International Journal of Systematic Bacteriology (1998), 48,475-479 Printed in Great Britain

Desulfurella karnchatkensis sp. nov. and Desulfurella propionica sp. nov., new sulf urlrespiring thermophilic from Kamchatka thermal environments

M. L. Miroshnichenko,' F. A. Rainey,' H. Hippe,2 N. A. Chernyh,' N. A. Kostrikina' and E. A. Bonch-Osmolovskaya'

Author for correspondence: M. L. Miroshnichenko. Tel: +7 95 1350131. Fax: +7 95 1356530. e-mail : [email protected]

1 Institute of Microbiology, Two strains of moderately thermophilic bacteria, which reduce elemental Russian Academy of sulfur to hydrogen sulfide, were isolated from volcanic sources in Kamchatka. Sciences, Prospect 60 Letiya Oktyabrya 712, Strain K-llgTwas obtained from a thermophilic microbial community 11781 1 Moscow, Russia associated with Thermothrix thiopara, and strain U-8Twas isolated from a

2 German Collection of cyanobacterial mat inhabiting a sulf ide-rich hot spring. Cells of both strains Microorganisms and Cell are short Gram-negative rods, motile with one polar flagellum (strain K-1193 Cu Itu res, B ra unsc hw e i g, or non-motile (strain U-83. Both strains are obligate anaerobes, have Germany temperature optima of 54-55 "C and pH optima of 69-702. Molecular hydrogen, acetate, fumarate, malate, pyruvate, lactate and long-chain saturated fatty acids served as growth substrates for both ; strain U-8Twas also able to grow on propionate. All substrates were oxidized completely, H,S and CO, being the only metabolic products. Elemental sulfur was obligately required for growth of strain K-llgT,whereas strain U-8l was able to grow also with thiosulfate as electron acceptor and on pyruvate without an external electron acceptor. The DNA G+C contents of strains K-11gT and U-8Twere 31.6 and 32.2 molo/o, respectively. Phenotypic features and the results of 165 rRNA sequencing indicate the affiliation of the new isolates to the genus Desulfurella. The DNA-DNA hybridization with Desulfurella acetiworans was 40 O/O for strain K-11gT and 55 O/O for strain U-8l; the DNA-DNA hybridization between the new isolates was 32%. Based on the results of morphological, physiological and phylogenetic studies the following two new species are proposed: Desuhrella kamchatkensis sp. nov. with the type strain K-1lgT (= DSM 104093 and Desulfurelia propionica sp. nov. with the type strain UaT (= DSM 104103.

Keywords : Desulfurella kamchatkensis sp. nov., Desulfurella propionica sp. nov., sulfur-respiring bacteria, thermophilic bacteria

INTRODUCTION olism predominates, being found in the extremely thermophilic genera Thermotoga and Thermosipho (6, The ability to reduce elemental sulfur in the course of 12), as well as in moderately thermophilic organo- growth is common among thermophilic micro- trophic anaerobes (4). Moderately thermophilic, organisms. Extremely thermophilic archaea comprise sulfur-respiring bacteria are represented so far only by both lithotrophic and organotrophic sulfur-respiring the genus Desulfurella (3, 8). The type species of this organisms, as well as fermentative organisms which genus, , is an obligate anaerobe use sulfur only as the external electron sink (13). capable of lithotrophic growth with molecular hy- Among thermophilic bacteria, the latter type of metab- drogen and elemental sulfur; it also oxidizes acetate and saturated fatty acids via sulfur reduction. Here we present the description of two new strains, isolated The EMBLaccession numbersfor the 165 rDNAsequences of D. multipotens, from continental hot vents of the Kamchatka pen- strain K-I lgTand strain U-8Tare Y16943, Y16941 and Y16942, respectively. insula.

