Halococcus Dombrowskii Sp. Nov., an Archaeal Isolate from a Permian Alpine Salt Deposit

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1807–1814 DOI: 10.1099/ijs.0.02278-0

Halococcus dombrowskii sp. nov., an archaeal isolate from a Permian alpine salt deposit

1 Institut fu$ r Genetik und Helga Stan-Lotter,1 Marion Pfaffenhuemer,1 Andrea Legat,1 Allgemeine Biologie, 2,3 1 1 Hellbrunnerstrasse 34, Hans-Ju$ rgen Busse, Christian Radax and Claudia Gruber A-5020 Salzburg, Austria

2 Institut fu$ r Bakteriologie, Author for correspondence: Helga Stan-Lotter. Tel: j43 662 8044 5756. Fax: j43 662 8044 144. Mykologie und Hygiene, e-mail: helga.stan-lotter!sbg.ac.at Veterina$ rplatz 1, A-1210 Wien, Austria 3 Institut fu$ r Mikrobiologie Several extremely halophilic coccoid archaeal strains were isolated from pieces und Genetik, Universita$ t of dry rock salt that were obtained three days after blasting operations in an Wien, Dr Bohr-Gasse 9, A-1030 Wien, Austria Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225–280 million years ago). On the basis of their polar-lipid composition, 16S rRNA gene sequences, cell shape and growth characteristics, the isolates were assigned to the genus Halococcus. The DNA–DNA reassociation values of one isolate, strain H4T, were 35 and 38% with Halococcus salifodinae and Halococcus saccharolyticus, respectively, and 658–678% with Halococcus morrhuae. The polar lipids of strain H4T were

C20–C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate. Whole-cell protein patterns, menaquinone content, enzyme composition, arrangements of cells, usage of carbon and energy sources, and antibiotic susceptibility were sufﬁciently different between strain H4T and H. morrhuae to warrant designation of strain H4T as a new species within the genus Halococcus. It is proposed that the isolate be named Halococcus dombrowskii, and the type strain is H4T (l DSM 14522T l NCIMB 13803T l ATCC BAA-364T).

Keywords: archaea, Halococcus dombrowskii sp. nov., salt mine, subsurface, longevity

INTRODUCTION may be interesting candidates to be considered when targeting the search for extraterrestrial forms of life, In recent years, viable halophilic archaea have been particularly since halite has been detected in outer isolated from various ancient salt deposits of the space (Gooding, 1992; McCord et al., 1998; Zolensky Permian and Triassic ages (see review by McGenity et et al., 1999; Whitby et al., 2000). To be able to identify al., 2000). The ﬁrst such strain to be described and potential signatures of micro-organisms in extraterres- validated as a novel species was Halococcus salifodinae, trial materials, it is necessary to have as extensive a which was isolated from rock salt from an Austrian record of terrestrial forms of extremophilic life as salt mine (Denner et al., 1994). Further work, which possible. This record should also include prokaryotes included the culturing of strains as well as the analysis isolated from subsurface materials, such as evaporites. of uncultured samples (Norton et al., 1993; Grant et Here we describe a coccoid haloarchaeal species that al., 1998; Stan-Lotter et al., 1999; Radax et al., 2001), was isolated from Permian rock salt and which is revealed vast biodiversity within the viable halo- distinct from the known representatives of the genus archaeal community in rock salt, but this biodiversity Halococcus. has yet to be fully comprehended. In view of the potential longevity of these haloarchaeal organisms (Grant et al., 1998), it has been suggested that they METHODS Isolation of haloarchaea from rock salt. Samples of freshly ...... blasted rock salt taken from a depth of about 650 m below Published online ahead of print on 1 March 2002 as DOI 10.1099/ the Earth’s surface were obtained from a salt mine near Bad ijs.0.02278-0. Ischl in Austria, three days after blasting operations for the The GenBank/EMBL accession number for the 16S rDNA sequence of creation of new tunnels had been done. The geological Halococcus dombrowskii is AJ420376. setting of the salt mine has been described previously, as

