated photosynthetically at 52°C more than 1 year. yielded a bright red-pigmented culture within 72 h. The Morphology. C. tepidum consists of rod-shaped cells spring which was sampled smelled of sulfide and was devoid which measure 1.2 by 2.8 to 3.2 p,m and occasionally form of cyanobacteria. Other rod-shaped bacteria that were free short chains of two to four cells (Fig. 1). Cells in young of refractile globules also were present in the mat sample. cultures (Fig. 1A) contain two or more sulfur globules, while The primary enrichment culture was observed microscopi- cells in stationary phase (Fig. 1B) are generally sulfur free. cally, and a 5% inoculum was transferred to fresh medium. Although motile cells are occasionally observed, pure cul- The predominant organism was a highly motile rod-shaped tures of the new organism have not been observed to be as bacterium containing sulfur globules, which displayed a highly motile as the original enrichment cultures. Electron distinct phobophototactic response (24) when the micro- microscopy of thin sections of C. tepidum (Fig. 2) revealed scope field was darkened. The major contaminant was a long features that are typical of Chromatium species. The intra- thin rod-shaped organism that was devoid of sulfur globules. cellular membranes of C. tepidum are of the vesicular The transferred enrichment grew within 24 h and was kept at (chromatophore) type (Fig. 2). Large holes in the cell periph- 4°C for 2 weeks before pure culture isolation was performed. ery are frequently observed, and these presumably represent A pure culture of the new organism was eventually ob- the locations of elemental sulfur or poly-P-hydroxybutyrate tained following successive applications of the agar shake granules before the embedding and dehydration process; culture technique (17). Bacto-Agar (Difco Laboratories) was large amounts of poly-P-hydroxybutyrate are produced by the solidifying agent used. By the third shake culture series cells of C. tepidum that are grown photoheterotrophically on the only remaining contaminant was the long thin rod-shaped acetate. A fibrous material arranged in clumps (Fig. 2) was organism mentioned above. In shake cultures prepared observed in the cytoplasm of thin sections of C. tepidurn, thereafter the long thin rod-shaped organism was present in and these structures appeared to be loosely attached via low numbers until shake tubes were prepared by using agar membrane connections to the cytoplasmic membrane; their that had been washed with deionized water, followed by function, if any, is unknown. washes in ethanol and then acetone. The washes converted The cell wall of C. tepidum is morphologically distinct. the slightly yellow agar preparation into a much whiter Outside what appears to be a typical lipopolysaccharide crystalline powder. Media prepared from this “purified” layer, a dense, darkly staining area of unknown chemical agar readily yielded pure cultures of the thermophilic composition is present (Fig. 2). Although chemical assays of phototroph following two shake tube applications. Appar- the cell wall of C. tepidurn have not been performed, the 224 MADIGAN INT. J. SYST.BACTERIOL.

aoa

FIG. 2. Electron micrograph of thin sections of C. tepidum cells. 400 500 600 700 800 900 The arrow indicates the intracellular membrane of the chromatophore type. Note the darkly staining outer cell wall layer. Wavelength (nm) Bar = 0.5 pm. FIG. 3. Absorption spectra of intact cells of C. tepidurn. The cells were suspended in 30% bovine serum albumin. organism is highly susceptible to penicillin and therefore presumably contains peptidoglycan. However, the thickness including vitamin B12, are required for growth. The sulfide of this outer layer is not typical of peptidoglycan in gram- tolerance of strain MCT is fairly high; cultures routinely negative bacteria and the layer may consist of other poly- grow in the presence of 2 to 3 mM sulfide, and the upper limit saccharide material. In addition to penicillin, which inhibits is near 4 mM sulfide. Acetate and pyruvate are photoassimil- the growth of C. tepidum at a concentration of 10 pg/ml, the ated (in the presence of sulfide) and substantially increase cell wall inhibitors vancomycin and cycloserine also inhibit cell yields. No other organic compounds tested (various the growth of C. tepidum at the same concentration. The organic and amino acids, fatty acids, sugars, and complex protein synthesis inhibitors chloramphenicol and oxytetra- cycline 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 sus- pended 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 770 esterifying alcohol of the bacteriochlorophyll a of C. tepidum is phytol, based on high-pressure liquid chromatog- 477 raphy-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 propor- tions of the various components are not typical of any known Chromatium species. The major carotenoid, as kindly deter- mined by Karin Schmidt, Universitat Gottingen, is rhodovibrin (or a rhodovibrinlike compound), and this pig- ment 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 chem- ical structure is produced by C. tepidum. Physiology and biochemistry. C. tepidum is an obligately 1 1 phototrophic bacterium. This organism requires substrate 400 500 600 700 800 levels of sulfide for growth under any nutritional conditions WAVELENGTH (nm) and grows photoautotrophically in mineral media containing FIG. 4. Absorption spectra of acetone-methanol (7:2) extracts of HC03- and C02 as sole carbon sources. No vitamins, C. tepidurn cells. VOL. 36, 1986 THERMOPHILIC CHROMA TZACEAE 225

I I I I I 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. 1 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 cyanobacte- rial mat material and therefore may represent a less sulfide- tolerant variant of C. tepidum. Like C. tepidum, however, 57O 1 the New Mexico strain grows at 50"C, oxidizes sulfide, and 4 deposits elemental sulfur intracellularly . I I I I 1 0 10 20 30 40 50 DISCUSSION Most of the properties of the thermophilic photosynthetic Time (h) bacterium described in this paper readily identify it as a FIG. 5. Growth rate of C. tepidum as a function of temperature. member of the genus Chromatiurn, including (i) the rod- Cells were grown photoheterotrophically (acetate, COz, and sulfide) shaped cells, (ii) bacteriochlorophyll upas the sole bacteri- at 2,000 lx at different temperatures and counted as described previously (8, 9). ochlorophyll, (iii) carotenoids of the normal spirilloxanthin series, (iv) intracytoplasmic membranes of the vesicular type, and (v) photoautotrophic growth with sulfide as the substrates, such as yeast extract and Casamino Acids) electron donor and accumulation of elemental sulfur stimulate the growth of C. tepidum. The only nitrogen intracellularly. When the properties of C.tepidum are com- sources utilized by C. tepidum are ammonia, urea, and pared with those of other small-celled species of glutamine; N2 does not support growth as a sole nitrogen Chromatiurn, however, there are several differences (Table source when it is tested at either 37 or 48°C. 1). The most obvious difference relates to the optimum The sulfide requirement of C. tepidum does not simply temperature for growth. C. tepidum grows optimally at 48"C, reflect a need for a biosynthetic sulfur source. No growth of whereas all other Chromatium species grow optimally at C. tepidum is obtained in growth tests when sulfide is temperatures between 25 and 35°C (16). Therefore, the replaced with either cysteine (2.5 to 5 mM), thiosulfate (2 to thermophilic character alone makes C. tepidum unique 8 mM), or sulfite (0.5 to 2 mM) in a medium containing among species of the Chromatiaceae. It should be pointed acetate as the carbon source. Hence, sulfide probably serves out that some of the extremely halophilic Ectothiorhodo- a dual role in the metabolism of C. tepidum (as a photosyn- spira species isolated from African saline lakes by Imhoff thetic electron donor and as a biosynthetic sulfur source). and Triiper also show a rather high optimum temperature. Hydrogen and thiosulfate do not serve as electron donors in For example, Ectothiorhodospira halochloris grows opti- place of sulfide, nor do they stimulate the growth of C. mally at 45 to 48°C (5). However, the optimum growth tepidum above the level achieved on sulfide alone. When the temperature of E. halochloris is very close to its maximum gradient technique of Kampf and Pfennig (7) was used, no growth temperature (-50°C); C. tepidum is the only purple dark growth of C. tepidum was obtained in a medium sulfur bacterium that is capable