International Journal of Systematic and Evolutionary (2002), 52, 1961–1967 DOI: 10.1099/ijs.0.02173-0

Carboxydocella thermautotrophica gen. nov., sp. nov., a novel anaerobic, CO-utilizing from a Kamchatkan hot spring

Institute of Microbiology, T. G. Sokolova, N. A. Kostrikina, N. A. Chernyh, T. P. Tourova, Russian Academy of Sciences, Prospect 60 Let T. V. Kolganova and E. A. Bonch-Osmolovskaya Oktyabrya 7/2, 117811 Moscow, Russia Author for correspondence: Tatyana Sokolova. Tel: j7 095 135 4458. Fax: j7 095 135 6530. e-mail: sokolova!inmi.host.ru

A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41T, was isolated from a terrestrial hot vent on the Kamchatka Peninsula. Strain 41T was found to be a Gram-positive bacterium, its cells being short, straight, motile rods. Chains of three to five cells were often observed. The isolate grew only

chemolithoautotrophically on CO, producing equimolar quantities of H2 and

CO2 (according to the equation COMH2O ! CO2MH2). Growth was observed in the temperature range 40–68 SC, with an optimum at 58 SC, and in the pH range 65–76, with an optimum at pH 70. The generation time under optimal conditions for chemolithotrophic growth was 11 h. The DNA GMC content was 46O1 mol%. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. On the basis of the phenotypic and phylogenetic features, it is proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen. nov., sp. nov. (type strain 41T l DSM 12356T l VKM B-2282T).

Keywords: terrestrial hot vents, carbon monoxide utilization, thermophilic anaerobe, Carboxydocella thermautotrophica

INTRODUCTION (Svetlichny et al., 1994). Carboxydobrachium pacificum was the first isolate found to originate from the deep- Anaerobic oxidation of CO coupled to equimolar sea hot vents of the Okinawa Trough (Sokolova et al., hydrogen and CO# formation was first found in the 2001). In addition to being capable of anaerobic CO purple non-sulfur, mesophilic, phototrophic oxidation\H# formation, it could also ferment diverse Rubrivivax gelatinosus (Willems et al., 1991) (pre- organic substrates. All of these are obligate viously Rhodocyclus gelatinosus; Uffen, 1976, 1983) or facultative lithotrophs, but have a requirement for and Rhodospirillum rubrum (Bonam et al., 1989). Later, yeast extract for growth. Here, we report the isolation it was shown that the reaction CO H O ! CO H of a novel CO-utilizing, H -producing, thermophilic ! j # #j # # (∆G lk20 kJ) might be performed by several phylo- bacterium (from a thermal spring of the Geyzer Valley, genetically diverse thermophilic bacteria. Among Kamchatka Peninsula) that is an obligate litho- them, Carboxydothermus hydrogenoformans, isolated autotroph. from terrestrial hot springs of the Kuril Islands, was the first taxonomically described representative (Svetlichny et al., 1991). Except for CO, the only METHODS substrate utilized by this was pyruvate. Sampling. Samples of hot water, mud and cyanobacterial Another organism, ‘Carboxydothermus restrictus’, mat from a hot spring (60 mC, pH 8n6) near the Giant Geyser was isolated from a terrestrial hot vent on Raoul (Geyser Valley, Kamchatka Peninsula) were taken Island (Kermadek Archipelago, New Zealand) anaerobically in tightly stoppered bottles and transported (at ambient temperature) to the laboratory.

...... Enrichment and isolation. For enrichment and isolation of The GenBank accession number for the 16S rDNA sequence of strain 41T is anaerobic carboxydotrophic bacteria, the following medium −" AY061974. (medium 1) was used (g l ): NH%Cl (1); MgCl# ; 6H#O

