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1 Department of Microbiology, University of Georgia, Athens, GA 30602-2605, USA
2 Department of Environmental Science and Engineering, Technical University of Denmark, 2800 Lyngby, Denmark
Correspondence
Juergen Wiegel
jwiegel{at}uga.edu
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using xylan as the substrate. This bacterium, a Gram-type positive (Wiegel, 1981), low-G+C content rod, has many characteristics of a typical member of the Firmicutes (Gibbons & Murray, 1978). Based on its physiological properties alone, it was placed in the genus Thermoanaerobium, the type species of which is Thermoanaerobium brockii (Zeikus et al., 1979).
Because the 16S rRNA gene sequence for Thermoanaerobium acetigenum X6BT had not been determined previously, the classification of Thermoanaerobium acetigenum X6BT was therefore based only on some physiological similarities. Although the type species of Thermoanaerobium, Thermoanaerobium brockii, was reclassified as Thermoanaerobacter brockii by Lee et al. (1993) and, subsequently, as Thermoanaerobacter brockii subsp. brockii (type strain HTD4T) by Cayol et al. (1995), Thermoanaerobium acetigenum X6BT was not transferred to the genus Thermoanaerobacter (Wiegel & Ljungdahl, 1981) because of the lack of 16S rRNA gene sequence analysis. Here we report on the assignment of the type strain of Thermoanaerobium acetigenum to the genus Caldicellulosiruptor as Caldicellulosiruptor acetigenus comb. nov., based on 16S rRNA gene sequence, DNA–DNA hybridization analysis and retesting of its properties. Special attention was given to cellulose degradation, as all other presently known Caldicellulosiruptor species are cellulolytic, whereas strain X6BT has been described as being non-cellulolytic.
Strain X6BT was obtained as a freeze-dried culture of strain DSM 7040T from the DSMZ (Braunschweig, Germany). To determine the 16S rRNA gene sequence, Thermoanaerobium acetigenum DSM 7040T was grown under anaerobic conditions (Ljungdahl & Wiegel, 1986; Angelidaki et al., 1990). A basal salts medium (final pH 7.3–7.4) was prepared as described by Nielsen et al. (1993). Strain DSM 7040T was grown in basal salts medium supplemented with yeast extract (0.3 %), tryptone (1.0 %) and glucose (0.5 %), and subjected to two rounds of isolation of single colonies using yeast extract, tryptone, glucose salts medium solidified with 2.2 % Gelrite (colonies became visible after incubation at 65 °C for 48–72 h). Because the initial 16S rRNA gene sequence analysis yielded two different 16S rRNA species, which bracketed another Caldicellulosiruptor species, it became necessary to confirm the purity of the culture further. Therefore, strain DSM 7040T was grown using three different media (substrate conditions as described below), and each culture was then subjected to three subsequent rounds of single-cell colony isolation. To establish three lines of cultures, strain DSM 7040T was grown in the above-described basal salts medium, supplemented with yeast extract, tryptone, glucose and brain heart infusion (0.2 %) (termed BYTG medium). From this culture, three parallel cultures were inoculated (0.1 % inoculum) using the following media: (i) basal salts plus 0.2 % arabinose medium, (ii) basal salts plus 0.2 % raffinose medium and (iii) BYTG medium. Arabinose- and raffinose-supplemented basal salts media were used because the closest Caldicellulosiruptor species to strain X6BT on the phylogenetic tree (Fig. 1) are unable to use these substrates (Table 1). After checking microscopically that the cultures were suspensions of individual cells and did not contain any clumps or associations of cells, each of the above cultures was used to inoculate dilution series of Gelrite shake-roll tubes (Ljungdahl & Wiegel, 1986), with 2.2 % (w/v) Gelrite, to obtain single-cell colonies. The Gelrite shake-roll tubes were incubated at 65 °C for 48–72 h before colonies became visible. Colonies were picked in an anaerobic chamber (Coy Products) and resuspended in a tube containing 0.3–0.4 ml of the corresponding medium, which was then used to inoculate the next round of Gelrite shake-roll tubes. This process of colony picking was repeated for three rounds of colony isolation with three colonies being picked from each of the arabinose, raffinose and BYTG media after the third and final round. Each of the picked final colonies was reinoculated into a fresh tube of the medium from which it was isolated, resulting in nine cultures: three with the arabinose medium, three with the raffinose medium and three with BYTG medium.