02278 # 2002 IUMS Printed in Great Britain 1807 H. Stan-Lotter and others have the methods for sterile dissolution of the salt pieces and extract, 1 mM NH%Cl, 27 mM KCl, 100 mM MgCl#,1n4mM the initial steps for the culturing of isolates (Radax et al., CaCl#,0n1% trace elements solution SL-6 (Malik, 1983) and 2001; Stan-Lotter et al., 1993). 1% of the respective carbohydrates or 0n01% of the Culture conditions and bacterial strains. Initial streaking of respective amino acids. Incubation was done in test tubes at dissolved rock salt was done onto solidified M2 medium 37 mC without shaking for 10–12 weeks. The most un- (Tomlinson & Hochstein, 1976) that had been prepared with ambiguous results were obtained by determining the size of commercially available NaCl. In later experiments, dissolved cell sediments, following growth of the strain on carbon rock salt was occasionally used as an additive to all growth sources. The diameter of the sedimented inoculum was media at a final concentration of 10%, since it enhanced cell approximately 3 mm and did not change over the course of yields considerably (M. Pfaffenhuemer, unpublished data). the experiments. Diameters of cell pellets were considered The pH of the growth medium was 7n4, unless indicated positive evidence of archaeal growth when they otherwise. Growth of strains in liquid culture was monitored were " 6 mm; some pellet diameters ranged between 7 and spectrophotometrically at 600 nm. " 10 mm. Acidification was determined with special pH indicator test strips (Merck) for the range pH 6n0–10n0. All The following haloarchaeal strains were obtained from the carbon utilization experiments were done at least twice. DSM (Deutsche Sammlung von Mikroorganismen und T Zellkulturen, Mascheroder Weg 1B, D-38124 Braunschweig, Determination of the antibiotic susceptibility of strain H4 Germany): Halobacterium salinarum DSM 668; Halococcus was tested by spreading cell suspensions onto agar plates morrhuae DSM 1307T, DSM 1308 and DSM 1309; Halo- and then applying filter-paper discs (6 mm in diameter) on coccus saccharolyticus DSM 5350T ; Halococcus salifodinae which the antibiotics were dispensed in 20 µg amounts, with BIpT DSM 8989T and BG2\2 DSM 13045; Natronococcus the exception of novobiocin, which was used in 10 µg occultus DSM 3396T. Halococcus morrhuae NRC 16008 amounts. Zones of inhibition were measured following 2 ( l NCIMB 746) was obtained from the NCIMB (National weeks incubation at 37 mC; sensitivity was deemed strong Collection of Industrial and Marine Bacteria, Aberdeen, when the zone of inhibition extended more than 3 mm Scotland, UK). Halorubrum saccharovorum M6T (ATCC beyond the antibiotic disc. At least three determinations 29252T), Halobacterium salinarum (R1), Haloferax denitrifi- were carried out for each antibiotic. T cans (ATCC 35960 ) and Haloarcula vallismortis (ATCC Analysis of menaquinones and polar lipids. All steps were 29715T) were obtained from L. I. Hochstein, formerly of the done under a nitrogen atmosphere. Menaquinones were NASA Ames Research Center, USA. extracted from about 200 mg of cells (wet weight) with Experiments for the description of the new halobacterial methanol\hexane (2:1, v\v) as described by Tindall (1990), taxon. All tests for determining the phenotypic properties of except that pellets of growing cells were used instead of strain H4T were carried out as specified in the proposed lyophilized cells. The hexane phase contained the mena- minimal standards for the description of new taxa in the quinones, which were purified by TLC on silica-gel plates order Halobacteriales (Oren et al., 1997), with some addi- using hexane\diethylether (85:15, v\v) as the developing tions and exceptions as outlined below. solvent, and then separated by HPLC (Denner et al., 1994). Identifications and comparisons were done with the mena- Physiological and biochemical tests. The range of salt T T T quinones from strain H4 , Halococcus salifodinae BIp DSM concentrations that permitted growth of strain H4 was 8989T and Halococcus morrhuae DSM 1307T. The methanol determined by spreading 100 µl of a growing culture of the phase contained the polar lipids, which were further purified strain onto agar plates containing M2 medium supplemented by extraction with chloroform\methanol\0n3% NaCl (1:2: with NaCl to a final concentration of 7n5, 10, 12n5, 15, 17n5, 0n8, v\v) and heating of the mixture to 80 mC for 15 min. 20, 22n5, 25, 27n5 or 30%. Similarly, the requirement of the #+ Following removal of the cell debris by centrifugation, the strain for Mg was tested with M2 agar plates containing extraction with chloroform was repeated. The dried polar MgCl# at a final concentration of 0, 5, 10, 20, 100 or 500 mM. T lipids were resuspended in chloroform\methanol (2:1, v\v) Agar plates inoculated with strain H4 were incubated for and separated by two-dimensional TLC (Ross et al., 1985). 