02173 # 2002 IUMS Printed in Great Britain 1961 T. G. Sokolova and others

(0n33), CaCl# ; 6H#O(0n1), KCl (0n33), KH#PO% (0n5), ward primer and 5h-TACGGTTACCTTGTTACGACTT- resazurin (0n001), 1 ml trace-mineral solution (Kevbrin & 3h as the reverse primer (Lane, 1991). The PCR was carried Zavarzin, 1992) and 1 ml vitamin solution (Wolin et al., out in 100 µl reaction mixture containing 1 µg DNA tem- 1963). After being boiled, the medium was flushed with N# plate, 200 µM (each) primers, 200 µM (each) dNTPs and 3 U −" −" and cooled. NaHCO$ (0n5gl ) and Na#S ; 9H#O(1n0gl ) Tet-z polymerase (BioMaster) in reaction buffer (100 mM were then added and the pH was adjusted to 6n8 with 0n5M Tris\HCl pH 8p3, 500 mM KCl, 20 mM MgCl#). The PCR HCl. Aliquots (10 ml each) of the medium were placed into was performed using the following programme: 30 ampli- 50 ml bottles and the headspaces filled with 100% CO at fication cycles of denaturation at 94 mC for 1 min, annealing atmospheric pressure. The incubation temperatures were 55 at 42 mC for 1 min and extension at 72 mC for 1 min. The final and 70 mC. Growth was determined using light microscopy extension was carried out at 72 mC for 6 min. The PCR and GLC detection of CO utilization and the formation of products were purified using a PCR-Prep kit (Promega) as gaseous growth products. Positive enrichments were used recommended by the manufacturer. The 16S rDNA was for further purification. After a number of serial dilution sequenced in both directions by using a set of forward and transfers, colonies were obtained in roll-tubes prepared in reverse universal primers (Lane, 1991). DNA sequencing 15 ml Hungate tubes on the same medium solidified with was performed by using Sequenase version 2 of the VSB kit 5% agar, with CO in the gas phase. Well-separated colonies (USB). were transferred into the same liquid medium as that used The 16S rDNA sequence was aligned with a representative for enrichments. set of 16S rRNA sequences obtained from the Ribosomal Light and electron microscopy. Light microscopy was carried Database Project or from recent GenBank releases by using out using a phase-contrast microscope with an oil-immersion . Positions that had not been sequenced in one or objective (90\1n25). For electron microscopy, negative stain- more reference organisms were omitted from the analysis. ing of whole cells was done with 2% phosphotungstic acid. Pairwise evolutionary distances were computed by using the For the preparation of thin sections, cells were fixed with correction of Jukes & Cantor (1969). Phylogenetic trees were 5% glutaraldehyde for 2 h and 1% OsO% for 4 h at 4 mC, constructed by the neighbour-joining method using the embedded in Epon-812 resin, thin-sectioned and stained programs of the  package (Van de Peer & De with uranyl acetate and lead citrate. Electron micrographs Wachter, 1994), by the maximum-likelihood method using were taken with a JEM-100C electron microscope. the  program (Strimmer & von Haeseler, 1996) and by the maximum-parsimony method using program Physiology studies. Growth of the novel isolate was tested on  of the  package (Felsenstein, 1989) with different substrates in the same liquid mineral medium, with bootstrap analysis of 100 trees. N in the gas phase. Possible substrates were added in- # " dividually at a concentration of 2 g l− (final concentration). " Possible electron acceptors were added at 2 g l− , and RESULTS AND DISCUSSION " elemental sulfur at 10 g l− . Enrichment and isolation CO, CO# and H# were analysed using a Chrom-5 (CSFR) gas chromatograph equipped with a thermal conductivity de- For the enrichment of anaerobic, carboxydotrophic, tector and a 1n2 m glass column filled with activated carbon thermophilic bacteria, bottles with liquid anaerobic AG-3. The carrier gas was argon and the temperature in the medium and a CO gas phase were inoculated with incubator was 25 mC. Volatile fatty acids were determined on about 1 g sample. After 2 days incubation at 55 mC, a Chrom-5 gas chromatograph with a flame-ionization two of the bottles showed an increase in pressure from detector: the column was filled with Chromosorb 10 (Sigma), 140 to 160–170 kPa. Growth of small rod-shaped the temperature was 170 mC and the carrier gas was argon at −" bacteria was observed. The CO content in the gas a flow rate of 40 ml min . phase had decreased to 40%, and about 30% H# and The effect of temperature on growth was studied in medium 30% CO# had appeared. After several transfers in the 1 under a CO atmosphere. The effect of pH on growth was same medium, the enrichments were serially diluted studied under a CO atmosphere in the same medium, except 10-fold and transferred to solidified medium in roll- that the phosphate component of the medium was added in tubes. After 4 days incubation at 60 mC, round, white the form of 0n01 M phosphate buffer with the required pH. colonies, about 0n5 mm in diameter, were observed. Single colonies were isolated into liquid medium and Temperature and pH optima were determined from the T growth rates. The cell density was measured by direct cell two pure cultures were obtained. Strain 41 was counting. selected for further characterization. " Sensitivity of the novel isolate to penicillin (100 µgml− ), " " ampicillin (100 µgml− ), streptomycin (100 µgml− ), kana- Morphology " " mycin (50 µgml− ) and neomycin (50 µgml− ) was tested in T the same medium under a CO atmosphere. Cells of isolate 41 were straight rods of various lengths from 1 to 3 µm and about 0n4–0n5 µm in width, DNA isolation and base composition. DNA was prepared as arranged singly or in short chains of three to five cells. described by Marmur (1961). The GjC content of the Cells were motile due to lateral flagella (Fig. 1a). DNA was determined by melting-point analysis (Marmur & Electron microscopy of ultrathin sections revealed that Doty, 1962) using Escherichia coli K-12 DNA as a reference. the cell envelope was of the Gram-positive type, being 16S rDNA sequence determination and analysis. 16S rDNA composed of a cytoplasmic membrane and a double- was selectively amplified from genomic DNA by a PCR layer cell wall comprising an inner electron-dense layer using 5h-AGAGTTTGATCCTGGCTCAG-3h as the for- and outer lighter layer. Cell membrane invaginations