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). PCR was carried out as described previously (Lee et al., 2005). The PCR products were purified using a QIAquick PCR Purification kit (Qiagen) and sequenced by Macrogen (Seoul, Korea). PCR products from the colonies were cloned using a TOPO TA Cloning kit (Invitrogen). Clones were randomly chosen, from which plasmid DNA was extracted by using an Eppendorf FastPlasmid Mini kit (Brinkman). The DNA was subsequently amplified, purified and sequenced. The sequence similarities were determined using Sequencher v4.1.4 (Gene Codes). Ten clones were sequenced, resulting in two similar sets of sequences (Fig. 1
; termed T6 and T4, GenBank accession nos AY772477 and AY772476, respectively). Three clones with the same 16S rRNA gene sequence were never obtained from a single culture, suggesting that an even distribution of clones with the different sequences existed. Analysis of the 16S rRNA gene sequence using nucleotide to nucleotide BLAST (BLASTN) at NCBI (http://www.ncbi.nlm.nih.gov/blast/) to retrieve the most significant homologues of the query 16S rRNA gene sequence revealed that the most significant sequence alignments of Thermoanaerobium acetigenum X6BT were with Caldicellulosiruptor species (Sissons et al., 1987; Rainey et al., 1994), with Caldicellulosiruptor lactoaceticus 6AT (Mladenovska et al., 1995) and Caldicellulosiruptor kristjanssonii I77R1BT (Bredholt et al., 1999) as the closest relatives (Fig. 1). Subsequently, 16S rRNA gene sequence-based phylogenetic trees were generated using CLUSTAL_X (Thompson et al., 1997) for sequence alignments, and phylogeny inference package (PHYLIP) software (Felsenstein, 1989) and neighbour-joining algorithms (Saitou & Nei, 1987) to look at differing tree constructions and to generate distance matrices. TreeExplorer (Kumar et al., 1994), a supplemental program of MEGA, was used to view the tree. The phylogenetic trees generated (the neighbour-joining tree is shown in Fig. 1) showed clearly that Thermoanaerobium acetigenum X6BT belongs to the clade of Caldicellulosiruptor and not to the genus Thermoanaerobacter or Thermoanaerobacterium. Each of the repurified strains exhibited the two different 16S rRNA sequences. In contrast to other reported cases, e.g. Clostridium paradoxum (Rainey et al., 1996), the two sequences were not juxtaposed, but rather separated by a sequence of another species, Caldicellulosiruptor kristjanssonii. A comparable situation has been reported by Amann et al. (2000), i.e. single-cell-derived pure cultures contained two different 16S rRNA genes with about 5 % inferred difference in substitutions and bracketing a different species. The relative distance between the two identified Thermoanaerobium acetigenum X6BT 16S rRNA gene sequences was about 2 % and the distance to the closest neighbours Caldicellulosiruptor lactoaceticus 6AT and Caldicellulosiruptor kristjanssonii I77R1BT was around 1 %. Therefore, DNA–DNA hybridization experiments were performed using the method described by De Ley et al. (1970) and modified by Huß et al. (1983). Chromosomal DNA for DNA–DNA hybridization was extracted and purified using a Maxi Genomic [0]DNA Prep kit (A&A Biotechnology).
DNA–DNA hybridization values between Thermoanaerobium acetigenum DSM 7040T and Caldicellulosiruptor owensensis OLT, Caldicellulosiruptor lactoaceticus 6AT and Caldicellulosiruptor kristjanssonii I77R1BT were 34.3, 50.9 and 53.1 %, respectively. These values are all significantly below the 70 % relatedness mark that would indicate a relationship at the species level (Wayne et al., 1987) and clearly distinguish Thermoanaerobium acetigenum X6BT from these three Caldicellulosiruptor species that are its closest neighbours in the 16S rRNA gene-based phylogenetic tree (Fig. 1). The repeated isolation of single-cell colonies after growth in different media, the observed homogeneity of the colony and cell morphologies with and among cultures (using microscopy) and the fact that all isolated colonies gave rise to the two different 16S rRNA sequences indicate that it is unlikely that the two sequences are due to a mixed culture having been analysed. These results indicate that Thermoanaerobium acetigenum X6BT belongs in the genus Caldicellulosiruptor, a member of the order Clostridiales, and not in the genus Thermoanaerobacter, order ‘Thermoanaerobacteriales’ (Garrity et al., 2002).