00580 Q 1998 IUMS 475 M. L. Miroshnichenko and others

METHODS acceptor. In both cases, growth was accompanied by abundant sulfide production. In acetate-utilizing en- Sampling sites. Samples used in this work were obtained from Kamchatka thermal areas in 1994. Samples from one richment cultures from the Therrnothrix-associated of the Pauzhetka hot springs consisted of a Thermothrix community, short motile rods predominated. From thiopara-like organism (9,filaments of which were covered this enrichment, a pure culture of an acetate-utilizing with precipitated elemental sulfur. The pH of the water was organism, designated K- 11 9T, was isolated by serial around 6.5, and the temperature of the sampling site was dilution. In propionate-utilizing enrichments from the 72-78 "C. A sample of the cyanobacterial mat was taken cyanobacterial mat, large coccoid cells and short rods from Uzon caldera hot pond, which had a pH of 6.2 and a were present. In subsequent enrichments, only the high sulfide concentration (up to 38 mM). Elemental sulfur short rods remained. Propionate-utilizing rods from was suspended in the water and precipitated between the this enrichment were isolated by serial dilution, and layers of cyanobacteria (2). The temperature of the mat was the strain was designated U-ST. The purity of both 48-63 "C. cultures was checked by microscopy of cells after the Enrichment, isolation and physiological tests. Enrichment incubation in non-selective glucose- and peptone- and pure cultures of thermophilic sulfur-reducing bacteria containing media. Further checks of the purity were were obtained using anaerobically prepared medium of the following composition (mg 1-l): NH4C1, 330; KC1, 330; made during the physiological studies of growth on a KH,PO,, 330; CaC1,. 2H,O, 330; MgCl, .6H,O, 330; yeast variety of substrates as well during the molecular extract, 100; Na,S . 9H,O, 500; NaHCO,, 1500; resazurin, studies. 2; trace elements solution (l), 1 ml 1-l; vitamins (16), 1 ml 1-l. Elemental sulfur was added as flowers of the sulfur Morphology of the new isolates aqueous suspension at a final concentration of 10 g 1-l. Organic growth substrates were added at 0.5% (w/v). Cells of strain K-1 19T were short rods, single or in Possible electron acceptors were added at 0.2 % (w/v). The pairs, motile with one polar flagellum (Fig. la). The pH was adjusted with 5 M H,S04 or with 5 M NaOH. After size of the cells varied in the range 1-5-2.0 x 0-5-0.8 pm. dispension to 15 ml Hungate tubes with screw caps, the head Spores were never observed. The cell wall had a typical space (5 ml) was filled with N,/CO, gas mixture (8:2, v/v). Gram-negative structure. Cells of strain U-ST were When molecular hydrogen was used as substrate, the head short rods measuring 1.5-2.0 x 1.0 pm and were devoid space (10 ml) was filled with H,/CO, gas mixture (8 :2, v/v). of flagella (Fig. 1b). However, numerous pilus-like Inoculated tubes were incubated at 55 "C. structures were observed on the surface of the cells Tests for growth with ferric iron as an electron acceptor were (Fig. lc). done in sulfide-free media with 15 mM ferric citrate replacing the elemental sulfur. A 10 YO(w/v) solution of ferric citrate Growth characteristics pentahydrate was separately autoclaved under N,. 0.5 ml were added to 10 ml medium and the medium pH readjusted Both strain K-119T and strain U-ST grew only after to 6.2 with sterile 2 M NaOH. No reducing agent was added. reduction of the medium with sodium sulfide. Growth Analytical methods. The cell density was determined by of the strain K-1 19T occurred between 40 and 70 "C direct cell counting in a light microscope. Gaseous and liquid with an optimum at 54 "C; the pH optimum was at fermentation products were detected by GLC (8). Hydrogen 6.9-7.2. Strain U-ST grew in the temperature range sulfide was measured by a colorimetric method (14). 33-63 "C with an optimum at 55 "C and at the same Morphological and ultrastructural studies. The morphology pH optimum as for strain K-1 19T.Strain K- 1 19Twas of cells was studied with a light microscope (MBI-11). found to be able to grow on H,/CO, gas mixture, Electron microscopy methods were described previously (3). acetate, pyruvate, lactate, fumarate, malate, palmitate Genome characterization. Determination of the G + C con- or stearate as electron donor and carbon source in the tent of the DNA and DNA-DNA hybridization were carried presence of elemental sulfur. The only products out according to previously described methods (8). detected were H,S and CO,. Glucose, sucrose, starch, 165 rDNA sequence determination. Genomic DNA extrac- peptone, yeast extract, formate, propionate, butyrate, tion, PCR mediated amplification of the 16s rDNA and caproate, benzoate, hexadecane, succinate, ethanol or purification of PCR products was carried out as described by methanol did not support growth in the presence or the Rainey et al. (10). Purified PCR products were sequenced absence of elemental sulfur. Strain U-8T could utilize using the Taq DyeDeoxy Terminator Cycle Sequencing Kit propionate in addition to all the substrates utilized by (Applied Biosystems) as directed in the manufacturers strain K- 11 9T. protocol. Sequence reactions were electrophoresed using the Applied Biosystems 373A DNA Sequencer. The 16s rDNA No growth of strain K-119T on any energy substrate sequences were aligned manually against the previously occurred in the absence of elemental sulfur. Sulfate, published 16s rDNA sequence of D. acetivorans (1 1). sulfite, thiosulfate, nitrate, malate, fumarate, ferric iron were tested as possible electron acceptors. None RESULTS of them allowed significant growth. Enrichment and isolation In contrast to strain K-119T, strain U-ST was able to use thiosulfate as an electron acceptor. It was also able After 3-5 d incubation at 55 "C, bacterial growth was to grow on pyruvate in sulfur-free medium. Both detected with acetate and propionate as organic strains grew well on mineral medium without the growth substrates and elemental sulfur as electron addition of yeast extract.

