INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,July 1997, p. 657-660 Vol. 47, No. 3 0020-7713/97/$04.00 + 0 Copyright 0 1997, International Union of Microbiological Societies

Reclassification of the Crenarchaeal Orders and Families in Accordance with 16s rRNA Sequence Data

S. BURGGRAF,* H. HUBER, AND K. 0. STETTER Lehrstuhl fur Mikrobiologie und Archaeenzentrum, Universitat Regensburg, 93053 Regensburg, Germany

A phylogenetic analysis of all validly published members of the , including several new isolates from our laboratory, suggests three orders within this archaeal . The consist of both the rod-shaped, hyperthermophilic, neutrophilic representatives of the and the members of the new family .The harbor all thermoacidophilic, coccoid organisms. The neutro- philic, hyperthermophilic cocci are members of a new order tentatively named “Igneococcales.” This order comprises two families, the , characterized by maximal growth temperatures of up to lOO”C, and the new family , for which optimal growth occurs at temperatures above 100°C.

Phylogenetic analyses based on 16s rRNA sequence com- 3’), 890aR (5’ TITCAGYCmGCGRCCGTAC 3’), 1044aR (5‘ GGCCATG parisons demonstrated that the archaeal of life consists CACCWCCTCTC 3’), 1lOlaR (5‘ GGYRSGGGTCTCGCTCGTT 3’), 1206aR (5‘ GCCCSGGGGDTTCGGGGC 3’), 364aF (5’ CGGGGYGCASCAG of two kingdoms, the Euiyarchaeota and the Crenarchaeota GCGCGAA 3’), 533F (5’ TGBCAGCMGCCGCGGTAA 3’), 797aF (5’ (34). All Crenarchaeota are thermophilic or hyperthermophilic GCRAASSGGATTAGATACCC 3‘), and 1060aF (5’ GAGAGGWGGTG organisms, and they have been isolated from continental or CATGGCC 3’). The derived sequences were aligned with a set of representative submarine hydrothermal systems or from hot anthropogenic archaeal sequences (obtained from the Ribosomal Database Project [21]) by using the ARB program from the Department of Microbiology of the Technical biotopes such as smoldering refuse piles (3, 11, 13, 18, 27, 30, University in Munich (31) (kindly provided by W. Ludwig and 0. Strunk). For 38). Their classification has been mainly based on physiological the calculations, only positions present in all analyzed sequences (57 to 1370) and and biochemical properties, since only a few 16s rRNA se- with unambiguous alignments were used, for a total of 1,240 positions. Pairwise quences from these organisms have been available. Two orders evolutionary distances were calculated from percent similarities with a modified Jukes and Cantor correction (19, 32). Dendrograms were calculated with the within this group have been described, the Sulfolobales (29) DeSoete program (10) provided by the Ribosomal Database Project and with the and the Thermoproteales (35). The latter comprises two validly neighbor-joining, maximum-parsimony (Phylip package), and maximum-likeli- published families, the Thermoproteaceae (38) and the Desul- hood (fastDNAml) programs included in the ARB package. To check for arti- (37). The genera ficially deep branchings due to a high G+C content of the analyzed 16s rRNA firococcaceae Fyrodictium, “,” sequences, all calculations were also performed with only transversions (33). and Staphylothemus were tentatively placed in the Themzopro- Nucleotide sequence accession numbers. The sequences of the organisms were teales (35). A 16s ribosomal-DNA (rDNA)-based classification deposited in the EMBL Nucleotide Sequence Database with the following ac- of the order Sulfolobales, including all described thermoacido- cession numbers: Pyrodictium ubyssi, X99559; H. butylicus, X99553; S. marinus, philic species, was published recently (12), demonstrating the X99560; “Thennosphuera agreguns,” X99556; “D. saccharovorans,” X99558; “Thermodiscus maritimus,” X99554; “ librum,” X99557; Pyrolobus group to be monophylogenetic. Here we present a complete fumurii, X99555; “I. islandicus,” X99562; strain OC11, X99561; strain CB9 phylogenetic analysis of all cultivated members of the Crenar- X99563; and strain SlOTFL, X99564. chaeota, including several new isolates. Based on our results we reclassify the orders and families within this archaeal kingdom. RESULTS AND DISCUSSION