1962 International Journal of Systematic and Evolutionary Microbiology 52 Carboxydocella thermautotrophica gen. nov., sp. nov.

(a)

(b)

...... Fig. 2. Growth of strain 41T at 60 mC in mineral medium under a CO atmosphere. Cell density ($), CO consumption ( ), H2 production (>) and CO2 production (=) were monitored.

the equation COjH#O ! CO#jH# (Fig. 2). Neither methane, acetate nor any other metabolic product was detected. The generation time of strain 41T under (c) optimal conditions was 1n1h. Strain 41T did not grow on peptone, yeast extract, starch, cellobiose, sucrose, lactose, glucose, maltose, galactose, arabinose, fructose, acetate, formate, pyruvate, ethanol or methanol. A H#\CO# gas mixture (4:1, v\v) did not support growth. No growth was observed on acetate, ethanol or lactate in the presence or absence of elemental sulfur or sulfate. Neither sulfur nor sulfate was reduced during growth with CO. No ...... growth or H#S production occurred in the medium T Fig. 1. Electron micrographs of cells of strain 41 . (a) Negative with elemental sulfur or sulfate and H# in the gas staining. (b, c) Thin sections. Bars, 0n5 µm. phase. Penicillin, ampicillin, streptomycin, kanamycin and neomycin completely inhibited CO utilization and the were often observed (Fig. 1b, c). The cells divided by growth of strain 41T. binary transverse fission (Fig. 1b).

