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Ignicoccus hospitalis sp. nov., the host of ‘Nanoarchaeum equitans’

Lehrstuhl für Mikrobiologie und Archaeenzentrum, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany

Correspondence
Harald Huber
harald.huber{at}biologie.uni-regensburg.de

	ABSTRACT

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A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for ‘Nanoarchaeum equitans’, the only cultivated member of the ‘Nanoarchaeota’. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/I^T=DSM 18386^T=JCM 14125^T).

	INTRODUCTION

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Both prokaryotic domains, the Bacteria as well as the Archaea, contain hyperthermophilic micro-organisms, with an optimal growth temperature above 80 °C (Stetter, 1989). The cultivated hyperthermophiles cluster around the root in 16S rRNA-based phylogenetic trees (Stetter, 1992). Within the Bacteria, only members of the phyla Thermotogae and Aquificae can be characterized as hyperthermophiles, whereas hyperthermophilic organisms are found within all archaeal phyla, the Crenarchaeota, Euryarchaeota and ‘Nanoarchaeota’. Until recently, all cultivated crenarchaeotes were exclusively extremely thermophilic or hyperthermophilic (Huber, 2005). Interestingly, a mesophilic, autotrophic, ammonium-oxidizing, marine crenarchaeote was described recently (Könneke et al., 2005). Within the Crenarchaeota, three orders have been described so far: the Sulfolobales, the Thermoproteales and the Desulfurococcales. The Desulfurococcales can be subdivided into two families: the Pyrodictiaceae and the Desulfurococcaceae (Huber & Stetter, 2001). Within the Desulfurococcaceae, only members of the genus Ignicoccus harbour obligately chemolithotrophic sulfur reducers (Huber et al., 2000). All strains of this genus exhibit a cell envelope unique among the Archaea: it consists of a cytoplasmic membrane, a periplasmic space with a variable width of between 20 and 400 nm and, as the outermost sheath, an outer membrane (Rachel et al., 2002).

Here we report on the isolation and properties of a novel species of the genus Ignicoccus, which represents the host for ‘Nanoarchaeum equitans’, the only cultivated representative of the recently described phylum ‘Nanoarchaeota’ (Huber et al., 2002).

	METHODS

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Source of sample.
On a diving excursion to the hydrothermal system at Kolbeinsey Ridge, to the north of Iceland (67° 03' 46'' N 18° 42' 64'' E), samples of hot rocks and gravel were taken in a depth of about 106 m (Fricke et al., 1989).

Strains and culture conditions.
The new isolate and Ignicoccus type strains (obtained from our culture collection) were cultivated in strictly anaerobic SME medium (Stetter et al., 1983; Pley et al., 1991; modified by Huber et al., 2006), prepared according to Balch & Wolfe (1976). The medium contains the following components (l^–1): NaCl, 13.85 g; MgSO₄.7H₂O, 3.5 g; MgCl₂.6H₂O, 2.75 g; KH₂PO₄, 0.5 g; CaCl₂.2H₂O, 0.38 g; KCl, 0.33 g; (NH₄)₂SO₄, 0.25 g; NaBr, 0.05 g; H₃BO₃, 0.015 g; NaHCO₃, 0.16 g; SrCl₂.6H₂O, 7.5 mg; KI, 25 µg; elemental sulfur, 5.0 g. Reduction of the medium was carried out by addition of 0.5 g Na₂S; afterwards, the pH was adjusted at room temperature to pH 5.5–6.0 with sulfuric acid. Growth conditions were similar to those described for the other two described Ignicoccus species (Huber et al., 2000): routinely, the organisms were grown in 120 ml serum bottles containing 20 ml SME medium pressurized with H₂/CO₂ (250 kPa; 80 : 20, v/v). Incubation was carried out at 90 °C under shaking (100 r.p.m.). Heterotrophic growth was tested under a gas phase of N₂/CO₂ (200 kPa, 80 : 20, v/v), using the following substrates: yeast extract, Casamino acids (Difco), gelatin (Sigma Aldrich), formate, acetate, meat extract, starch, peptone and glucose (VWR). Unless otherwise noted, substrates were added at final concentrations of 0.1 %. The same concentration was used for the alternative electron acceptors thiosulfate, sulfite, sulfate, nitrate and nitrite. Resistance to ampicillin, rifampicin and vancomycin was tested at final concentrations of 50 µg ml^–1 each. For investigations of the pH dependence of growth, the pH was adjusted with diluted sulfuric acid as indicated; additional buffers were not used. Batch cultures were grown in a 50 l enamel-protected fermenter (HTE Bioengineering) at 90 °C under stirring (150 r.p.m.) and gassing with H₂/CO₂ (80 : 20, v/v; 2 l min^–1).