of growth at temperatures containing sulfide and acetate as energy sources. above 50°C. On the other hand, the thermophilic property of A major difference between recognized species of C. tepidum is not extreme enough to suggest that the Chromatium and C. tepidum concerns the temperature re- organism is an archaebacterium. Indeed, the antibiotic sus- quirements for growth. Reflecting its hot spring origin, C. ceptibility of C. tepidum indicates it is not an archae- tepidum grows optimally at temperatures of 48 to 50"C, but bacterium, and a preliminary analysis of the 16s ribosomal it also grows at reasonable rates at temperatures as low as ribonucleic acid sequences clearly identified C. tepidum as a 37°C and as high as 55°C (Fig. 5). The minimum generation eubacterium (Carl Woese, personal communication). time at the optimum growth temperature is about 3.5 h (8). The color of mass suspensions of C. tepidum resembles The mass doubling times at 55 and 38°C are 25 and 10 h, the colors of suspensions of Chromatium vinosum and respectively. Slow growth of C. tepidum occurs at 56 to Chromatium gracile (i.e., brownish red), but cultures of C. 57°C; however, cells grown at this temperature are consid- tepidum are distinctly more red than cultures of the other erably smaller than cells grown at 48°C and produce long two species. This may be due to the predominance in C. chains, suggesting that the organism is experiencing prob- tepidum of rhodovibrin, a carotenoid that is essentially lems with cell division. The minimum growth temperature absent from C. vinosum and C.gracile (22). Rhodovibrin is for C. tepidum has not been determined precisely, although not a common carotenoid in photosynthetic bacteria. It is cultures incubated photosynthetically at 33 to 34°C did not found in trace amounts in a variety of purple bacterial develop, while cultures at 37°C did. Fully grown cultures of species, but in substantial amounts only in the nonsulfur C. tepidum remain viable for days at room temperature if purple bacterium Rhodospirillum photometricum and in cer- they are left in an unopened, anaerobic state; cultures tain strains of Rhodopseudomonas palustris and Rhodo- exposed to oxygen lose viability more quickly. Cells of C. spirillum rubrum (22). C. tepidurn is morphologically similar 226 MADIGAN INT. J. SYST.BACTERIOL.

TABLE 1. Characteristics of C. tepidum and other small-celled Chromatium species“

Cell size (pm) Species COlOP Width Length

C. tepidum 1.2 2.8-3.2 Red Rhodovibrin, spirilloxanthin 61.5 - Yes - C. vinosum 2 2.5-6 Brownish red Spirilloxanthin, lycopene, rhodopin 64.3 + No + C. minus 2 2.5-6 Purplish red Okenone 52 - Yes + C. purpuratum 1.2-1.7 3 Purplish red Okenone 68.4 + No - C. violascens 2 2.5-6 Purplish violet Rhodopinal 62.2 + No + C. gracile 1.2 2-6 Brownish red Spirilloxanthin, lycopene, rhodopin 69.9 + 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, experiences little competition from Chloroflexus in such Chromatium purpuratum, and Chromatium violascens, but springs, because both the temperature and the pH are clearly differs from these species in carotenoid content and substantially below the optimum values for Chloroflexus. guanine-plus-cytosine base ratio (Table l), as well as in A thermophilic filamentous photosynthetic bacterium that thermal characteristics . resembles Chlorojlexus but lacks bacteriochlorophyll c, (or C. tepidum shares the following interesting physiological any other type of chlorobium chlorophyll) and chlorosomes characteristic with large-celled species of Chromatium has been described recently (20). Although not yet in pure (, Chromatium weissei, and Chromatium culture, this organism, which has been given the provisional buderi) and with Thiospirillum jenense: the only organic name “Heliothrix,” contains bacteriochlorophyll a and ap- compounds photoassimilated are acetate and pyruvate. Ac- pears to be physiologically similar to the nonsulfur purple cording to the classification of Triiper (25), C. tepidum photosynthetic bacteria and Chlorojlexus (18). “Heliothrix” should therefore be included with the species listed above in grows in cocultures with the filamentous aerobic heterotroph the nutritionally restricted Chromatiaceae I group. By con- Zsocystis pallida and photoassimilates organic compounds trast, the