14–20 days. The pH range for growth was determined in M2 Polar-lipid extracts were spotted onto the corner of a thin- medium, which was made with 50 mM of the following layer 10i10 cm silica-gel plate (60 F254; Merck). These buffer substances and adjusted to the desired pH values (pH were first developed in chloroform\methanol\water (65:25: values as measured after autoclaving are in parentheses): 4, v\v) and then in chloroform\methanol\glacial acetic MES (pH 5n13); MES (pH 5n83) and MOPS (pH 6n79); Tris acid\water (80:12:15:4, v\v). Total lipids were visualized (pH 8n09); Tris (pH 9n01); CAPS (pH 9n77). Tubes con- by spraying the chromatograms with phosphomolybdic acid taining 20 ml of the media were inoculated in duplicate with and then heating them to 160 mC. Phospholipids were 100 µl of a washed culture and then incubated at 37 mC detected with molybdenum blue, amino groups were de- without shaking for 4 weeks. tected with ninhydrin, carbohydrates were detected with α- Standard tests (cytochrome oxidase production, catalase naphthol and carbohydrates and sulfate groups were de- production, nitrate reduction and gelatin liquefaction) were tected with Bial’s reagent (orcinol ferric chloride spray performed on strain H4T as described by Smibert & Krieg reagent). The equivalence of spots was determined by co- (1994). The Analytical Profile Index systems API ZYM and chromatography of extracts of known haloarchaea in two API 20NE (both from bioMe! rieux) were used for analysis of dimensions and by comparison with published data. additional enzyme activities (Humble et al., 1977) and for Analysis of whole-cell-protein patterns. SDS-PAGE of assimilation tests, respectively. Strips were inoculated with a whole-cell proteins was performed as described previously cell suspension in Tris-buffered 4 M NaCl and incubated for (Stan-Lotter et al., 1989, 1993). Briefly, approximately 50 mg " up to 24 h (API ZYM) or 3 weeks (API 20NE). All API tests wet weight cells ml− were lysed by boiling them in SDS were done at least three times. sample buffer (Laemmli, 1970) for 10 min. The lysate was The utilization of carbohydrates or amino acids by strain then centrifuged at 10000 g for 15 min, to remove any H4T was tested in a semi-defined medium (M2A) which precipitates. The gel system of Laemmli (1970) was used to consisted of 50 mM Tris\HCl, 4 M NaCl, 0n02% yeast separate the proteins. Proteins were visualized by staining

1808 International Journal of Systematic and Evolutionary Microbiology 52 Halococcus dombrowskii sp. nov. with Coomassie blue. Marker proteins of 2n5 to 200 kDa in 3.5.1c; Felsenstein, 1993) and   (Thompson et al., size (Mark 12) were from Novex. Electrophoresis of the 1997). The confidence of the branching pattern was assessed whole-cell proteins from strain H4T and from the type strains by analysis of 100 bootstrap replicates. used in this study was repeated more than four times. Other methods. Unstained haloarchaeal cells were observed DNA base composition. Cells of strain H4T were harvested in with a Zeiss Axioskop microscope using phase-contrast. the early-stationary phase of growth. The determination of Gram-staining of cells was carried out as described by the GjC content of this strain was carried out by the DSM Dussault (1955). Assays to determine the lysis of haloar- Identification Service. DNA was isolated on hydroxyapatite chaeal cells in water and the isoelectric focusing of whole-cell by the procedure of Cashion et al. (1977), following proteins were performed as described previously (Stan- disruption of cells by passage through a French press, and Lotter et al., 1989; Denner et al., 1994). Carotenoid pigments degraded as described by Mesbah et al. (1989). The resulting were extracted from freeze-dried cells with methanol\ deoxyribonucleosides