Light and electron microscopy.
Cells were routinely observed with an Olympus BX 60 phase-contrast microscope with a UPlanFl 100/1.3 oil-immersion objective. Bacterial growth was followed by direct cell counting using a Thoma chamber (depth 0.02 mm). Samples were prepared for transmission electron microscopy as described previously (Rieger et al., 1997; Huber et al., 2000; Rachel et al., 2002). Electron micrographs were digitally recorded using a slow-scan CCD camera (Tietz) attached to a CM12 transmission electron microscope (FEI Co.) with an acceleration voltage of 120 kV.

H₂S determination.
Formation of H₂S was monitored qualitatively by dripping the sample onto lead acetate tape, yielding a dark brown colouring.

Nitrogen fixation.
The capacity of isolate KIN4/I^T for nitrogen fixation was tested in SME medium without (NH₄)₂SO₄. This medium was pressurized with N₂/H₂/CO₂ (300 kPa; 65 : 15 : 20, by vol.). Experiments were carried out with and without Na₂MoO₄ and VOSO₄ (1 µM each).

DNA isolation and DNA base composition.
DNAs were prepared as described previously (Wildgruber et al., 1982). The G+C content of genomic DNA was determined by melting-point analysis (Marmur & Doty, 1962) and by direct analysis of the nucleotides after digestion of the DNA with nuclease P1 and separation by HPLC (Völkl et al., 1993). Calf thymus DNA (G+C content of 42 mol%) was used as a reference.

16S rRNA gene sequence analysis.
The nearly complete 16S rRNA gene sequence of the new isolate was amplified by PCR. The primers used in the amplification corresponded to positions 8–23 (5'-TCYGGTTGATCCTGCC; Eder et al., 1999) and 1406–1390 (5'-ACGGGCGGTGTGTRCAA; Lane, 1991) in the 16S rRNA sequence of Escherichia coli (Brosius et al., 1981). Both strands of the PCR product were sequenced directly as described by Burggraf et al. (1997). The sequence was aligned with a set of representative archaeal 16S rRNA gene sequences using the ARB program (Ludwig et al., 2004). Dendrograms were computed with the neighbour-joining, maximum-parsimony and maximum-likelihood methods included in the ARB package.

Outer-membrane protein composition.
After homogenizing the cells using a glass homogenizer, outer membranes were purified by gradient centrifugation on a continuous sucrose gradient, as described previously (Näther & Rachel, 2004). Protein gel electrophoresis under denaturing conditions was performed according to Schägger & von Jagow (1987).

	RESULTS

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Enrichment and isolation
Enrichment attempts were carried out with samples of hot rocks and gravel taken at the hydrothermal system at Kolbeinsey Ridge. About 1 g of the material was added to 20 ml anoxic, organic compound-free culture medium. Enrichment attempts were incubated under shaking at 90 °C. After 2 days, cocci about 1–2 µm in diameter were visible in the enrichment attempt of sample KIN4 and large amounts of H₂S could be detected qualitatively in the culture medium. Surprisingly, several cells were covered by very tiny cocci. These tiny cocci had meanwhile been described as ‘Nanoarchaeum equitans’, the first representative of the novel archaeal phylum ‘Nanoarchaeota’ (Huber et al., 2002). A pure culture of the new isolate could be obtained by the use of optical tweezers (Huber et al., 1995). Separations were carried out with and without attached ‘Nanoarchaeum’ cells. As expected, cells without ‘Nanoarchaeum’ cells always led to ‘Nanoarchaeum’-free subcultures (designated KIN4/I^T). Surprisingly, the same result was obtained for cells with attached ‘Nanoarchaeum’ cells (only one co-culture was obtained from about 50 separations). In these subcultures, ‘N. equitans’ was also no longer detectable by specific PCR amplifications of the 16S rRNA gene. These cultures were initially designated KIN4/AH, but later turned out to be identical to strain KIN4/I^T.

