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1 Thermophile Research Unit, University of Waikato, Private Bag 3105, Hamilton, New Zealand
2 Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand
Correspondence
Thomas D. Niederberger
tdn{at}waikato.ac.nz
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-cyclodextrin as carbon sources. The G+C content was 52.9 mol%. Based on 16S rRNA gene sequence analysis and physiological features it is proposed that isolate AQ1.S1T (=DSM 17230T=JCM 13409T) represents the type strain of a novel species of a new genus within the Crenarchaeota, Ignisphaera aggregans gen. nov., sp. nov.
Published online ahead of print on 23 December 2005 as DOI 10.1099/ijs.0.63899-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Ignisphaera aggregans AQ1.S1T and strains Tok10A.S1, Tok37.S1 and Tok1 are DQ060321, DQ060322, DQ060323 and DQ060320, respectively.
Present address: St Andrews University of Biomolecular Science, St Andrews, Fife KY16 9AJ, UK. 
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). The Thermoproteales are rod-shaped organisms that group into the families Thermoproteaceae and Thermofilaceae, whereas members of the Sulfolobales are coccoid-shaped, lobed acidophiles. The Desulfurococcales are all coccoid to disc-shaped, neutrophilic or weakly acidophilic and strict anaerobes with fermentative metabolism or anaerobic respiration, with the exception of Aeropyrum pernix and Aeropyrum camini (Sako et al., 1996; Nakagawa et al., 2004). Phenotypic and 16S rRNA gene phylogenetic analyses divide the members of the Desulfurococcales into two families, the Pyrodictiaceae and the Desulfurococcaceae. The Pyrodictiaceae forms a distinct group [genera Pyrodictium (Stetter et al., 1983), Hyperthermus (Zillig et al., 1991) and Pyrolobus (Blöchl et al., 1997)] within 16S rRNA phylogenetic trees, whereas the Desulfurococcaceae consists of a diverse range of cultured representatives (Burggraf et al., 1997). These two families can also be distinguished phenotypically by growth temperatures, where the Pyrodictiaceae consists of organisms with optimal temperatures above 100 °C and members of the Desulfurococcaceae are characterized by maximal growth temperatures of up to 100 °C. The Desulfurococcaceae consists of cocci to disc-shaped micro-organisms of the genera Desulfurococcus (Zillig et al., 1982), Aeropyrum (Sako et al., 1996; Nakagawa et al., 2004), Ignicoccus (Huber et al., 2000), Staphylothermus (Fiala et al., 1986; Arab et al., 2000), Stetteria (Jochimsen et al., 1997), Sulfophobococcus (Hensel et al., 1997), Thermodiscus (Stetter, 2003), Thermosphaera (Huber et al., 1998) and the distantly related ‘Acidilobus group’ consisting of Acidilobus aceticus (Prokofeva et al., 2000), Caldisphaera lagunensis (Itoh et al., 2003) and strain NC12 (‘Caldococcus noboribetus’; Aoshima et al., 1996).
As part of a project investigating the microbial ecology of New Zealand's high-temperature geothermal habitats, a novel coccoid-shaped archaeum (isolate AQ1.S1T) was isolated into pure culture from co-culture with a strain of Pyrobaculum. The isolated coccus was similar in morphology and 16S rRNA gene sequence to a previously identified archaeon, which was also in co-culture with a strain of Pyrobaculum and was obtained from a New Zealand hot spring (Götz, 1998). Other thermal habitats of New Zealand and Yellowstone National Park (WY, USA) were screened for the presence of these novel cocci by using culturing methods. In this study, we describe the novel isolate AQ1.S1T and, based on 16S rRNA nucleotide sequence and phenotypic analysis, propose that it represents a novel genus and species, Ignisphaera aggregans gen. nov., sp. nov., within the Crenarchaeota.
