Huber, Gertrud

Huber, Robert

Seger, J.

Segerer, Stephan

Stetter, Karl Otto

English

University of Regensburg Publication Server

In: Microbial Growth on Compounds. Proceedings of the 5th International Symposium. / (H.W. van Verseveld, J.A. Duine eds.) pp. 44-51, Nijhoff Publ., Dordrecht (1987). AEROBIC AND A N A E R O B I C EXTREMELY THERMOPHILIC A U T O T R O P H S R. H U B E R G H U B S * . A . S E G E R E R J . S E G E R and K.O. STETTER Lehrstuhl für MkrobJoiogie, Urwers/iát Regensburg, 8400 Regensburg, Federal Repubßc of Germany. 1. I N T R O D U C T I O N During the last years, various extremely themioprÄ badend 8 0 X r w e been i»latedfTOT 15,17} .Alof these organisms belong to the archaebacteria, the third kingdom of Be[19] . The mode of nutrition is Hthoautotrophlc or organotrophic, depenolng o n the isolates. In this paper, limoautotrophic extreme thermophlles including some very recent isolates are described which are the primary producers of organic matter at high temperatures. 2 . B I O T O P E S Up t a now, aft extremely therrnophfe autotrophic b ^ fieWs and submarine hydPotherinalsysteni& about 400 to 2000 m w M the floor (10]. From tf^^ mainly C0 2 . SO2 and H^S. escape and are heal the s o l and surface water. The examination of s o * profiles wiminsotfatarlcfields s h o w e d ^ the top, there is an o x y p e n ^ n t a i o i n g strongly aocfcs lay er of about 15 to 30 cm in thickness, which shows an ochre coto i r due to the presence of feró to neutraJ btoish-Wack 20m. which contains ferrous suTxJe. Submarine hydrcmermaJ syalems contain seawater of neutral to s f igMy acxScpH which remains Squid even above 100*C due to the prevenfion of boHmg by the h y d r o s ! ^ sulfur which ts formed b y the oxidation of H^S ana by the reaction of H2S with S0 2 - Seawaler contains atwutjfnmotes of sufate per fitre. 3. A C I D O P H I L I C A E R O B I C A N O F A C U L T A T I V E L Y A N A E R O B I C A U T O T R O P H S The upper oxkfced layer within soff marie fields inducing solfataric mudholes contains coocoid lobe-shaped strictly aerobic organisms which belong to the genus Svffofoöus which was described by T O . Brock (4J> The type species Suffotobus ac&xaxiaws and most other isolates can gain their energy i% .  8 + c w < X : 9 i » O » Ul i IS CO Sä if. 5? 8 I • • • • <• + .+ • + .+ • Tí "O "O TJ T> c c cí «£ c • • • • 3 « N S 8 S N tO IA 0» ^ *- O ^ c*> co ^ co o w «¡o co 8 x S ui m > F > > X chemo lit hotroph»caiiy by oxidation oí S°, forming H 2 S 0 4 . Some isolates (Tab. 1) are able to grow iithoautotrophicaJly in S°-containing mineral medium in the presence of C 0 2 and oxygen, others require the addition of traces of yeast extract (e.g. 0.01%) , the function of which is not yet clear (source of minerals or of organic carbon). Alternatively both Suifclobus-speties and many Sulfolobus-shaped isolates (Tab. 1) grow without S°* organotrophtcally on yeast extract, sugars, peptone and amino acids [3. 