Morphology
Cells of isolate KIN4/I^T were regular to irregular cocci, usually occurring singly or in pairs. They exhibited cell diameters of 1–4 µm, rarely up to 6 µm. The organisms stained Gram-negative. Like I. islandicus, up to nine flagella-like appendages, each with a diameter of 14 nm, were observed to be anchored at one pole into the cell. The cell architecture of isolate KIN4/I^T was quite similar to that of the type strains of I. islandicus and I. pacificus (Huber et al., 2000; Rachel et al., 2002). The densely packed cytoplasm was enclosed by a membrane (Fig. 1a). The width of the periplasm varied between 20 and 500 nm and contained round or elongated membrane-coated vesicles, about 50 nm in diameter and up to 300 nm long. They were seen to be released from the cytoplasmic membrane and to come into close proximity with the outer membrane, the outermost sheath of the cells. It had a weak double-layer appearance in sections; the two leaflets were seen to become separated in freeze-etched samples (Fig. 1b). As in I. islandicus, this membrane contained numerous tightly packed proteins (Näther & Rachel, 2004) and lipids which, according to LC-MS analyses, are derivatives of archaeol (2,3-di-O-phytanyl glycerol diether) (Jahn et al., 2004). In addition, caldarchaeol (glyceroldibiphytanyl glycerol tetraether) was found in the cytoplasmic membrane in small but significant amounts. The predominant polar headgroups of the core lipids consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group (Jahn et al., 2004). The quantitatively dominant protein of the outer membrane is Imp1227 (Ignicoccus outer-membrane protein; molecular mass 6.23 kDa), which occurs in several thermostable oligomeric complexes, with a total mass of more than 50 kDa (Burghardt et al., 2007).

View larger version (92K): [in this window] [in a new window]   Fig. 1. Transmission electron micrographs of strain KIN4/IT. (a) Ultrathin section; (b) freeze-etching. Cy, Cytoplasm; P, periplasm; V, vesicles; OM, outer membrane; *, fracture face through the outer membrane. Bars, 1 µm.

Optimal growth conditions
Minimal doubling times for isolate KIN4/I^T of around 1 h were observed at 90 °C, 1.4 % NaCl and pH 5.5 (Fig. 2). It grew at temperatures of 73–98 °C, salt concentrations of 0.5–5 % NaCl (w/v) and pH 4.5–7.0 in the medium. No growth was obtained at or below 70 °C or at or above 100 °C or at or below pH 4 or at or above pH 7.5. Furthermore, strain KIN4/I^T was not able to propagate when NaCl was omitted from the medium or when its concentration exceeded 6.0 %.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Optimal growth conditions of strain KIN4/IT during growth on H2 and sulfur. Doubling times were calculated from the slopes of the growth curves. (a) Effect of temperature at pH 5.5 and 1.4 % NaCl. (b) Effect of NaCl concentrations at 90 °C and pH 5.5. (c) Effect of pH at 90 °C and 1.4 % NaCl.

DNA base composition
The G+C content of genomic DNA of isolate KIN4/I^T was 56 mol%, calculated by melting-point analysis and by direct analysis of the mononucleotides.

Phylogenetic analysis
Comparison of the 16S rRNA gene sequences of isolate KIN4/I^T and the two other representatives of the genus Ignicoccus (I. islandicus and I. pacificus; Huber et al., 2000) clearly showed that the genus Ignicoccus represents a separate branch within the Desulfurococcales (Fig. 3) (phylogenetic distances to all other members of the Desulfurococcales at least 6.1 %). Isolate KIN4/I^T exhibits phylogenetic distances of 4.0 % from I. islandicus Kol8^T and 2.8 % from I. pacificus LPC33^T. Investigations were also carried out on the phylogenetic distances between isolate KIN4/I^T, isolate KIN4/AH and the host organism of the co-culture with ‘N. equitans’. Identical 16S rRNA gene sequences were obtained for all three strains, demonstrating that strain KIN4/I^T is the host organism for ‘N. equitans’.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Phylogenetic position of Ignicoccus type strains Kol8T, LPC33T and KIN4/IT calculated by the neighbour-joining method using the Jukes and Cantor correction, based on 16S rRNA gene sequences. Bar, 10 estimated substitutions per 100 nucleotides.