Samples from hot springs and mud pools situated in the Rotorua and Tokaanu thermal areas of New Zealand were collected in sterile containers and transported back to the laboratory under ambient conditions. The medium used for enrichment and isolation contained (g l–1): (NH4)2SO4, 1.3; CaCl2, 0.074; MgSO4.7H2O, 0.28; KH2PO4, 0.28; yeast extract, 0.1; trypticase peptone, 2.0; soluble starch, 2.0; and cystine, 0.6. Resazurin (0.1 %, w/v), FeCl3 (0.28 g l–1) and trace elements (mg l–1: MnSO4, 2.2; ZnSO4.7H2O, 0.5; H3BO4, 0.5; CuSO4, 0.016; Na2MoO4.2H2O, 0.025; and CoCl2.6H2O, 0.046) were added at 1 ml l–l. The pH of the medium was adjusted to 7.0 at room temperature, and the medium was then boiled and dispensed into Hungate tubes under an N2 atmosphere, autoclaved and reduced with 10 % Na2S.9H2O as required. Growth was monitored using phase-contrast light microscopy. Approximately 1 ml pool water from hot springs in Yellowstone National Park was used to inoculate media which were incubated in situ in the pool from which the inoculum originated. The media used included the enrichment medium outlined above and a modified enrichment medium, where cystine was removed and starch was replaced with konjac glucomannan (Shintoa Koeki Kaisha). The tubes were incubated in the pools for 4–7 days, after which they were transported to the laboratory under ambient conditions and microbial growth was checked using phase-contrast light microscopy.
Cells were examined under phase-contrast light microscopy. For scanning electron microscopy, cells were grown to late exponential phase, filtered through a 0.22 µm filter and fixed using 2.5 % glutaraldehyde. The filter was then exposed to four changes of 0.1 M sodium cacodylate buffer, rinsed in water and dehydrated in increasing concentrations of ethanol (50, 75 and 90 %, respectively), followed by four changes of absolute ethanol. The filter was then critical point dried, sputtered with platinum and viewed using a Hitachi S-4100 field emission scanning electron microscope.
For metabolic studies, all experiments were undertaken at 90 °C unless otherwise stated and growth was documented using phase-contrast microscopy with a Thoma counting chamber (depth 0.02 mm). Unless otherwise stated, carbon utilization studies involved the addition of substrates at 0.2 % (w/v) final concentration to modified enrichment medium. The modified enrichment medium had the starch supplement omitted, reduced concentrations of yeast extract and trypticase peptone (0.05 and 1.0 g l–1, respectively), MOPS added to a final concentration of 25 mM and the pH adjusted to approximately 7.0 at room temperature. A positive growth response to a substrate was only recorded when a similar cell density was achieved on three successive transfers in the same medium. For the growth response to pH, buffers were added to the medium to a final concentration of 25 mM. The buffers used and the corresponding pH ranges were: MES, pH 4.5–6.5; MOPS, pH 6.5–7.5; Tris, pH 7.5–9.0; CHES, pH 9.0–10.0; and CAPS, pH 10.0–10.5. The pH was adjusted at a temperature of 75 °C and the corresponding pH value (at 90 °C) was extrapolated using the appropriate d(pKa)/dt coefficient. Various electron acceptors were tested by replacing the cystine of the standard enrichment medium. Alternative electron acceptors were used at final concentrations of both 2.5 and 10 mM in medium containing 25 mM MOPS. Elemental sulphur (approx. 20 mg) was added to individual Hungate tubes containing 9 ml medium prior to autoclaving. Utilization of electron acceptors was recorded as positive when the same cell density was achieved after at least two transfers in the medium. Salt tolerance was determined by adding NaCl directly to the standard enrichment medium. Autotrophic growth was tested in modified enrichment medium (starch omitted and both trypticase peptone and yeast extract at 0.05 g l–1) under an H2/CO2 atmosphere (80 : 20, v/v). Autotrophic medium without an electron acceptor was tested and cystine, sulphite, thiosulphate and elemental sulphur were trialled as electron acceptors.