4,15]. Many Sulfolobus isolates are able to oxidize ferrous iron [2, 5]. However, due to extensive precipitates of jarosite, even at pH 2 and in uninoculated controls, no significant growth could be observed [3, Huber and Stetter, unpublished]. Sulfolobus bherleyi, which has now been re named Adolanus brierieyi [12] and some still unnamed Suffolobus-shaped isolates [8] are moderate thermophiles, growing at temperatures up to 70°C. They are able to grow on surfkjic ores» forming sulfuric acid and soiubilizing heavy metals. New ore-leaching bacteria could be isolated aerobicafly in mineral medium in the presence of ore mixture G1 , consisting of pyrite, chalcopynte. sphalerite and pitchblende [Tab. 1; 6]. tepyes HV5 and VE2 from samples of Hveragerthi and Hveraveflir in Iceland are strictly dependent on ores and can not grow on S°. Strains HV5 and VE6 are the most extremely ore-leaching bacteria known, growing at temperatures up to 95°C (Tab. 1). in contrast, Sulfolobus acidocaldanus and Sulfolobus solfatahcus are unable to grow by the oxidation of suffidic ores. Recently, the new genus Acktianus was recognized (tentatively named 'Acidothermus', [14,17]. Members have been isolated from acidic soffatara fields in Italy, Iceland, the Azores and the United States, and from a submarine hydrothermal vent in Italy [11, 12]. Although the cefts of these thermoacidophfles have a coccoid, lobed shape similar to Sulfolobus (Fig. 1), they are different in DNA composition (G+C-content; DNA homology) and in metabolic properties. Figure 1 .Thin section of AckManus internus contrasted with lead citrate and uranyl acetate.Bar 1 urn. Z7 CO CO ft ^ S CO N 3 3 S= 10 • in eo 1 •5 5 S 5, 8 S f 3 2 1 P 0) O .2 I 8 i I 1 "2 3 I Tí O Adátanos mfemus, the first species to be recognized (12], is able to grow aerobicaöy by S°-oxidation, forming sulfuric acid. Surprisingly, this organism is also able to grow under extremely anaerobic conditions by S°-reduction: H^S is formed from ^ and S° (Tab, 1. 2). Sulfolobus bnerteyi also processes this previously unrecognized metabolic versatility, and was therefore considered to also belong to the genus Aadianus (11, 12]. A further isolate, which contains a prophage (W. Zfllig, pers. comm.) and had been tentatively named "Sulfolobus ambtvalensT [21] exhibits 60% DNA homology with the type species, Adóianus infernas, indicating that it is another species of the genus AckSanus. 4. N E U T R O P H I L I C A N D S L I G H T L Y ACIDOPHILIC A N A E R O B I C A U T O T R O P H S From the lower reduced zone of solfataric fields, extremely thermophilic methanogens and S°-dependent autotrophs could be isolated. Previously, the methanogens could be only obtained from Icelandic solfataric fieids. They are rod-shaped. Gram-positive lithoautotrophs, growing by formation of methane from H 2 and C 0 2 at temperatures up to 97°C. Until now, the two species Methanothermus fervidus and Methanothermus sociabilis ( F ig . 2) are the only known representatives of Methanoathermaceae [7.8]. Figure 2. Electron micrograph of Methanothermus sociabilis, platinum-shadowed. Bar 1 urn. The S°-dependent autotrophs are rod-shaped organisms which divide by budding [20]. Unta now, members of the two new genera Thermoproteus and "Pyrobacukjm" (Fig. 3) could be isolated growing iithoautotrophicairy on S°, H 2 and C 0 2 . gaining energy by H¿S formation (Tab. 2). *Pyrobacukim* has a G-kC-content of onry 46 mof% which is 10% lower than that of Thermoproteus [20]. In addition, isolates belonging to "Pyrobacvlum' show an optimal growth temperature of 10O°C which is 12°C higher than that of the Thermoproteus-isolales [17,20]. Alternatively. "Pyrobaculum islandicum" and Thermoproteus tenax are able to grow