members of the Chromatiaceae I1 group (C. under both aerobic and anaerobic conditions (20). In one vinosum, C. violascens, C. gracile, C. purpuratum, and important respect “Heliothrix” resembles the recently de- others) photoassimilate, in addition to acetate and pyruvate, scribed aerobic marine pigmented bacteria (23), because it C-4 citric acid cycle intermediates, fatty acids with longer synthesizes bacteriochlorophyll under fully aerobic condi- chains than acetate, and certain sugars (25) (Table 1). tions (20). This is in contrast to what is observed in nonsulfur Representatives of the Chromatiaceae I group also lack an purple bacteria and in Chloroflexus, in which oxygen serves assimilatory sulfate reduction pathway and are unable to as a potent repressor of bacteriochlorophyll synthesis (4, grow under any nutritional conditions in darkness. These 19). Additionally, “Heliothrix” requires unusually high light properties are also observed in C. tepidum (Table 1) and intensities for growth and synthesizes carotene derivatives serve to further separate this species from the remaining (as does Chloroflexus [18]) as carotenoids (20). Hence, small-celled Chromatium species. “Heliothrix” shares properties with a variety of photo- The observations of Castenholz (2) suggest that the trophic bacteria, but shares relatively little with C. tepidum. thermophilic chromatia are widely distributed in high-sulfide Interestingly, however, the upper temperature limit for neutral-pH springs in the Mammoth Hot Springs region, but photoassimilation of acetate (and, presumably, also for are generally absent from the springs dominated by stands of growth) for “Heliothrix” is about 56°C (20), the same as the Chloroflexus. Strains of Chloroflexus isolated from these upper limit of C. tepidum. Perhaps this is the upper temper- springs are dark green in color, reflecting their high bacteri- ature limit for photosynthetic purple bacteria in general. ochlorophyll contents, and are unable to grow by respiratory Based on the novel assemblage of properties shown by means (R. W. Castenholz and S. J. Giovannoni, Abstr. pure cultures of the thermophilic purple sulfur bacterium Annu. Meet. Am. SOC. Microbiol. 1981, 179, p. 100). described above, this organism, provisionally assigned to the Anaerobic strains of Chloroflexus predominate in these genus Chromatium and referred to previously (8) as springs probably because the source temperature (-70°C) Chromatium sp. strain MCT, is proposed as a new species of excludes the development of C. tepidum-like organisms and the genus Chromatium, Chromatium tepidum. the sulfide content (1 to 2 mglliter) excludes the development Description of Chromatium tepidum. Chromatium tepidum of high-temperature species of cyanobacteria, such as (tepi.dum. L. neut adj. tepidum lukewarm) cells are rod Synechococcus (2). Around 50 to 55°C thermophilic shaped and 1 to 2 pm wide by 2.8 to 3.2 pm long. Gram chromatia begin to appear along with Spirulina, a major negative, occasionally motile. Photosynthetic intracytoplas- cyanobacterial representative in sulfide-containing waters mic membrane system of the vesicular type. with temperatures below 50°C (2). In high-sulfide springs at Pigments. Absorption spectra of intact cells show peaks at temperatures of 45 to 50°C it is likely that Chromatium is the 858, 808, and 599 nm, which are typical of bacteri- favored photosynthetic bacterial species, since pure cultures ochlorophyll a. The esterifying alcohol of bacteri- of C. tepidum grow optimally near 50°C and pH 7 and ochlorophyll a is phytol. Carotenoids of the normal spiril- Chloroflexus grows optimally at 55°C and at a more alkaline loxanthin series are present; rhodovibrin and spirilloxanthin pH (18, 19). In the spring from which C. tepidum was predominate. isolated, for example, the temperature was 44”C, the pH was Physiology. Obligately phototrophic. Growth occurs 7, and no cyanobacteria were present. C. tepidum probably photoautotrophically in mineral media supplemented with VOL. 36, 1986 THERMOPHILIC CHROMATIACEAE 227 sulfide as electron donor. Sulfur is formed as an intermediate 1:61-69. product, which is further oxidized to sulfate. Hydrogen and 7. Kampf, C., and N. Pfennig. 