To minimize temperature degradation in experiments to test for sensitivity to antibiotics (Peteranderl et al., 1990), antibiotics were added to cells in exponential growth (90 °C) to a final concentration of 100 µg ml–1.
H2S was determined qualitatively (Huber et al., 1986). The breakdown products of growth on konjac glucomannan were tested by size exclusion chromatography through a Biogel P2 (2.6x95 cm) size exclusion column. Size standards of monosaccharide (glucose) and disaccharide (maltose) were run through the column at 1 % (w/v) in pure water (Milli-Q; Millipore). Growth medium containing konjac glucomannan with and without an inoculum of AQ1.S1T cells was incubated at 90 °C. The medium was removed after approximately 3 days incubation, growth in the inoculated tubes was recorded by using light microscopy and the control tubes were confirmed as sterile. The medium was then passed through a 0.22 µm filter and 10 ml of the filtrate was snap-frozen in liquid N2 and freeze-dried. It was then resuspended in 4–5 ml 100 % methanol, incubated for 30 min in an ultrasonic cleaner and then centrifuged at 2000 g for 7 min. The supernatant was transferred to a clean tube and the methanol was removed by exposure to N2 gas flow, with the tube in a 40 °C heat block. The residue was resuspended in 2 ml Milli-Q water, sonicated for 5 min and passed through a 0.22 µm filter. Finally, 2 ml was loaded onto the column for analysis. In addition, konjac glucomannan (1 %, w/v) suspended in Milli-Q water was processed as described for the growth medium (without the 90 °C incubation) and passed through the column.
Determination of G+C content was undertaken in duplicate as described by Gonzalez & Saiz-Jimenez (2002) using a Smart Cycler II (Cepheid). DNA from Escherichia coli (strain DH5
) was used as a control.
The 16S rRNA gene was amplified by PCR utilizing the primers A5F (5'-CCGTTGATCCTGCCGG-3') and U1522R (5'-AAGGAGGTGATCCARCCGCA-3'). Each PCR consisted of 1 µM of each primer, 200 µM dNTPs, 1x PCR buffer, 1.5 mM MgCl2, 1.25 units Taq polymerase and approximately 20 ng template DNA, in a final reaction volume of 25 µl. The thermocycling conditions consisted of initial denaturation at 94 °C for 3 min and 30 s, then 32 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min, with a final extension at 72 °C for 6 min. The 16S rRNA gene was sequenced by using the MegaBACE DNA analysis system (Amersham Bioscience) using the primers A5F, A347F (5'-CCAGGCCCTACGGGGCGCA-3'), A915F (5'-AGGAATTGGCGGGGGAGCAC-3'), A907R (5'-CCGTCAATTCCTTTGAGTTT-3'), 519R (5'-GWATTACCGCGGCKGCTG3-'), A1335R (5'-GTGTGCAAGGAGCAGGGAC-3') and U1522R. The 16S rRNA gene sequences for the coccal members of the Tok10A and Tok1 consortia were determined by using DNA extracted from a coccal-dominated culture for Tok10A.S1 and gene-cloning for Tok1 (Götz, 1998). Both strands of the 16S rRNA gene were sequenced for the Tok37.S1 and AQ1.S1T isolates, with single-strand sequencing being undertaken for Tok10A.S1 and Tok1. Sequences were checked for chimeric artefacts using the CHIMERA_CHECK online tool of the Ribosomal Database Project (Maidak et al., 2001). Phylogenetic analysis and alignment of 16S rRNA gene sequences were performed using the ARB software package (Ludwig et al., 2004). The phylogenetic positions of the sequences were determined using the PHYLIP package with analysis of sequences undertaken using the programs DNADIST, DNAML, DNAPARS, FITCH, NEIGHBOR and SEQBOOT (Felsenstein, 1993).