without H 2 organotrc^.icaliy on yeast extract in the presence of S°, S2O3 2- and S O 3 2 - (Tab. 2). Figure 3. Electron micrograph of 'Pyrobaculum islancScur-'Geo 3, platinum-shadowed. Bar 1 urn. During organotrophic growth, onry tow amounts of H^S are formed compared with lithotrophic growth of these organisms, tn the case of "Pyrobaculum tslanöcum~. organotrophic growth is also obtained when S° is replaced by cysteine. It is most Beefy, these bacteria are growing organot rophcaüy by an Electron micrograph of PyrocScäum occuJtum. aaimum-shadowed. Bar 5 urn. unknown fermentation rather than by sulfur respiration. From the submarine hydrothermal vents of VuJcano. Italy, the most extremely thermophilic organisms known until now have been isolated- They have an optimal growth temperature of 105°C, and a maximum at around 110°C [under slight overpressure, 13]. These d isc - to dish-shaped cells form unusual networks of fibers connecting the celts (Fig.4). The new genus m PyroaTctíum" currently contains the species Pyroofctium occuftum and Pyroäctium brockS, which are strict H^S-aiitotrophs (Tab. 2). During growth, the fiber networks cover the sulfur like cobwebs [13.16]. 5. I S O L A T I O N O F E X T R E M E L Y T H E R M O P H I L I C S U L F A T E - R E D U C I N G A R C H A E -B A C T E R I A Until now, no archaebacteria thriving by o^imüatory sulfato reduction were known [9J. During our recent to situ enrichments wimin the hydrothermal systems close to Vulcano, Italy, coccoid to disc-shaped irregular ca fe could be isolated. These grew anaerobtcaty wihout S ° at temperatures between 65 and 96°C with an optimum at around 83°C. They use formate, L(+) and D(-) lactate and, less efficiently, glycogen, starch, yeast extract, meat extract and bacterial homogenates as substrates. Sulfate, thtosuifate and sulfite are used as electron acceptors, while S° is toNbitory tor growth. As end products, H2S, CO2. and traces of methane (about 4 mmotos/l) could be detected. Growth is inhibäed by 0.1 mmoies/l of Na-motybdate. Under the UV microscope, the cells show a blue-green fluorescence due to the presence of F420 PI - , n addtion, rnethanopcerin, but not F430 [A. Pfaitz, pets, commjor coenzyme M could be detected. The RNA polymerase of isolate VC-16 shows homology to subunito B and B" of the rrtothanogens, but is unique to serological cross-reaction of the heaviest subunit with components A and C of methanogens and S-dependent archaebacteria. indicating an outstanding position for the new isolates within the phylogenetic tree of the archaebacteria. R e f e r e n c e s [1] Balch, W E . G E . Fox, L J . Magrum, C.R. Woese, and R.S . Wolfe. (1979) Methanogens: Reevabation of a unique b i c ^ o g ^ c ^ p . M k ^ c ^ L Rev. 43:260-296. [2] Briertey, C . L , and J . A . Br ier ley. (1973) A (^ernolithoautotrophic and thennophäfc microorc^nisrrMsototed from an acidic hot spring. Can. J . Microbiol. 19:183-188. [3] Brock. T.D. (1981) Thermophilic rnicroorganiaros and Me at high temperatures. Springer, Beruft Heidetoerg, New York. [4] Brock, T.D.. KM. Brock. R.T. Befly, and R . L Weiss. (1972) SuKobbus. a new genus of sufttf-oxkSzing bacteria »Ving at low pH and high ternperature. Arch. Microbiol. 