MATERIALS AND METHODS For a phylogenetic analysis of the Crenarchaeota, we deter- Strains and culture conditions. Pyrodictium abyssi DSM 615gT (24), Hyper- mined nearly complete 16s rDNA sequences for the already- thermus butylicus DSM 5456T (36), marinus DSM 3639T (11), described species S. marinus, H. butylicus, and Fyrodictium “Thermosphaera aggregans” M11TL (17), “ sacchurovoruns” abyssi. In addition the 16s rDNAs from the new organisms V24K (28), “Thermodiscusmaritimus” S2 (28), and “Thermofilum librum” V24N Pyrolobus fumarii, “Thermosphaera aggregans,” “D. saccharo- (28) were grown as described previously. Pyrolobus fumarii 1A (4), “Igneococcus islandicus” Ko18 (6), isolate OCll (15), isolate CB9 (16), and isolate SlOTFL voran~,~’“Themzodiscus maritimus,” “I. islandicus,” and (16) are novel, hyperthermophilic isolates characterized in our laboratory. “Themofilumlibrum” and from the isolates SlOTFL, CB9, and Isolation of nucleic acids and amplification of the 16s rRNA gene. DNAs of OCll from our laboratory were sequenced. all organisms were prepared as described previously (1,22). The nearly complete All of the algorithms utilized to derive the phylogenetic tree 16s rDNA was amplified by PCR (25, 26) in a Perkin-Elmer Gene Amp PCR System 9600 with Perkin-Elmer AmpliTaq DNA polymerase and a standard (neighbor joining, maximum parsimony, and maximum likeli- PCR protocol. The primers used in the amplification corresponded to positions hood) yielded the same tree topology (Fig. 1). Calculations, 8 to 23 (7) and either 1390 to 1407 (23) or 1492 to 1511 (20) in the 16s rRNA considering only transversions, to detect artificial clustering sequence of Escherichia coli (5).The amplified PCR products were purified and due to similarities in the G+C contents of the sequences (33) concentrated in a Microcon 100 column (Amicon). Sequencing and data analyses. Both strands of the PCR products were se- revealed no significant differences in the tree topologies, with quenced directly by using an AmpliCycle sequencing kit (Perkin-Elmer, devel- the exception of the isolate CB9 (see below). According to the oped and manufactured by Roche Molecular Systems, Inc., Branchburg, N.J.) phylogenetic tree, the kingdom Crenarchaeota is composed of with [33P]dCTPfor internal labeling. The following forward and reverse sequenc- three major branches corresponding to three orders, the Ther- ing primers, identical or complementary to conserved sequences in archaeal 16s rRNA, were used: 338aR (5’ CTGSTGCRCCCCGTAGGGCC 3‘), 524aR (5’ moproteales, the Sulfolobales, and a new order tentatively GSNGCYGGTRTTACCGCGGC 3‘), 750aR (5’ TTCGBCCCTCACCGTCGG named “Igneococcales.” The rod-shaped organisms of the order Thermoproteales form a distinct cluster within the Crenarchaeota. Two branches * Corresponding author. Mailing address: Lehrstuhl fur Mikrobiolo- are evident within the Thermoproteales, one which can be as- gie und Archaeenzentrum, Universitat Regensburg, Universitatsstrasse signed to the family Themzoproteaceae and one which can be 31, 93053 Regensburg, Germany. assigned to the new family Themzofilaceae.This separation is in

657 658 BURGGRAF ET AL. INT.J. SYST.BACTERIOL.

1 Euyarchaeota - Methanobacteriumfonnicicum Methanococcus thermolithotrophicus Methanococcusjannaschii 7 L---L___ Thermococcus celer

Pyrodictiuna occulturn Hypertherrnus butylicus Pyrodictium abyssi Pyrodictiaceae Pyrolobus fumarii Desulfurococcus mobilis I “Desulfurococcus saccharovorans “ isolate OCllLL “Igneococcales”

Desulfurococcaceae pernix

“Igneococcus islandicus “ isolate CB9LL Metallosphaera sedula Acidianus brierleyi Crenarc haeota

Sulfolobus metallicus Sulfolobales Sulfolobus solfataricus Sulfolobus acidocaldarius Stygiolobus azoricus J islandicum --I 1 Thermoproteaceae tenax Thermoproteales Thermofilum pendens I “Thermophilum librum” isolate SlOTFL 1Thermofilaceae

FIG. 1. Phylogenetic tree derived from 165 rDNA sequence data for the members of the Crenarchaeota and for some representatives from the major euryarchaeal groups. The tree was derived with the maximum-parsimony program. The scale bar indicates 10 estimated changes per 100 nucleotides.