were analysed by HPLC according to acetone (1:1, v\v) and their absorption spectra were de- Mesbah et al. (1989) and Tamaoka & Komagata (1984). termined (Gochnauer et al., 1972) with a Beckman DU-650 Non-methylated λ DNA was used for calibration (Mesbah spectrophotometer in the range 330–600 nm. et al., 1989). DNA–DNA hybridization. DNA was isolated as described by RESULTS AND DISCUSSION Cashion et al. (1977). Levels of DNA–DNA hybridization between strain H4T and three haloarchaeal type strains were Isolation and selection of strains from rock salt determined spectrophotometrically by the renaturation method of De Ley et al. (1970), with the modifications by Following incubation of the dissolved-rock-salt Huss et al. (1983) and Escara & Hutton (1980). Renaturation samples on M2 medium at 37 mC for 5–6 weeks, light- rates were computed by the program . (Jahnke, red or pink colonies were obtained. Smears taken from 1992). These experiments were carried out by the DSM the pure cultures of these colonies were examined by Identification Service. phase-contrast microscopy. Two different arrange- Sequencing of the 16S rRNA genes of the rock-salt isolates ments of coccoid cells could be recognized among the and phylogenetic analyses. For each strain isolated from the strains. One consisted mainly of cells growing in rock salt, the 16S rRNA gene was amplified by PCR using Sarcina-like packets, which were often in very large primers Archae21F (DeLong, 1992) and 1525R (McGenity clusters; several representative strains with this ar- et al., 1998). Template DNA was purified from cells grown rangement of cells (e.g. strains H1 and N2) were on M2 agar plates as follows. A loopful of cells was identified recently as belonging to the species Halo- suspended in 500 µl water and an equal volume of buffer- coccus salifodinae (Stan-Lotter et al., 1999). The other saturated phenol (pH 8n0) was added to the suspension. The arrangement consisted of cells that appeared as single DNA from the aqueous phase was then precipitated with ethanol. After enzymic treatment with RNase A (Sigma) and cells, diplococci or small clusters of only a few cells. Proteinase K (Sigma), the DNA was extracted with phenol\ For further differentiation of the strains isolated from chloroform\isoamyl alcohol (24:24:1), reprecipitated, rock salt, the whole-cell protein patterns of the strains washed and dissolved in water. Amplification reactions were were examined (see below). In addition, partial se- performed in a 50 µl reaction volume in a programmable quences (about 400 bp long) for the 16S rRNA genes thermal cycler (Biometra). The reaction mixtures were of the strains (i.e. a total of eight strains) were composed of 50 pmol of each primer, 12n5 nmol deoxy determined (see Methods). On the basis of this pre- nucleotides, 1n5 mM MgCl#,1UTaq DNA polymerase liminary characterization, several of the strains iso- (Promega), appropriately diluted DNA polymerase buffer B lated from the rock salt were found to be similar to (Promega) and 0n1–1 ng template DNA. Thermal cycling Halococcus salifodinae. On the basis of the sequence consisted of a primary heating step (94 mC for 5 min), data, some strains were more similar to Halococcus followed by 30–35 cycles of denaturation at 95 mC for 1 min, annealing at 55 mC for 1 min and extension at 72 mC for morrhuae than to other halococcal species, but they 2 min. The amplified PCR product was purified from appeared as single cells, diplococci or small clusters ethidium-bromide-stained agarose gels by using a com- upon microscopic examination rather than in Sarcina- mercially available kit (NucleoTrap; Macherey & Nagel). like packets. These strains are the subject of the present Sequences were determined by automated dideoxynucleo- study. tide methods using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 310 Cell and colony morphology Genetic Analyser (both from Applied Biosystems), according to the manufacturer’s instructions. Primer 1525R and One strain isolated from the rock salt, strain H4T,was primers described by Radax et al. (2001) were used as a coccoid organism of about 0n8–1n2 µm in diameter. sequencing primers. When grown in M2 medium or on M2 agar, cells of Sequences used for comparison with the 16S