DNA base composition Growth characteristics T T The GjC content of the DNA of strain 41 was Growth of strain 41 occurred within the temperature 46p1mol%. range 40–68 mC, with an optimum at 58 mC. No growth T was observed at 37 or 70 mC. Strain 41 grew over a pH range of 6n5–7n6, with an optimum at 7n0, but no Phylogenetic analyses growth was detected at pH 6n2or7n8. We determined a partial 16S rDNA sequence (1456 nt) Strain 41T grew only under strictly anaerobic con- for strain 41T, corresponding to positions 37–1467 of ditions. It did not grow in medium without reducing the E. coli numbering. Preliminary comparisons (using agents, nor under a CO\air mixture (4:1, v\v). It grew ) with 16S rDNA sequences available in in an atmosphere of 100% CO (0 23 mmol CO in the GenBank revealed that the novel isolate 41T was a " n gas phase ml− medium) on the mineral medium member of the Bacillus\Clostridium subphylum of the without yeast extract or other organic compounds Gram-positive bacteria. The highest score was found except vitamins. CO oxidation was coupled to H# and with 16S rDNA sequences of the Thermo- CO# formation in equimolar quantities according to anaerobacter–Syntrophomonas group, but it was no http://ijs.sgmjournals.org 1963 T. G. Sokolova and others more than 89%, so there were no sequences that were closely related to that of strain 41T. Several phylogenetic trees were constructed by chang- ing the composition of reference organisms belonging to the Clostridium group. Regions of alignment un- certainty resulting from the presence of long inserts in 16S rDNA sequences of some members of this group (Rainey et al., 1993; Slobodkin et al., 1999) were omitted from the sequence analysis. A final com- parison was carried out of 1322 nt of 16S rDNA sequences of strain 41T and 57 of the closest reference strains of the Thermoanaerobacter–Syntrophomonas group, combined members of clusters V–VIII of the Clostridium spectrum (Collins et al., 1994). This group, including species of the genera Desulfotomaculum, Thermoanaerobacter, Thermoanaerobacterium, Moorella, Anaerobranca and some others, was used for the reconstruction of a phylogenetic tree and the calculation of sequence similarity. The level of sequence similarity of strain 41T was relatively low and almost equal for all reference strains (85n3–90n7%). In the phylogenetic tree constructed by using the neighbour-joining algorithm (Fig. 3), strain 41T was not clustered exactly with any genus or species of the Clostridium group. Trees constructed using other treeing algorithms had the same topology (data not shown). A variety of diverse bacteria possess CO-oxidizing enzymes, termed CO dehydrogenases, which allow them to assimilate CO as a sole source of carbon and energy. Aerobic carboxydotrophic bacteria grow heterotrophically on various organic compounds or chemolithoautotrophically on CO; they are taxo- ...... nomically diverse, consisting of more than 15 described Fig. 3. Phylogenetic tree showing the position of strain 41T.A species from eight genera (Zavarzin & Nozhevnikova, consensus 16S rDNA sequence of the actinomycete lineage was chosen as the outgroup. The scale bar represents the expected 1977; Meyer et al., 1986, 1993). number of changes per sequence position. Bootstrap values In anaerobes, CO dehydrogenases are key enzymes of (expressed as percentages of 100 replications) are shown at branch points; values greater than 80% were considered the acetyl-CoA pathway found in the following phylo- significant. Triangles include the following sequences: Thermo- genetically distant micro-organisms: acetogenic bac- anaerobacter thermohydrosulfuricus DSM 567T (L09161); teria (Wood & Ljungdahl, 1991; Ljungdahl, 1994; Thermoanaerobacter sulfurophilus L-64T (Y16940); Thermoan- Drake, 1994), sulfate-reducing bacteria (Schauder et aerobacter wiegelii Rt8.B1T (X92513); Thermoanaerobacter acetoethylicus ATCC 33265T (L09163); Thermoanaerobacter kivui al., 1987; Janssen & Schink, 1995; Oude Elferink et al., DSM 2030T (L09160); Thermoanaerobacter brockii subsp. 1999; Fukui et al., 1999), photosynthetic bacteria brockii DSM 1457T (L09165); Thermoanaerobacter mathranii A3T (Uffen, 1983; Ensign, 1995), methanogenic (Y11279); Thermoanaerobacter thermocopriae JT3-3T (L09167); (Deppenmeier et al., 1996; Ferry, 1999) and non- Thermoanaerobacter ethanolicus JW 200T (L09162); Thermo- T methanogenic archaea (Mo$ ller-Zinkhan & Thauer, anaerobacter brockii subsp. lactiethylicus SEBR 5268 (U14330); Thermoanaerobacter siderophilus SR4T (AF120479); Caldicellulo- 1990; Vorholt et al., 1995, 1997). Some of them can siruptor owensensis OLT (U80596); Caldicellulosiruptor kristjans- grow on CO as the only energy source. During growth sonii DSM 12137T (AJ004811); Caldicellulosiruptor lactoaceticus on CO, homoacetogenic and methanogenic bacteria DSM 9545T (X82842); Caldicellulosiruptor saccharolyticus DSM respectively produce acetate and methane. CO is a 8903T (AF130258); Thermoanaerobacterium thermohydrosul- furicus E100-69T (L09161); Thermoanaerobacterium aotearoense common component of volcanic gases in terrestrial JW/SL-NZ613T (X93359); Thermoanaerobacterium saccharoly- and submarine hot springs. Thus, the ability to utilize ticum DSM 7060T (L09169); Thermoanaerobacterium xylanoly- CO might be assumed to be a widespread capacity of ticum DSM 7097T (L09172); Thermoanaerobacterium thermosac- thermophilic micro-organisms. However, only a lim- charolyticum ATCC 7956T (M59119); Moorella thermoauto- T ited number of thermophilic species are capable of trophica JW 701/5 (X58354); Moorella glycerini DSM 11254 (U82327); Moorella thermoacetica DSM 521T (X58352); Syntro- anaerobic CO utilization. Among the thermophilic phomonas sapovorans DSM 3441T (AF022249); Syntrophomonas prokaryotes, CO-utilizing are repre- wolfei LYB (AF022248); Desulfotomaculum halophilum SEBR sented only by Methanothermobacter thermauto- 3139T (U88891); Desulfotomaculum nigrificans NCIMB 8395 trophicus (Zeikus & Wolfe 1972; Wasserfallen et al., (X62176); Desulfotomaculum ruminis DSM 2154T (Y11572);