agreement with morphological characteristics. The cell diam- All representatives of the third cluster (order) are coccoid or eters of members of the Themzoproteaceae are at least 0.4 pm, disc-shaped anaerobic (with the exception of Aeropyrum per- while the filamentous cells of members of the Thermofilaceae nix) hyperthermophiles that grow at neutral pHs. Therefore, exhibit cell diameters of less than 0.35 pm. Despite the large we propose the name “Igneococcales” for the new order. Two phylogenetic distance between these families of more than 10 families, the Pyrodictiaceae and the Desulfurococcaceae, are estimated changes per 100 nucleotides (data not shown), most evident. The family Pyrodictiaceae, a phylogenetic group of treeing programs show a common root. Only the calculation organisms characterized by optimal growth temperatures with the DeSoete program results in a separate branching above 100°C, consists of the genera Pyrodictium and Hyperther- point for the two lineages. While Themzofilumpendens and mus and the new Pyrolobus (4). The maximum phyloge- “Thermofilum librum” have identical 16s rDNA sequences, the netic distance between these genera of only 1.5 estimated new isolate SlOTFL, which has been isolated from Obsidian changes per 100 nucleotides demonstrates their close relation- Pool, a hot spring at Yellowstone National Park, exhibits a ship (data not shown). However, they exhibit significant differ- phylogenetic distance from these organisms of 2.2%. More ences in morphology and metabolism. The two Pyrodictium interestingly, its 16s rDNA sequence is identical to the se- species are coccoid cells connected by a network of hollow quence of clone pJP6 obtained by phylogenetic analysis of fibers. This network is missing in Hyperthemzus and Pyrolobus. DNA directly extracted from sample material of this hot spring Pyrodictium and Hyperthermus grow strictly anaerobically by (1). chemolithoautotrophic or heterotrophic reduction of elemen- A detailed analysis of the members of the second branch, the tal sulfur. Pyrolobus gains energy by H, oxidation with nitrate, order Sulfolobales, has been described recently (12) and is in S2032-, or a low concentration of 0, as the electron acceptor. full agreement with our results. All members of this order are The family Desulfurococcaceae is phylogenetically clearly regularly or irregularly coccus-shaped thermoacidophiles. separate from the Pyrodictiaceae (Fig. 1). The coccoid or disc- However, the present of the Sulfolobales does not shaped organisms of the Desulfurococcaceae are hyperthermo- always coincide with the results of the 16s rDNA-based phy- philes with an optimal growth temperature above 85°C and, in logenetic investigations. The phylogeny, especially of members contrast to the Pyrodictiaceae, a maximum growth temperature of the genus Sulfolobus,would justify splitting this group into not higher than 100°C. The organisms grow chemolithoauto- three different genera or even families (Fig. 1). However, due trophically by sulfur reduction to H,S with CO, as the sole to the lack of physiological or biochemical characteristics sig- carbon source or heterotrophically by sulfur respiration of nificant for defining these taxa, it has been suggested that the various organic substrates or by fermentation or aerobic genus not be reclassified at present (12). respiration. One branch of this family harbors S. marinus, VOL. 47, 1997 CRENARCHAEAL ORDER AND FAMILY RECLASSIFICATION 659