rDNA sequence T T strain H4 were non-motile and appeared as single from strain H4 were obtained from the European Molecular cells or diplococci. This was a different arrangement Biology Laboratory (EMBL) web interface or from the than that seen with cells of Halococcus morrhuae DSM Ribosomal Database Project II (RDP) (release 8; Maidak et 1307T, which exhibited predominantly classical Sar- al., 2000) and fitted in a subset of aligned archaeal sequences cina-like packets when grown in M2 medium or on M2 obtained from the RDP, using the software BioEdit (Hall, T 1999). The alignment was subjected to phylogenetic analyses agar. Cells of strain H4 stained uniformly Gram- by distance-matrix (Jukes–Cantor correction; Jukes & Can- negative, whether they were taken from 5-day-old or 14-day-old cultures; endospores were not produced by tor, 1969), maximum-likelihood and maximum-parsimony T methods using programs of the  package (version the strain. Colonies of strain H4 on solidified complex http://ijs.sgmjournals.org 1809 H. Stan-Lotter and others medium (pH 7n4) were circular, convex, had entire Table 1. Comparison of phenotypic characteristics of margins, were about 1–2 mm in diameter following 14 Halococcus morrhuae DSM 1307T and strain H4T days of incubation at 37 mC, light red in colour and had ...... a shiny surface. In contrast, colonies of Halococcus Tests were performed at salinities of at least 20% NaCl (see morrhuae DSM 1307T grown under the same condi- Methods). Enzyme assays were done with the API ZYM tions as strain H4T were irregular and dark red in system. Both strains were positive for alkaline phosphatase, colour. esterase (C4), esterase lipase (C8) and leucine arylamidase activity; no activity was detected for arginine dihydrolase, urease, β-glucosidase or β-galactosidase in either strain. Both Physiological and biochemical characteristics of strain strains liquefied gelatin. Neither strain was able to utilize H4T (j)arabinose, (j)raffinose, (j)trehalose, ,-Phe nor - T Trp, but they were both able to utilize (j)galactose. j, Strain H4 grew aerobically with doubling times of Positive reaction or growth; k, no reaction or growth. 25–28 h at 37 mC when grown in liquid M2 medium (4 M NaCl, pH 7n4) with shaking. Optimum growth of Characteristic DSM 1307T H4T strain H4T occurred when it was grown in the presence of NaCl concentrations ranging from 20 to 30%; it did Gram reaction j\k* k not grow in medium containing ! 15% NaCl. Strain T #+ pH Range for growth 5n5to! 8n0* 5n2–8n0 H4 required at least 2n5mMMg to be present in Catalase * growth medium that included 4 M NaCl; optimum j j #+ Oxidase j* j growth of the strain occurred when Mg was present Nitrate reduction * in the medium at a concentration of 20–60 mM. Slight j j #+ Enzymes assayed for: but noticeable differences in the Mg requirements of Cystine arylamidase Halococcus morrhuae DSM 1307T (optimum growth in kj # Acid phosphatase kj + Halo presence of between 2n5 and 5 mM Mg ) and - Utilization of: coccus saccharolyticus DSM 5350T (optimum growth # (k)Fructose kj in presence of 60 mM Mg +) were observed, when #+ T (j)Glucose jk compared to the Mg requirements of strain H4 . The ( )Xylose growth of strain H4T was supported by M2 media of j kj -Arg jk pH 5n2, 5n8, 6n8or8n0. No growth of this strain -Glu occurred in the media at pH values above 9 0 or below jk n -Met jk 5n1. Upon their examination under phase-contrast -Ser microscopy, cells of strain H4T grown at lower pH jk values (i.e. pH 5n8 and 6n8) appeared larger and with a * Data taken from Grant & Larsen (1989). All other data were somewhat irregular shape when compared with strain determined in this work. H4T cells grown at pH 7–8 (not shown). Compared to Halococcus salifodinae BIpT, which grew between pH 6 8 and 9 5, strain H4T grew well at lower pH n n Growth of strain H4T was strongly inhibited by the values. Halococcus morrhuae and Halococcus sac- antibiotics bacitracin, novobiocin and rifampicin. charolyticus have both been reported to grow at pH Moderate susceptibility of the strain to erythromycin values as low as 5 5or60, respectively (Larsen, 1989; n n and trimethoprim was observed. No inhibition of Montero et al., 1989). Similar to cells of other strain H4T growth was observed when