1964 International Journal of Systematic and Evolutionary Microbiology 52 Carboxydocella thermautotrophica gen. nov., sp. nov.

Table 1. Characteristics of anaerobic, thermophilic, carboxydotrophic bacteria ...... Data for reference species were taken from Svetlichny et al. (1991) (Carboxydothermus hydrogenoformans 2901T), Svetlichny et al. (1994) (‘Carboxydothermus restrictus’ R1) and Sokolova et al. (2001) (Carboxydobrachium pacificum JMT).

Characteristic Carboxydothermus ‘Carboxydothermus restrictus’R1 Carboxydobrachium pacificum JMT Strain 41T hydrogenoformans 2901T

Terrestrial or marine Terrestrial Terrestrial Marine Terrestrial Morphology Short, slightly curved rods with Short, slightly curved rods with Non-motile, long, thin rods, sometimes Straight, short rods lateral flagella peritrichous flagella branching with lateral flagella Cell-wall structure Gram-positive, globular S-layer Gram-positive, globular S-layer Gram-positive with S-layer Gram-positive Substrates CO, pyruvate CO CO, pyruvate, peptone, yeast extract, starch, CO cellobiose, glucose, galactose, fructose Temperature for growth (mC): Range 40–78 45–80 50–80 40–68 Optimum 70–72 70 70 58 pH for growth: Range 6n6–8n06n6–8n05n8–7n66n5–7n6 Optimum 7n07n06n97n0 Minimum doubling time (h) 2n08n310n21n1 GjC content of DNA (mol%) 39–41 39 33 46