476 International Journal of Systematic Bacteriology 48 Two new Desulfurella species

DNA hybridization between strain U-8T and D. acetivorans was 55 'YO. Cross-hybridization of DNA between strains K-1 lgT and U-8T was 32 %.

165 rDNA sequence analysis Full 16s rDNA sequences comprising 1526 nucleotides were determined for D. multipotens, strain K- 11 9T and U-8T. Comparison of these sequences with the pre- viously determined sequence of D. acetivorans showed D. muZt@otens and the new strains to have high 16s rDNA sequence similarity both with each other and D. acetivorans. All of the 16s rDNA sequence similarity values are greater than 99 Yo.

DISCUSSION For many years, the reduction of elemental sulfur as the major process of energy conservation was con- sidered to be a feature of only one bacterial genus, Desulfuromonas (9). Recently, three representatives of a new mesophilic genus, Desulfuromusa, which are able to oxidize a wide range of organic acids and alcohols in the course of elemental sulfur reduction, were de- scribed (7). Elemental sulfur is a common component of volcanic environments and plays an important role in the anaerobic carbon cycle. Moderately thermophilic sulfur-reducing bacteria are widespread in submarine and continental hot vents (4). However, so far only two isolates have been characterized in detail; these are D. acetivorans, the type species of the genus Desul- furella (3), isolated from a Kamchatkan cyanobacterial mat, and Desulfurella multipotens (8), obtained from bottom deposits of Green Lake (Raoul Island, New Zealand). The ability of D. multipotens to utilize butyrate was the main phenotypic feature differen- tiating it from D. acetivorans. The DNA-DNA hybri- dization of the two organisms was found to be 69 %, which is borderline for the designation of a new species.

Fig. 1. Electron micrographs of the cells of new thermophilic The new isolates from Kamchatka possess several sulfur-reducing bacteria. Negatively stained whole cells of strain features common to the genus Desulfurella : their cells K-1 lgT (a) and strain U-8T (b). Platinum-shadowed whole cells of are Gram-negative short rods, they are obligate strain U-8T (c). Bars, 0-5pm. anaerobes and neutrophilic moderate thermophiles ; their main catabolic reaction is the reduction of elemental sulfur to hydrogen sulfide via the complete In optimal conditions, with acetate as the growth oxidation of several characteristic growth substrates : substrate, strain K-1 19Tgrew up to a cell density of lo8 molecular hydrogen, acetate and saturated fatty acids. cells ml-l, with a doubling time of around 2.5 h. Strain Comparison of 16s rDNA sequences of D. multipotens, U-8T growing in optimal conditions on propionate- the new isolates and D. acetivorans confirms their containing medium reached the same cell density, but affiliation to the genus Desulfurella. All strains have its doubling time was 5 h. very high 16s rDNA sequence similarity and are members of the distinct lineage previously represented Genome characteristics by the single species D. acetivorans (1 1). In spite of the high 16s rDNA similarities between these strains, The G + C content of the DNA of strain K- 1 19Twas DNA-DNA hybridization studies show that they 3 1-6mol %. The DNA-DNA hybridization between represent distinct species (1 5). DNA-DNA cross- strain K-1 19T and D. acetivorans was 40 %. The G + C hybridization showed that the new isolates differ content of strain U-8T was 32.2 mol 'YO. The DNA- significantly from D. acetivorans (40-55 YO hybridi-