the strain was halococci, cells of strain H4T did not lyse within 1–2 h grown in the presence of ampicillin, anisomycin, when suspended in distilled water (Grant & Larsen, aphidicolin, chloramphenicol, gentamicin, kanamycin, 1989). Strain H4T grew between 28 and 50 C, with its m nalidixic acid, streptomycin, tetracycline or vanco- optimum temperature for growth between 37 and mycin. The susceptibility pattern of strain H4T was 40 C. Its growth in test tubes with broth occurred as m similar to that described for Halococcus morrhuae sediment. T (Larsen, 1989; Tindall, 1992), except that Halococcus Further phenotypic characteristics of strain H4 are morrhuae is moderately susceptible to anisomycin shown in Table 1, along with some characteristics of (Larsen, 1989), a result corroborated in this work. Halococcus morrhuae DSM 1307T. The growth of T strain H4 on various carbon sources was tested in Gel electrophoresis of whole-cell proteins semi-defined media containing 0n02% yeast extract and, due to the known long generation times of SDS-PAGE of whole-cell proteins is a rapid method members of the genus Halococcus (Grant, 2001), for distinguishing bacterial species and has a similar incubation was carried out for up to 12 weeks. level of discrimination to DNA–DNA hybridization Acidification of the media was, in general, weak or not (Jackman, 1987). Strain H4T had a unique protein detectable. Halococcus salifodinae BIpT has a similar profile following SDS-PAGE that did not resemble the complement of enzymes as strain H4T, except that profiles of any of the halococcal representatives ana- lipase (C14), leucine arylamidase and cystine arylami- lysed in this study (Fig. 1). In addition, the protein dase have not been detected in this strain (Stan-Lotter pattern of strain H4T was also dissimilar to the protein et al., 1999). patterns of Halorubrum saccharovorum, Halobac-

1810 International Journal of Systematic and Evolutionary Microbiology 52 Halococcus dombrowskii sp. nov.

with eight isoprenoid units in the side chain, and dihydromenaquinone MK-8(H#) (84% of total quinones), in accordance with the results of Collins et al. (1981). Traces of dihydromenaquinone MK-7(H#) were also present (1% of total quinones). Strain H4T possessed all of these menaquinones, although the bulk quinone (94% of total quinones) was MK-8(H#), while MK-8 and MK-7(H#) were present in small amounts (6 and 1% of total quinones, respectively). For comparison, an extract of Halococcus salifodinae BIpT was also analysed; this consisted almost ex- clusively of MK-8(H#) (98% of total quinones). The presence of MK-8(H#) as the dominant menaquinone type in strain H4T indicated that this strain was more closely related to Halococcus salifodinae BIpT than to Halococcus morrhuae DSM 1307T,asHalococcus salifodinae BIpT was also characterized by the presence of MK-8(H#) as its major component, MK-8 (2%) as a minor component and MK-7(H#) as a trace component. Two-dimensional TLC of lipids of strain H4T revealed the presence of C#!C#! and C#!C#& archaeal core lipids, ...... as detected by double spots. Phosphatidylglycerol and phosphatidylglycerol methylphosphate were present Fig. 1. Whole-cell proteins from coccoid haloarchaeal strains, T including several type strains and strain H4T, following in strain H4 in greatest abundance, similar to Halo- separation by SDS-PAGE. Approximately 20 µg of protein were coccus morrhuae DSM1307T and Halococcus salifo- loaded per lane. Proteins were stained with Coomassie blue. dinae BIpT; no phosphatidylglycerol sulfate was pres- Lanes: 1, molecular mass markers of (from top) 200n0, 116n0, ent. Sulfated mannosylglucosylglycerol diether was 97n4, 66n3, 55n4, 35n5, 31n1, 21n5 and 14n4 kDa; 2, Halococcus T morrhuae NRC 16008; 3, Halococcus morrhuae DSM 1307T ;4, detected among the lipids of strain H4 , as well as two Halococcus morrhuae DSM 1308; 5, Halococcus morrhuae DSM unknown glycolipids. The overall phospholipid pat- T T T T 1309; 6, strain H4 , DSM 14522 ; 7, strain H4 , NCIMB 13803 ;8, tern of strain H4T was characteristic of members of the Halococcus salifodinae BIpT DSM 8989T ;9,Halococcus salifodinae BG2/2 DSM 13045; 10, Halococcus saccharolyticus genus Halococcus (Ross et al., 1985; Wainø et al., DSM 5350T. 