1980. Capacity of Chromatiaceae thiosulfate are not utilized as electron donors. Acetate and for chemotrophic growth. Specific respiration rates of Thiocystis violacea and Chromatiurn vinosum. Arch. Microbiol. pyruvate are photoassimilated in the presence of sulfide; 127:125-135. citric acid cycle intermediates, fatty acids other than acetate, 8. Madigan, M. T. 1984. A novel photosynthetic purple bacterium and sugars are not utilized. Ammonia, urea, and glutamine isolated from a Yellowstone hot spring. Science 225313-315. serve as nitrogen sources. No growth factors are required. 9. Madigan, M. T., J. C. Cox, and H. Gest. 1980. Physiology of Optimum pH, 7. Optimum growth temperature, 48 to 50°C; dark fermentative growth of Rhodopseudomonas capsulata. J . no growth below 34°C or above 57°C. NaCl is not required Bacteriol. 142:908-915. for growth and is growth inhibitory at concentrations above 10. Madigan, M. T., and H. Gest. 1979. Growth of the photosyn- 1% (wthol). Catalase negative. thetic bacterium Rhodopseudomonas capsulata chemoautotro- Storage material. Poly-P-hydroxybutyrate is a storage phically in darkness with H2 as the energy source. J. Bacteriol. 137 :5 265 30. material. 11. Mandel, M., E. R. Leadbetter, N. Pfennig, and H. G. Truper. DNA base composition. The DNA contains 61.5 mol% 1971. Deoxyribonucleic acid base compositions of phototrophic guanine plus cytosine, as determined by thermal denatur- bacteria. Int. J. Syst. Bacteriol. 21:222-230. ation. 12. Marmur, J., and P. Doty. 1962. Determination of the base Habitat. The habitat of C. tepidum: sulfide-containing hot composition of deoxyribonucleic acid from its thermal denatur- springs of neutral to alkaline pH at temperatures below 60°C. ation temperature. J. Mol. Biol. 5109-118. Type strain. The type strain is strain MC, which was 13. Miyoshi, M. 1897. Studien iiber die Schwefelrasenbildung und isolated from Stygian Springs, located in the Upper Terraces die Schwefelbakteriein der Thermen von Yumoto bei Nikko. of Mammoth Hot Springs, Yellowstone National Park. This Centralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 strain has been deposited with the American Type Culture 3526-527. 14. Pfennig, N. 1965. Anreicherungskulturen fur rote und griine Collection as strain ATCC 43061T. Schwefelbakterien. Zentralbl. Bakteriol. Parasitenkd. Infek- tionskr. Hyg. Abt. 1 Suppl. 1:179-189. ACKNOWLEDGMENTS 15. Pfennig, N. 1974. Rhodopseudomonas globiformis, spa., a new This work was supported by grant DMB-8505492 from the National species of the Rhodospirillaceae. Arch. Microbiol. 100:197-206. Science Foundation and by the Coal Research Center of Southern 16. Pfennig, N., and H. G. Truper. 1974. The phototrophic bacteria, Illinois University. p. 24-64. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey’s I thank the National Park Service, U.S. Department of the manual of determinative bacteriology, 8th ed. The Williams & Interior, for permission to sample in Yellowstone National Park. I Wilkins Co., Baltimore. especially thank Dave Ward for help in field sampling, Rudy Turner 17. Pfennig, N., and H. G. Truper. 1981. Isolation of members of the for taking the electron micrographs, Joseph Mayers, Jr., for helpful families Chromatiaceae and Chlorobiaceae, p. 279-289. In discussions, Sharon Cox for technical assistance, and Hans Truper M. P. Starr, H. Stolp, H. G. Triiper, A. Balows, and H. G. for suggesting the species epithet. Schlegel (ed.), The prokaryotes, a handbook on habitats, isola- tion, and identification of bacteria. Springer-Verlag, Berlin. ADDENDUM IN PROOF 18. Pierson, B. K., and R. W. Castenholz. 1974. A phototrophic gliding filamentous bacterium of hot springs, Chloroflexus Near infra-red spectroscopy of membrane preparations aurantiacus gen. and sp. nov. Arch. Microbiol. 1005-24. from cells of C. tepidum shows a distinct absorption peak at 19. Pierson, B. K., and R. W. Castenholz. 1974. Studies of pigments 910 nm due to an unusual antennae form of bacterio- and growth in Chlorojexus aurantiacus, a phototrophic filamen- chlorophyl a (Andre VermCglio, personal communication). In tous bacterium. Arch. Microbiol. 100:283-305. addition, “Heliothix’ ’ has been given formal taxonomic 20. Pierson, B. K., S. J. Giovannoni, and R. W. Castenholz. 1984. standing with the description of Heliothrix oregonensis, gen. 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