From an in-depth study of the microbial ecology of a hot pool (designated AQ1) situated in Kuirau Park, Rotorua, New Zealand, a consortium of rod- and coccoid-shaped organisms was obtained as an enrichment from pool water incubated at 90 °C. Typically, rods dominated the cultures within the first days of incubation, with an overgrowth of cocci occurring after 4–7 days. Therefore, serial dilution (1 : 10) of a nascent consortium culture was undertaken to obtain a pure culture of the rod-shaped organism. A pure culture of the coccoid-shaped organism was eventually achieved after many successive dilutions (1 : 10) from an older consortium culture in which cocci dominated. Both cultures were maintained by weekly transfer of serial dilutions (1 : 10) for nearly a year prior to characterization. Sequencing of the 16S rRNA gene identified the rod as a strain of Pyrobaculum (results not shown) and the coccus (isolate AQ1.S1T) matched very closely (16S rRNA gene sequence and morphology) another previously identified coccus (Tok1) that had been isolated from a hot spring in Tokaanu, New Zealand (Götz, 1998). Tok1 was also enriched as a co-culture with a strain of Pyrobaculum; however, extensive attempts to obtain a pure culture of Tok1 failed (Götz, 1998). Other geothermal habitats within New Zealand and Yellowstone National Park were then screened by enrichment for the presence of these novel cocci (Table 1). Samples from New Zealand hot springs and mud pools were inoculated into anaerobic enrichment medium and incubated at 93 °C. A duplicate enrichment was incubated at 80 °C if the in situ pool temperature was below 90 °C (results not shown). No cocci were enriched from springs in Yellowstone National Park. However, for the New Zealand springs, as was the case for the AQ1 consortium, rod-shaped organisms were commonly observed in the medium after 2–4 days incubation, followed by an overgrowth of cocci (Table 1). Conversely, cells with a coccoid morphology dominated rod-shaped organisms in the Tok10A enrichment (coccus member designated Tok10A.S1) and an enrichment containing only cocci was obtained from pool Tok37 (coccus designated Tok37.S1). Comparison of denaturing gradient gel electrophoresis (DGGE) profiles of the enrichments and AQ1.S1T suggested that all coccal-containing consortia contained AQ1.S1T-related members (results not shown). All attempts to isolate pure cultures of cocci from enrichments of pools AQ2, AQ5, Tok10A and Tok13 failed, including attempts at isolation using solid Gelrite medium (Hungate, 1969). The 16S rRNA genes of the coccal isolates from pools Tok10A and Tok37 were sequenced and were found to match closely those of AQ1.S1T and Tok1 (results presented below). However, the Tok37 coccal enrichment also contained an atypical 16S rRNA gene PCR product that contained introns, indicating that it may be a mixed culture of at least two different coccal species. Isolate AQ1.S1T was therefore used for further characterization.
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. A web-like array of extracellular material appeared to hold cells together when cell aggregates were examined using scanning electron microscopy (Fig. 2). Attempts to stain this material using Alcian blue- and copper sulphate-based capsule/polysaccharide staining methods were unsuccessful. Cells of Tok1 also formed aggregates, but this was not observed for either the Tok37.S1 or Tok10A.S1 cultures.