2000). Strain H4T produced carotenoid pigments characteristic of members of the haloarchaea, as determined terium salinarum (R1), Halobacterium salinarum and with methanol\acetone extracts. Absorption peaks at Haloferax denitrificans (data not shown). A high wavelengths of 388, 495 and 528 nm and a shoulder at degree of similarity between protein profiles of dif- 466–476 nm were observed with the extracts from ferent strains belonging to the same species is usually strain H4T, Halococcus morrhuae DSM 1307T and observed (Stan-Lotter et al., 1999). This phenomenon Halococcus saccharolyticus DSM 5350T; these peaks was observed in this study (Fig. 1), as four different corresponded to bacterioruberin (Gochnauer et al., strains of Halococcus morrhuae (lanes 2–5) had similar 1972; Oren, 1983). The absorption spectra for extracts protein patterns and two different strains of Halo- of Halococcus salifodinae BIpT showed less-well-de- coccus salifodinae (lanes 8 and 9) had similar protein fined peaks, with shoulders at 490 and 526 nm; this patterns. Haloarchaea are known to possess acidic was concomitant with the light-pink pigmentation it bulk proteins (Reistad, 1970) whose isoelectric points produced under the growth conditions used in this are between pH 3n6 and 5n0 (Stan-Lotter et al., 1989). study (not shown). Isoelectric focusing of whole-cell proteins from strain H4T and Halococcus morrhuae DSM 1307T revealed GjC content bulk proteins whose isoelectric points were between T pH 4n2 and 5n2, and which differed from each other in The GjC content of strain H4 was 61n3 mol%; this their overall pattern (data not shown). was similar to the values determined for Halococcus morrhuae (61–66 mol%; Larsen, 1989), Halococcus Menaquinones, polar lipids and carotenoids saccharolyticus (59n5 mol%; Montero et al., 1989) and Halococcus salifodinae (62 mol%; Denner et al., 1994). Upon its analysis by HPLC, the quinone extract of T strain H4 produced three peaks that had the same DNA–DNA hybridization retention times as those detected in a quinone extract of Halococcus morrhuae DSM 1307T. The predomi- The measurement of the levels of DNA–DNA hybridi- nant types of menaquinones in Halococcus morrhuae zation between strain H4T and representative Halo- were MK-8 (15% of total quinones), a menaquinone coccus spp. showed the following homology values: http://ijs.sgmjournals.org 1811 H. Stan-Lotter and others

T 35n1% between strain H4 and Halococcus saccharo- Halococcus salifodinae BG2/2 DSM 13045 (AJ131458) T T lyticus DSM 5350 ;38n2% between strain H4 and 66 T T T T Halococcus salifodinae BIp DSM 8989 ; and 65n8 and Halococcus salifodinae BIp DSM 8989 (Z28387) T 67n8% (two determinations) between strain H4 and 69 T Halococcus salifodinae Br3 DSM 13046 (AJ238897) Halococcus morrhuae DSM 1307 . A DNA homology 96 value of ! 70% for two strains usually justifies Halococcus salifodinae BIpT DSM 8989T (AB004877) designation of the strains to different species (Wayne et 100 T al., 1987); hence, strain H4 represented a new species Halococcus saccharolyticus ATCC 49257T (AB004876) of the genus Halococcus. The homology values ob- 100 T Halococcus morrhuae ATCC 17082T (X72588) served for strain H4 and Halococcus morrhuae DSM 67 1307T indicated that these two species had a closer T Halococcus morrhuae NRC 16008 (D11106) relationship at the DNA level than strain H4 had with 100 T Halococcus salifodinae BIp and Halococcus saccharo- Halococcus dombrowskii H4T (AJ420376) lyticus DSM 5350T, respectively. Haloarcula marismortui ATCC 43049T, rrnB (X61689) 100 Haloarcula marismortui ATCC 43049T, rrnA (X61688) Phylogenetic position of strain H4T T The full sequence (1472 bases) of the 16S rRNA gene Natronomonas pharaonis JCM 8858 (D87971) T of strain H4 was determined. Comparison of the 16S Natronococcus occultus NCIMB 2192T (Z28378) rDNA sequence of this strain with the 16S rDNA 57 sequences of members of the family Halobacteriaceae Halobaculum salinarum E12 (U68538) placed strain H4T into the genus Halococcus (Fig. 2). Strain H4T, as deduced from this analysis, was most Halobaculum gomorrense DSM 9297T (L37444) similar to Halococcus morrhuae. The identity of strain 52 T T Halorubrum saccharovorum JCM 8865T (U17364) H4 with Halococcus morrhuae ATCC 17082 and 