View larger version (71K): [in this window] [in a new window]   Fig. 4. SDS-PAGE of sucrose-gradient-purified outer membranes of strain KIN4/IT (lane 1), I. islandicus Kol8T (2) and I. pacificus LPC33T (3). Lane M, molecular mass markers (sizes in kDa).

	DISCUSSION

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The most important property of I. hospitalis in comparison with the other Ignicoccus species is its capacity to serve as a host for ‘N. equitans’. So far, not only no other Ignicoccus strain but no other hyperthermophilic archaeon can serve as a host organism. Since growth of ‘N. equitans’ is strictly dependent on propagating I. hospitalis cells, these two organisms form the only known host–parasite or host–symbiont system of two archaea. The reason for this exclusivity is still unknown, although the obvious differences in the outer-membrane proteins of the different Ignicoccus species may provide an explanation (Näther, 2003). The fact that ‘N. equitans’ has a highly reduced genome, lacking nearly all metabolic and biosynthetic pathways (Waters et al., 2003), makes it reasonable that its growth depends on a host organism. Indeed, it has already been demonstrated that its lipids are most likely to be synthesized in I. hospitalis cells (Jahn et al., 2004). In contrast, I. hospitalis can grow in pure culture as well as in co-culture with ‘N. equitans’, which raises the question as to whether ‘N. equitans’ is a symbiont of or a parasite on I. hospitalis. At present, this question cannot be answered satisfactorily. In principle, I. hospitalis shows identical growth parameters, such as doubling time or maximal cell density, whether it is cultivated with or without ‘N. equitans’. So far, no benefits for I. hospitalis could be detected in the co-culture, a characteristic for a symbiosis. On the other hand, ‘N. equitans’ does not kill its host; the two organisms live in a stable co-culture. Therefore, ‘N. equitans’ is neither a predator nor, in a strict sense, a parasite on I. hospitalis. It seems that the correct description of their relationship is between symbiosis and parasitism, which we hope to elucidate by our ongoing studies of this unique archaeal system.

Description of Ignicoccus hospitalis sp. nov.
Ignicoccus hospitalis (hos.pi.ta'lis. L. masc. adj. hospitalis relating to a guest, hospitable, referring to its ability to serve as a host for ‘Nanoarchaeum equitans’).

Slightly irregular cocci, about 1–6 µm in diameter, with extracellular flagella-like appendages which insert at one cell pole. Gram-negative. Occurring singly and in pairs. No evidence for a regular arrayed surface protein. Grows at 73–98 °C, pH 4.5–7.0 and 0.5–5.5 % NaCl (optima: 90 °C, pH 5.5, 1.4 % NaCl). Strictly anaerobic. Chemolithoautotrophic growth in the presence of H₂ and CO₂ with sulfur as electron acceptor. No chemo-organotrophic growth on meat extract, yeast extract, peptone, Casamino acids, gelatin, starch, formate, acetate or glucose. Sulfate, sulfite, sulfide, thiosulfate, tetrathionate, nitrate and oxygen are not used as electron acceptors. H₂S is formed during growth. Growth is stimulated by addition of 0.1 % (w/v) meat extract or 0.02 % (w/v) yeast extract. The G+C content of genomic DNA of the type strain is 56 mol%. The cell envelope consists of a cytoplasmic membrane, a periplasm of variable width and an outer membrane. The main core lipids are archaeol and caldarchaeol. Caldarchaeol does not appear in the purified outer membrane. The predominant polar headgroups consist of one or more sugar residues (mainly mannose and small amounts of glucose) attached either directly to the core lipid or via a phosphate group. Imp1227 (Ignicoccus outer-membrane protein; molecular mass 6.23 kDa) is the quantitatively dominant protein within the outer membrane, which occurs in several, stable oligomers. Host organism for ‘N. equitans’.

The type strain is isolate KIN4/I^T (=DSM 18386^T=JCM 14125^T), isolated from the Kolbeinsey Ridge, to the north of Iceland.

	ACKNOWLEDGEMENTS

 
The authors wish to thank Gabi Gmeinwieser, Peter Hummel, Kerstin Roth, Thomas Hader and Konrad Eichinger for technical assistance and Irith Wyschkony for initial electron microscopy analysis. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Hu 701/1-1 and 1-2 to H. H. and Ra 753/3-1 to R. R.) and by the Fonds der Chemischen Industrie (to K. O. S.).

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