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-cyclodextrin. Weak growth was evident on beef extract and sucrose. Growth was not observed on yeast extract, cellobiose, methanol, ethanol, trehalose, pyruvate, acetate, malate, Casamino acids (0.1, % w/v), carboxymethylcellulose, amylopectin (corn), xanthan gum, locust gum (bean), guar gum, dextran, xylan (oat spelts, larch or birch), xylitol, xylose or amylose (corn and potato). The strain could also grow in the standard growth medium without the addition of starch. Addition of a vitamin solution (Wolin et al., 1963) weakly promoted growth. Cultures grew in the presence of up to 0.5 % NaCl (optimally with no added NaCl), but not at 0.75 % NaCl. An electron acceptor was not required and the removal of cystine from the growth medium did not affect cell numbers significantly. Other electron acceptors trialled included thioglycolate, nitrate, nitrite, sulphite, thiosulphate and elemental sulphur. The electron acceptors were inhibitory at a concentration of 10 mM (including cystine) and at 2.5 mM both sulphite and thioglycolate also inhibited growth of AQ1.S1T. Growth of AQ1.S1T, Tok37.S1, Tok10.S1 and Tok1 was inhibited by sulphur. No growth was detected in serum bottles under autotrophic conditions. All isolates were strict anaerobes; even trace quantities of oxygen (just sufficient to turn resazurin pink) inhibited growth. Mono- and disaccharides accumulated in AQ1.S1T cultures grown in medium containing konjac glucomannan, but not in sterile medium that had been exposed to the same temperature as the inoculated medium or the stock of konjac glucomannan. This most probably indicates that the konjac glucomannan is being hydrolysed enzymically by AQ1.S1T into sugars for metabolism. Hydrogen sulphide was also detected in AQ1.S1T cultures grown in enrichment medium. Cells of AQ1.S1T were resistant to novobiocin and streptomycin but sensitive to erythromycin, chloramphenicol and rifampicin.
A near-full-length 16S rRNA gene sequence for strain AQ1.S1T (1491 bp) was obtained and had a G+C ratio of 66 %. The 16S rRNA gene sequences of the other novel coccal isolates matched very closely that of AQ1.S1T, being 99 % similar to Tok37.S1 (1423 bp), 98 % to Tok10A.S1 (1433 bp) and 98 % to Tok1 (1191 bp), and all sequences were free of chimeric artefacts. Sequence similarities between strain AQ1.S1T and members of the Pyrodictiaceae ranged from 91.5 to 94 % and from 87 to 94.5 % for the Desulfurococcaceae, with the closest sequence match being that with Staphylothermus marinus F1T (GenBank accession no. X99560). The G+C content of strain AQ1.S1T was 52.9 mol%. Phylogenetic analysis revealed that AQ1.S1T and the other novel coccal isolates formed an independent group within the Crenarchaeota (Fig. 3). The topology of the phylogenetic tree did not change when DNADIST (FITCH and NEIGHBOR), DNAML or DNAPARS analysis was used, or when the shorter 16S rRNA gene sequence of Tok1 (1195 bp) was removed. Likewise, the structure of the phylogenetic tree did not change with the inclusion of Methanopyrus kandleri DSM 6324T (GenBank accession no. M59932), Archaeoglobus fulgidus DSM 4304T (Y00275) and Methanocaldococcus jannaschii DSM 2661T (M59126) as additional outgroups.
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Commonly, the novel coccus-shaped organisms grew commensally with species of Pyrobaculum, with the exception of Tok37.S1, and, although the Pyrobaculum sp. could readily be obtained as a pure culture, this proved impossible with the coccus-shaped organisms (with the exception of AQ1.S1T) and may be indicative of a metabolic dependency of the cocci with Pyrobaculum. In fact, the outgrowth of cocci following the development of rods (Pyrobaculum sp.) within cultures may be due to the cocci utilizing a product excreted by the rod-shaped members of the consortium. Likewise, the presence of a second atypical 16S rRNA gene PCR product for Tok37.S1 may indicate another coccal member that Tok37.S1 might be dependent upon.