84:54-68. [5] Brock, T. D.. S . Cook, S. Peterson, and J . L Mosser. (1976) Biochemistry and bacteriology <* ferrous iron oxidation in geothermai habtats. Geochkn. Casrrocim. Acta 40:493-500. [6] Hubsr, a . ft Huber. arto K.O. S t e t t e n bacteria. Biotechnology and Bioengineering Symp. No. & J . W9ey & Sons. Ina, New York. London. Sydney, Toronto. [7] Lauerer, G . , J . K . Kristjansson. T.A. Ungworthy . H . König, and K. O. better . (198«J MmanothermjssoaabiSs sp. nov.. a second species within the Mtmtnotrwmaeea* yo*W* 97°C. System. Appl. Mksobiol. 8:100-105. [8] Marsh. R M , P R . Norris, and N.W. Le Roux. (1983) Growth and mineral oxidation studies-witn Suttotoöos, p. 71-81. i n : G . Rossi, and A.E. Torma (eds), Associazione Mineraria Sarda. Iglesias, Italy. . [91 Pfennig, N.. and F. WWrJel. (1382) The bacteria of the sulfur cyde. PhH. Trans. R S o c U n d . B 298: 433-441. [10] Pichler, H. (1981) Italienische Vulkan-Gebiete i l l . Sammlung Geologischer Führer. Vol. 69. Gebrüöer Borrtfraeger. Benin, Stuttgart. [11] Segerer, A., K.O. Sletter. and F. Ktink. (1985) Two contrary modes of criemolftfiotrophy in the same archaebactenum. Nature 313:787-789. [12] Segerer, A.. A. Neuner, J J C Kristjansson, and K.O. Stetter. (1986) Ackfianus infernos gen. nov., sp. nov., and AckManus brwfleyi comb, nov.: facultatively aerobic, extremely acidophilic trtermophfic sulur-rretaoofaing afchaebacteria. Int. J . Syst. Microbio!, (in press). [13] Stetter, K.O. (1982) Utrathin myceHa-forming organisms from submarine volcanic areas having an optimum growth temperature of 105°C. Nature 300:258-260. [14] Stetter, K.O. (1986) Diversity of extremely thermophific archaebacteria. p. 39-74. In: T.D. Brock (ed.), ThermophMes: General, Molecular and Applied Microbiology. J . Wiley & Sons, Inc.. New York, London, Sydney, Toronto. [15] Stetter, K.O.. and W. Z36g. (1985) Thermop/asma and the thermophilic sulfur-dependent archaebacteria, p. 85-170. far. & S . Worte and C.R. Woes* {eds.), The Bacteria. Vol. Vitt. Academic Press, New York. [16] Stetter, K.O.. H. König, and E. Stackebrandt. (1983) Pyrodctium gen. nov., a new genus of submarine disc-shaped suf t r - fedudng archaebacteria growing opumaly at 105°C. Syst. Appl. Microbiol 4:535-551. [17] Stetter, K.O.. A. Segerer. W. Z»g, G. Huber, G. Ba la , R. Huber, and H. König. (1986) Extremely t h e r n i o p r * s u i u r - m e t a b t * z ^ [18] Stetter, K.O.. II. Thomm. J . Winter, G . WSdgruber. H. Huber, W. ZIffig, D. Janecovic, H. König, P. Palm, and S. Wundert. (1981) Methanothermus fervidus, sp. nov., a novel extremely thermophüc methanogen isolated from an IcetanrJc hot spring. 2bi. Bakt. Hyg., I. Abt. Orig. C 2:166-178. [19] Woese, C R , L J . Magrum, and aE Fox. (1978) Archaebacteria. J . Mol. Evo l 11:245-252. [20] Zillig, W.. K.O. Stetter, W. Schafer, D. Janekovic. S. Wunden*, I. Holz, and P. Palm. (1981) Thermoprotaahs: a novel type of extremely thermoaciotophSc anaerobic archaebacteria isolated from k a f a f i d f c s o t f l M * 2bL BaH. Hy§., I. Abt. Orig. C 2:205-227. [21] Zfflig, W. .S . YeÄLMolz.A.Böck,F.Gropp, M. Rettenberger, and S . Lutz. (1985) Rasrrád-related anaerobic autottophy of the novel archaebacterium Sutfoioöus amtxvatens. Nature 313:789-791. 

AEROBIC AND ANAEROBIC EXTREMELY THERMOPHILIC
AUTOTROPHS

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AEROBIC AND ANAEROBIC EXTREMELY THERMOPHILIC AUTOTROPHS

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