“Thermosphaera aggregans,” the isolate OC11, and the se- genomic DNA, 46 to 57%. Widely distributed in solfataric quence-identical species Desulfurococcus mobilis and “D.sac- hot springs and submarine hydrothermal systems. The order so charovorans. ” ‘% islandicus” (6), “Thermodiscus maritimus” far comprises two families, the Thermoproteaceae and the (28), and the only obligate aerobic member of the Desulfuro- Thermofilaceae fam. nov. coccaceae, A. pemk (27), represent a separate branch. Only Emendation of the family Themzoproteuceue Zillig et al. one tree calculation, based on the least-squares distance 1981. Rigid rods, with lengths between 1.5 and 8 pm and method of DeSoete (lo), showed an affiliation of these organ- diameters of 0.4 to 0.5 pm. Energy produced chemolithoau- isms to the Pyrodictiaceae, and therefore the tree topology totrophically by reduction of elemental sulfur with H, by-using shown in Fig. 1 is more likely. CO, as the sole carbon source, by sulfur respiration utilizing The phylogenetic affiliation of the novel organism CB9, a various organic substrates, or by respiration with 0, or nitrate coccoid, hyperthermophilic, anaerobic, neutrophilic hetero- as the electron acceptor. So far the family harbors two genera, troph isolated from Lake Tanganyika (Africa) (16), is still Thermoproteus and Pyrobaculum. somewhat unclear. While analyses constrained by only trans- Description of the family Thermofilaceue fam. nov. Ther- versions and the normal DeSoete analysis show an affiliation of mofilaceae (Ther.mo.fi.la’ce.ae. M.L. neut. n. Thermofilum this organism with the “Igneococcales,” all other calculations type genus of the family; -aceae to denote a family; M.L. fem. show isolate CB9 as the deepest branch of the Sulfolobales pl. n. Thermofilaceae the Thermofilum family). Thin rods 0.15 (Fig. 1). Therefore, the exact determination of the phyloge- to 0.35 pm in diameter and 1to >lo0 pm in length. The family netic position of this isolate has to await detailed physiological comprises the single genus Thermofilum. and biochemical characterizations and/or the isolation of Emendation of the family Desulfurococcuceue Zillig et al. closely related strains. 1982. Coccoid to disc-shaped cells. Hyperthermophilic; opti- Due to the different rates of evolution in various branches mal growth temperature above 85”C, maximum growth tem- within the phylogenetic tree, it is very difficult to define specific perature up to 100°C. Anaerobic or aerobic. Chemolithoau- phylogenetic distances separating classes of taxa. rRNAs from totrophic growth by sulfur reduction to H,S with CO, as the hyperthermophiles have a high G+ C content, which stabilizes sole carbon source. Strictly heterotrophic growth by sulfur res- the secondary structure at high temperatures. This results in a piration of various organic substrates, aerobic respiration, or lower rate of sequence change than that observed for meso- fermentation. Six genera are known so far: Desulfirococcus, philic organisms. Therefore it is impossible to use the phylo- Staphylothermus, Aeropyrum, “Themosphaera,” “Thermodis- genetic distances which, e.g., separate the orders of the meth- cus,” and “Igneococcus.” anogens as criteria for separation of hyperthermophilic, Description of the family Pyrodictiuceue fam. nov. Pyrodicti- crenarchaeal taxa. Although there is a much smaller phyloge- aceae (Pyr.o.dic’ti.a.ce.ae. M.L. neut. n. Pyrodictium type genus netic distance between the “Igneoc~ccales~~and the Thermo- of the family; -aceae to denote a family; M.L. fem. pl. n. proteales than there is between, e.g., the and Pyrodictiaceae the Pyrodictium family). Coccoid to disc-shaped the , they form distinct phylogenetic clusters cells. Hyperthermophilic; maximum growth temperature above and are also well separated by different morphological, bio- 100°C. Either chemolithoautotrophic growth with H, by reduc- chemical, and physiological properties. tion of elemental sulfur or thiosulfate to H,S with CO, as the For a long time the Crenarchaeota were regarded as a branch sole carbon source or growth by fermentation. Some genera of the sulfur-metabolizing hyperthermophiles. Now, the isola- gain energy by respiration with 0, or nitrate as the electron tion of several new members of this kingdom demonstrates a acceptor. great variety of physiologically diverse organisms. Even organ- isms which are inhibited by sulfur, like Pyrobaculum aerophi- ACKNOWLEDGMENTS lum or erolobus fiman’i, are included in this kingdom. Re- cently an in situ analysis of a hot spring in Yellowstone We gratefully acknowledge W. Ludwig and 0. Strunk for providing National Park revealed a very rich diversity of crenarchaeal the ARB program and for instruction on its usage. Furthermore, we are indebted to Clifford Brunk for critically reading the manuscript and small-subunit rRNA sequences (1, 2). By the same method, Nicole Eis for helpful discussions. sequences from mesophilic Crenarchaeota, which might repre- This work was supported by the Deutsche Forschungsgemeinschaft sent a major fraction of the picoplankton in the oceans (9), (STE 297/10-2). were detected (8, 14). This indicates that there are many, as-yet-uncultivated, Crenarchaeota that may lead to the iden- REFERENCES tification of further families or orders within this archaeal 1. Barns, S. M., R. E. Fundyga, M. W. Jeffries, and N. R. Pace. 1994. Remark- kingdom. able archaeal diversity detected in a Yellowstone National Park hot spring Emendation of the order Thermoproteules Zillig et al. 1981. environment. Proc. Natl. Acad. Sci. USA 91:1609-1613. 2. Barns, S. 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