International Journal of Systematic Bacteriology 48 477 M. L. Miroshnichenko and others

Table 1. Comparative characteristics of type strains of genus Desulfurella and new isolates

Characteristic D. acetivorans D. rnultipotens K-119T U-8T

Motility + - Growth substrates :

H2 + + Acetate + + Propionate - + Butyrate - - Lactate + + Fumarate + + Malate + + Pyruvate + + Stearate + + Palmit ate + + Electron acceptors : Sulfur + + Thiosulfate - + Growth on pyruvate without - + external electron acceptor DNA G + C content (mol %) 31.6 32.2

zation) and from each other (32 % hybridization). On two new species : Desulfurella kamchatkensis, with the the basis of the strains investigated in this study, it is type strain K- 1lgT, and Desulfurella propionica, with clear that 16s rDNA sequence data are useful in the type strain U-8T. assigning new isolates to the genus Desulfurella but has limited use in the differentiation of strains at the species level. Emended description of the genus Desulfurella However, there are several important morphological Desulfurella. Cells are short rods, non-motile, or motile and physiological differences which distinguish the with one polar flagellum. Gram-negative, with outer S- new isolates from the type species D. acetivorans and layer. Domain Bacteria. Obligate anaerobe. Moderate from each other (Table 1). Strain K-1 19T is able to thermophile with a temperature range between 45 and utilize a much wider range of substrates than known 75-80 "C, and an optimum of 55-60 "C. Neutrophile for D. acetivorans and D. multipotens. Apart from with pH optimum of 6.8-7.0. Energy conservation by utilization of the typical substrates for Desulfurella, it sulfur or thiosulfate respiration or by pyruvate fer- also carries out the complete oxidation of lactate, mentation. Substrates utilized : molecular hydrogen, fumarate, malate and pyruvate. Differing from all the organic acids, fatty acids. Products: CO,, H,S. No other strains of thermophilic sulfur-reducing bacteria, cytochromes detected. Menaquinones present. Habi- cells of strain U-8T are non-motile, possess pilus-like tats : warm sediments, therrnophilic cyanobacterial or structures, but not flagella. This strain is able to utilize bacterial communities, rich in organic compounds and the same range of substrates as strain K-1 19Tand, in elemental sulfur, with temperature 50-70 "C. Type addition, propionate. Another important distinguish- species : Desulfurella acetivorans A-63T (= DSM ing feature of this strain is its ability to grow in sulfur- 5264T). free medium with thiosulfate as the electron acceptor, or even without any acceptor if pyruvate was present Description of Desulfurella kamchatkensis as growth substrate. Miroshnichenko 1996 The results indicate some morphological and physio- Desulfurella kamchatkensis (kam.chat .ken'sis. L. part. logical differences between moderately thermophilic originating from Kamchatka). sulfur-reducing bacteria, belonging to genus Desul- furella. Similarly, representatives of the mesophilic Cells are short rods, 1.5-2-0 x 0.5-0-8 pm, single or in genus Desulfuromusa, though closely related on the pairs, motile with one polar flagellum. Cell wall of 16s rDNA level, differ in phenotypic features and have Gram-negative type. Spores not observed. Obligate low levels of DNA-DNA hybridization (7). anaerobe. Moderate thermophile growing between 40 and 70 "C, optimally at 54 "C; neutrophile with a On the basis of the data presented we propose the pH optimum of 6.9-7.2. Growth substrates : H2/C02, emendation of the genus Desulfurella and to include acetate, pyruvate, lactate, fumarate, malate, palmitate,