56 Halococcus morrhuae NRC 16008 was 99n3 and 99n5%, Haloferax volcanii ATCC 29605T (K00421) respectively. To date, three species of haloneutrophilic Halococcus morrhuae Halococcus sac Methanococcus jannaschii [ (Larsen, 1989), - T charolyticus (Montero et al., 1989) and Halococcus 0·1 DSM 2661 (L77117) salifodinae (Denner et al., 1994)] and two species of ...... haloalkaliphilic [Natronococcus occultus and Natrono- Fig. 2. Dendrogram of haloarchaeal species, based on 16S rRNA coccus amylolyticus (Tindall, 2001)] archaeal cocci gene sequence data, indicating the phylogenetic position of strain H4T. The tree was constructed using the neighbour- have been recognized. On the basis of the 16S rDNA joining method of Saitou & Nei (1987). Methanococcus data presented here and data from previous reports jannaschii was used as an outgroup. The bar represents the (Ventosa et al., 1999; Stan-Lotter et al., 1999), the scale of estimated evolutionary distance (10 substitutions at any genus Halococcus appears to contain at least two nucleotide position per 100 nucleotide positions) from the lineages – one which contains Halococcus salifodinae point of divergence. Bootstrap percentages, based on 100 and Halococcus saccharolyticus, and one which con- replications, are shown at the nodes. sists of Halococcus morrhuae and other coccoid strains. Strain H4T forms a branch within the Halococcus morrhuae lineage (Fig. 2). The 16S rDNA sequences of T Halococcus salifodinae and Halococcus saccharolyticus H4 could be distinguished from Halococcus salifo- are 98n9% identical (Ventosa et al., 1999), but these dinae, Halococcus saccharolyticus and natronococci, as two halococcal organisms differ in numerous proper- described above. Its slow growth, phospholipid con- ties, supporting their separation into two distinct tent and 16S rRNA gene sequence were similar to characteristics of Halococcus morrhuae. However, species. These two species are also 63n6% related on T the basis of DNA–DNA hybridization data (A. Legat, strain H4 differed from Halococcus morrhuae with C. Gruber and H. Stan-Lotter, unpublished data). It is respect to its cellular morphology and arrangement, therefore reasonable to deduce that with a similar DNA–DNA similarity, whole-cell protein patterns, mean value (66n8%) for DNA–DNA hybridization menaquinone content, presence of enzymes, pigmen- between it and other members of the genus Halococcus, tation, susceptibility to some antibiotics, and its usage and on the basis of other differentiating characteristics, of carbohydrates and amino acids (see Table 1 and T Fig. 1). We believe that these data justify the proposal strain H4 does not belong to the species Halococcus T morrhuae. for assigning H4 to a new species of the genus Halococcus, Halococcus dombrowskii. T Conclusions Strain H4 and several strains related to it, as well as members of the species Halococcus salifodinae, were On the basis of its polar-lipid content, antibiotic isolated from subterranean salt mines in Austria. The sensitivity and acidic bulk proteins, coccoid strain H4T salt sediments are thought to have been deposited was identified as being a halophilic archaeon. Strain during the Permian period (Zharkov, 1981). Evidence

1812 International Journal of Systematic and Evolutionary Microbiology 52 Halococcus dombrowskii sp. nov. gathered from the data presented in this and earlier schweig, Germany, for determination of the GjC content studies (Norton et al., 1993; Stan-Lotter et al., 1993, and DNA–DNA hybridization experiments, and Mag. 1999, 2000, 2001; McGenity et al., 2000; Radax et al., Michaela Eder for expert technical assistance. 2001) further strengthens the notion that halophilic salt mine strains are the remnants of archaeal popula- REFERENCES tions that originally inhabited the Palaeozoic brines. Cashion, P., Hodler-Franklin, M. A., McCully, J. & Franklin, M. How these organisms survived is not known, but (1977). A rapid method for the base ratio determination of bacterial suggestions for the basis of their longevity have been DNA. Anal Biochem 81, 461–466. considered by Grant et al. (1998) and McGenity et al. Collins, M. D., Ross, H. N. M., Tindall, B. J. & Grant, W. D. (1981). (2000). 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