AQ1.S1T is a coccoid-shaped, anaerobic, moderately acidophilic, heterotrophic hyperthermophile, with maximum growth occurring below 100 °C. These morphological and phenotypic attributes fit within the description of the family Desulfurococcaceae (Burggraf et al., 1997). The ability of strain AQ1.S1T to grow in media with no exogenous electron acceptor indicates its capacity to ferment polysaccharides and complex proteinaceous substrates. The fermentation of polysaccharides and/or peptides has also been noted for other members of the family Desulfurococcaceae, such as Desulfurococcus (Zillig et al., 1982), Thermosphaera (Huber et al., 1998), Staphylothermus (Fiala et al., 1986), Sulfophobococcus (Hensel et al., 1997) and both Acidilobus (Prokofeva et al., 2000) and Caldisphaera (Itoh et al., 2003) of the ‘Acidilobus group’. The formation of cell aggregates that was typical of AQ1.S1T and Tok1 has also been noted in cultures of members of the Desulfurococcaceae, for example, cells of Caldisphaera lagunensis clump together (Itoh et al., 2003) and Thermosphaera aggregans (Huber et al., 1998), Staphylothermus marinus and Staphylothermus hellenicus grow as grape-like aggregates (Fiala et al., 1986; Arab et al., 2000). However, the growth inhibition by sulphur of the novel cocci does not accord with the description of the Desulfurococcaceae, as its members typically use elemental sulphur for reduction or respiration (Burggraf et al., 1997). However, Aeropyrum camini (Nakagawa et al., 2004), Thermosphaera aggregans (Huber et al., 1998) and Sulfophobococcus zilligii (Hensel et al., 1997) are already exceptions within the Desulfurococcaceae in this regard.
By themselves, the phenotypic characteristics of the novel cocci suggest that they represent members of the Desulfurococcaceae; however, based on 16S rRNA gene analysis the novel coccal isolates form an independent lineage within the Crenarchaeota with a 100 % bootstrap value and do not group within any other lineage within the Desulfurococcaceae. Consequently, the ‘Ignisphaera group’ constitutes a novel deep-branching genus of the Desulfurococcaceae, branching independently from both the families Desulfurococcaceae and Pyrodictiaceae. Therefore, inclusion of the ‘Ignisphaera group’ and the proposal of strain AQ1.S1T as representing a new genus and species within the Desulfurococcales may be controversial, as is the case for the ‘Acidilobus group’, because of its independent grouping based on 16S rRNA gene analysis and acidophilic growth optima (Itoh et al., 2003). Despite the above considerations, with regard to the phylogenetic placement of strain AQ1.S1T and related isolates, we propose a new genus within the Desulfurococcales as the most conservative origin for this lineage. However, as more representatives of the Crenarchaeota are cultured and characterized the position of the ‘Ignisphaera-type’ isolates may be resolved.
Description of Ignisphaera gen. nov.
Ignisphaera (Ig.ni.sphae'ra. L. n. ignis fire, L. fem. n. sphaera ball, N.L. fem n. Ignisphaera fire ball).
Cells are regular to irregular cocci that occur singly, in pairs or as aggregates. Hyperthermophilic anaerobe. Moderate acidophile. Heterotrophic. Electron acceptor is not absolutely necessary. Elemental sulphur and NaCl inhibit growth. The 16S rRNA gene groups within the Crenarchaeota. Habitat is terrestrial, near-neutral hot springs and mud pots. The type species is Ignisphaera aggregans.
Description of Ignisphaera aggregans sp. nov.
Ignisphaera aggregans (ag'gre.gans. L. part. adj. aggregans aggregate forming, aggregating clumping).
Cells are approximately 1–1.5 µm in size when grown on starch. Temperature and pH ranges for growth are 85–98 °C (optimum at 92–95 °C) and pH 5.4–7.0 (optimum pH 6.4). Obligate anaerobe. Ferments both poly- and disaccharides. Grows at low salinity (<0.5 % NaCl) and optimally without addition of NaCl. Resistant to novobiocin and streptomycin but sensitive to erythromycin, chloramphenicol and rifampicin. The G+C content of the DNA is 52.9 mol%.
The type strain is Ignisphaera aggregans AQ1.S1T (=DSM 17230T=JCM 13409T), which was isolated from a near-neutral, boiling spring in Kuirau Park, Rotorua, New Zealand.
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