2000). Thermophilic acetogens include Moorella Description of Carboxydocella gen. nov. thermoacetica (Fontaine et al., 1942; Collins et al., 1994) and Moorella thermoautotrophica (Wiegel et al., Carboxydocella (Car.bo.xy.do.celhla. N.L. neut. n. 1981; Collins et al., 1994). Anaerobic CO oxidation carboxydum carbon monoxide; L. fem. n. cella cell; coupled to molecular hydrogen formation has been N.L. fem. n. Carboxydocella carbon monoxide- shown for four thermophilic isolates. Like strain 41T, utilizing bacterium). none of these organisms grows on a mixture of H# and Cells are short, straight rods. Motile. Cell wall of CO#. Their comparative characteristics are given in Gram-positive type. Cells divide by binary transverse Table 1. All of them are anaerobic thermophilic fission. Bacterium. Obligate anaerobe. Thermophile. bacteria with Gram-positive cell-wall structures. Neutrophile. Grows chemolithoautotrophically on Phylogenetically, they all belong to the Gram-positive CO. Utilizes CO as the sole energy source, with bacteria with low GjC content, but they do not form equimolar formation of H and CO according to the a separate cluster. The novel isolate differs from them # # equation COjH#O ! CO#jH#. The GjC content in growth-temperature characteristics and in the range of the DNA of the type strain of the type species is of energy substrates. According to 16S rDNA analyses, T 46p1 mol%. The type species is Carboxydocella strain 41 forms a new, separate line of descent within thermautotrophica. the low-GjC-content Gram-positive subdivision of the Bacteria. On the basis of the 16S rRNA sequence data, it can not be assigned to any validly described genus. Description of Carboxydocella thermautotrophica sp. nov. On the basis of the physiological and phylogenetic features of strain 41T, we propose the description of a Carboxydocella thermautotrophica (therm.au.to.troh new genus and species, Carboxydocella thermauto- phi.ca. Gr. adj. thermos hot; Gr. adv. auto self; trophica gen. nov., sp. nov. Gr. adj. trophikos one who feeds; N.L. fem. adj. thermautotrophica thermophilic and autotrophic, indicating that the organism grows at elevated temperatures and uses CO as the sole source of carbon Desulfotomaculum aeronauticum DSM 10349T (X98407); ‘Desulfotomaculum reducens’ MI-1 (U95951); Desulfotoma- and energy). culum putei SMCC W459T (AF053929); Desulfotomaculum acetoxidans DSM 771T (Y11566); Desulfotomaculum kuznetsovii The following characteristics are observed in addition DSM 6115T (AF009646); Desulfotomaculum luciae SLTT to those given in the genus description. Cells are short, (AF069293); Desulfotomaculum australicum AB33T (M96665); straight rods, about 0 4–0 5 µm in width and varying in T n n Desulfotomaculum thermocisternum ST90 (U33455); Desulfo- length from 1 to 3 µm, arranged singly or in short tomaculum thermobenzoicum DSM 6193T (L15628); Desulfo- tomaculum thermoacetoxidans DSM 5813T (Y11573); Desulfo- chains of three to five cells. Motile due to lateral tomaculum geothermicum DSM 3669T (Y11567); Desulfotoma- flagella. On solid medium, produces round, white, culum thermosapovorans DSM 6562T (Y11575); Anaerobranca translucent colonies. Grows within the temperature T horikoshii JW/YL-138 (U21809); Anaerobranca gottschalkii DSM range 40–68 mC, with an optimum at 58 mC. The pH for 13577T (AF203703); Desulfosporosinus orientis DSM 765T (Y11570); [Desulfotomaculum] auripigmentum ORex (U85624); growth ranges from 6n5to7n6, with an optimum at Desulfitobacterium dehalogenans JW/IU-DC1T (U40078); Desul- pH 7n0. Growth and CO consumption are inhibited by fitobacterium frappieri PCP-1T (U40078). penicillin, ampicillin, streptomycin, kanamycin and http://ijs.sgmjournals.org 1965 T. G. Sokolova and others neomycin. Does not grow on peptone, yeast extract, Lane, D. J. (1991). 16S\23S rRNA sequencing. In Nucleic Acid starch, cellobiose, sucrose, lactose, glucose, maltose, Techniques in Bacterial Systematics, pp. 115–147. Edited by E. galactose, arabinose, fructose, acetate, formate, Stackebrandt & M. Goodfellow. New York: Wiley. Ljungdahl, L. G. (1994). The acetyl-CoA pathway and the chemi- pyruvate, ethanol, methanol or a H#\CO# gas mixture osmotic generation of ATP during acetogenesis. In Acetogenesis, pp. (4:1, v\v). Does not grow on H#, acetate, ethanol or 65–87. Edited by H. L. Drake. New York: Chapman & Hall. lactate in the presence or absence of elemental sulfur or Marmur, J. (1961). A procedure for the isolation of desoxyribonucleic sulfate. 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