478 International Journal of Systematic Bacteriology 48 Two new Desulfurella species

stearate. Does not utilize glucose, sucrose, starch, sp. nov. - a new thermophilic sulfur-reducing eubacterium. peptone, yeast extract, formate, propionate, benzoate, Arch Microbioll53, 151-1 55. hexadecane, succinate, ethanol or methanol. Elemen- 4. Bonch-Osmolovskaya, E. A., Miroshnichenko, M. L., Chernyh, tal sulfur obligately required. Yeast extract is not N. A., Kostrikina, N. A., Pikuta, E. V. & Rainey, F. A. (1997). required. Growth products : H,S, CO,. G + C content Reduction of elemental sulfur by moderately thermophilic of DNA: 31-6mol YO. Habitat: continental hot organotrophic bacteria and characterization of Thermo- springs, rich in elemental sulfur and organics. Source anaerobacter sulfurophylus sp. nov. Microbiology (English of isolation : ' Therrnothrix thiopara ' association from translation of Mikrobiologiya) 66, 58 1-587. Pauzhetka hot spring (Kamchatka). Type strain : K- 5. Caldwell, D. E., Caldwell, 5. J. & Laycock, 1. P. (1976). Thermo- 119T (= DSM 10409T). thrix thiopara gen. and sp. nov., a facultatively anaerobic, facultative chemolithotroph, living at neutral pH and high temperature. Can J Microbiol22, 1509-1 5 17. Description of Desulfurella propionica 6. Jannsen, P. & Morgan, H. W. (1992). Heterotrophic sulfur Miroshnichenko 1996 reduction by Thermotoga sp. strain FJSS3Bl. FEMS Micro- biol Lett 96, 2 13-2 18. Desulfurella propionica (pro.pi.o'ni.ca. N.L. fem. adj. 7. Liesak, W. & Finster, K. (1994). Phylogenetic analysis of five propionica propionate-utilizing). strains of Gram-negative, obligately anaerobic sulfur- reducing bacteria and description of Desulfuromusa gen. Cells are short rods, single or in pairs, non-motile with nov., including Desulfuromusa kisingii sp. nov., Desul- multiple pilus-like structures. Cell wall of Gram- furomusa bakii sp. nov., and Desulfuromusa succinoxidans negative type. Spores not observed. Obligate anaerobe. sp. nov. Int J Syst Bacteriol44, 753-758. Moderate thermophile growing between 33 and 63 OC, 8. Miroshnichenko, M. L., Gongadze, G. M., Lysenko, A. M. & with an optimum of 55 "C; neutrophile with a pH Bonch-Osmolovskaya, E. A. (1 993). Desulfurella multipotens optimum of 6-9-7-2. Growth substrates : H,/CO,, sp. nov., a new sulfur-respiring thermophilic eubacterium acetate, propionate, pyruvate, lactate, fumarate, mal- from Raoul Island (Kermadec archipelago). Arch Microbiol ate, palmitate, stearate. Does not utilize glucose, 161, 88-93. sucrose, starch, peptone, yeast extract, formate, ben- 9. Pfennig, N. & Biebl, H. (1976). Desulfuromonas acetooxidans zoate, hexadecane, succinate, ethanol, methanol. Elec- gen. nov. and sp. nov., a new anaerobic sulfur-reducing tron acceptors : elemental sulfur and thiosulfate which acetate-oxidizing bacterium. Arch Microbiol 110, 3-12. are reduced to H,S. All substrates are oxidized to CO,. 10. Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stacke- Able to grow on pyruvate in the absence of sulfur. brandt, E. (1996). The genus Nocardiopsis represents a Yeast extract is not required. G + C content of DNA: phylogenetically coherent taxon and a distinct actinomycete 32.2 mol%. Habitat : continental hot springs rich in lineage : proposal of Nocardiopsaceae fam. nov. Int J Syst elemental sulfur and organics. Source of isolation : Bacteriol46, 1088-1092. cyanobacterial mat from sulfide-rich hot pond, Uzon 11. Rainey, F. A., Toalster, R. & Stackebrandt, E. (1993). Desul- caldera, Kamchatka. Type strain: U-8T (= DSM furella acetivorans, a thermophilic, acetate-oxidizing and 104 1OT) . sulfur-reducing organism, represents a distinct lineage within the . Syst Appl Microbioll6,373-379. 12. Ravot, G., Ollivier, B., Patel, B. K. C., Magot, M. & Garcia, ACKNOWLEDGEMENTS J.-L. (1996). 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