Members of the genus Caldicellulosiruptor have the characteristic trait of coupling cellulose degradation to growth (Rainey et al., 1994). However, Thermoanaerobium acetigenum X6BT was characterized previously (Nielsen et al., 1993) as being incapable of cellulose degradation.
Thermoanaerobium acetigenum DSM 7040T was retested for the ability to degrade cellulose using Whatman no. 1 filter paper and carboxymethylcellulose (1.0 % w/v, CMC 7LT or 7M; Hercules). In addition, cellulase activity was determined by the use of the reducing sugar assay employing p-hydroxybenzoic acid hydrazide and glucose as a standard (Lever, 1973). Thermoanaerobium acetigenum DSM 7040T was incapable of degrading Whatman no. 1 filter cellulose with or without 0.05 % (w/v) yeast extract, but utilized CMC, exhibiting moderate growth with 1.0 % of the low substitution (substitution level 0.7 of 3) form Hercules 7LT or 7M. Eleven and 10 µmol ml–1, respectively, of reduced sugar residues was released from the cultures after 4 days of incubation, with a requirement for yeast extract (0.05 %, w/v) for growth. Growth was not observed with only CMC 7LT/7M present. More highly substituted (e.g. 1.2 out of 3) CMCs (Hercules 12M or 12L) did not serve as substrates.
The substrate utilization spectrum of Thermoanaerobium acetigenum DSM 7040T, as performed by Nielsen et al. (1993), was re-examined by adding various carbohydrates (to a final concentration of 2 g l–1) from autoclaved stock solutions (pyruvate was filter-sterilized) to the basal media. Cultures were incubated at 73 °C for 48–72 h. Growth of cultures with insoluble substrates was determined by cell counts (Olympus model Vanox microscope with a Petroff-Hausser counting chamber). The results confirmed the previously published data.
The 16S rRNA gene sequence analysis, CMC-cellulase activity and growth observed on low-substituted CMC indicate that Thermoanaerobium acetigenum belongs to the genus Caldicellulosiruptor, and we propose the name Caldicellulosiruptor acetigenus comb. nov.
Emended description of the genus Caldicellulosiruptor Rainey et al. 1995
The description is the same as that given by Rainey et al. (1994) with the addition that some members do not possess the capacity to degrade crystalline cellulose or filter paper and cannot use cellulose as a carbon and energy source, but can hydrolyse CMC.
Description of Caldicellulosiruptor acetigenus comb. nov.
Caldicellulosiruptor acetigenus (a.ce.ti.ge'nus. L. n. acetum vinegar; L. v. genere, gignere to produce; N.L. masc. adj. acetigenus vinegar- or acetic acid-producing).
Basonym: Thermoanaerobium acetigenum Nielsen et al. 1994.
The description is based mainly on that given by Nielsen et al. (1993). Cells stain Gram-negative but have a Gram-type positive cell wall structure, occur singly or in pairs, and are about 3.6–5.9 by 0.7–1.0 µm in size. Sometimes occur as chains of up to eight cells. On solidified xylan-containing medium, off-white, milky-coloured colonies are observed. Strictly anaerobic chemoorganoheterotroph. At pH 7.0, growth occurs between 50 and 78 °C (optimum 65–68 °C). Growth occurs at pH 5.2–8.6 (optimum 7.0). Doubling time under optimal conditions is approximately 4 h. Arabinose, cellobiose, fructose, D-galactose, D-glucose, lactose, maltose, mannose, raffinose, soluble starch, sucrose, trehalose, D-xylose and xylan support growth. Growth and CMC-cellulase activity is observed when grown on carboxymethylcellulose (Hercules CMC, 7LT or 7M) in the presence of traces of yeast extract, but not with filter paper or crystalline (Avicel) cellulose. Acetate, CO2, H2, ethanol and traces of isobutyric acid (but not lactate) are formed during growth with glucose or D-xylose. The DNA G+C content of the type strain is 35.7±0.8 mol% (chromatographic method).
The type strain is X6BT (=DSM 7040T=ATCC BAA-1149T), which was isolated from a combined biomat and sediment sample taken from a slightly alkaline hot spring at Hverðagerdi, Iceland.
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