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1 Department of Bioresource Science, Ibaraki University College of Agriculture, Ami-machi, Ibaraki 300-0393, Japan
2 Department of Environmental Science and Technology, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama 226-8503, Japan
3 Soils Research and Development Centre, Bureau of Soils and Water Management, Diliman, Quezon City, Philippines
Correspondence
Hiroyuki Ohta
hohta{at}mx.ibaraki.ac.jp
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A figure showing whole-cell protein electrophoretic profiling of the group P strains is available as supplementary material in IJSEM Online.
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). Consequently, rates of vegetation growth and other ecosystem processes are often constrained by the supply of nitrogen and carbon in early developing ecosystems on volcanic deposits. Under these conditions, carbon and energy sources for microbial community development derive from the colonization of phototrophs or lichens (Jackson & Keller, 1970; Kurina & Vitousek, 1999; Crews et al., 2001), dry and wet depositions of organic matter (King, 2003) and atmospheric inputs of trace gases (e.g. CO, H2 and CH4) (Conrad, 1996). Recently, King (2003) showed that atmospheric CO and hydrogen utilization contribute 2–4 % and 15–20 % of total respiratory reducing equivalent flow, respectively, in intact cores from recent Hawaiian volcanic deposits.
Previously, bacterial diversity of very recent volcanic deposits sampled from volcanic mudflow-affected areas around Mt. Pinatubo, the Philippines, has been analysed by quinone profiling (Ohta et al., 2003). Subsequently, pure bacterial cultures were isolated from such volcanic deposit samples and then subjected to comparative 16S rRNA gene sequence analysis. Analysis revealed that 25 % of total culturable bacteria were phylogenetically related to a hydrogen-oxidizing bacterium, Ralstonia eutropha [recently transferred to the novel genus Wautersia (Vaneechoutte et al., 2004) and further reclassified as a synonym of Cupriavidus necator (Vandamme & Coenye, 2004)], with similarities of 97.3–98.2 % (Sato et al., 2004). Furthermore, most of the C. necator-related bacterial strains from the mudflow deposits grew chemolithoautotrophically with H2 as electron donor and CO2 as carbon source and also possessed hydrogenase activity (Sato et al., 2004). In the present study, the C. necator-related bacteria were further characterized and described using physiological characterization, DNA–DNA hybridization, determination of DNA base composition and whole-cell protein profiles. On the basis of these data, the two representative strains (1245T and 1263aT) should be classified as the type strains of two novel species.
The ten strains under study (1243, 1244a, 1245T, 1246a, 1247, 1249, 1250, 1263aT, 1266 and 1278a) were isolated in 1998 from the 0.00–0.25 m depth layer of the volcanic deposits at two sites [site N (15° 15' 17'' N 120° 34' 59'' E) and site S1 (2 km east of site N)] that had been hit repeatedly by mudflows during successive rainy seasons after the violent eruption of 1991 on the east side of Mt. Pinatubo (Ohta et al., 2003; Sato et al., 2004). C. necator JCM 11282 (the type strain of W. eutropha), Cupriavidus gilardii JCM 11283T, Cupriavidus oxalaticus JCM 11285T and Cupriavidus pauculus JCM 11286T were obtained from the Japan Collection of Microorganisms, Wako-shi, Japan. Cupriavidus basilensis DSM 11853T was obtained from the DSMZ, Braunschweig, Germany, and Ralstonia sp. LMG 1197 (Pemberton et al., 1979), C. necator LMG 8453T, Cupriavidus campinensis LMG 19282T, Cupriavidus metallidurans LMG 1195T, Cupriavidus respiraculi LMG 21510T and Cupriavidus taiwanensis LMG 19424T were from BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Gent, Belgium. All strains were grown aerobically at 27 °C on nutrient broth (NB) [1 % (w/v) Ehrlich meat extract (Kyokuto Seiyaku), 1 % (w/v) Trypticase peptone (Becton Dickinson) and 0.5 % (w/v) NaCl, pH 7.0 with 1 M NaOH] or 100-fold diluted NB (10–2 NB), unless otherwise stated.
Classical phenotypic tests and analysis of isoprenoid quinones were performed as described by Ohta & Hattori (1983). Growth of the strains at 41 °C on NB was monitored for 7 days. The API 20NE kit system (bioMérieux) was used according to the recommendations of the manufacturer. The substrate utilization profile was tested in a 10–2 NB liquid medium supplemented with each of the following compounds: sucrose, D-fructose, acetate, formate, glutamate, lactate, benzoate, caffeate, catechol, ferulate, guaiacol, phenol, vanillate, 2-hydroxybenzoate, 3,4-dihydroxybenzoate and 4-hydroxybenzoate. All compounds were sterilized by filtration and added to autoclaved 10–2 NB medium. Sugars were added at a concentration of 0.2 % (w/v); organic acids and aromatic compounds were added at 0.02 % (w/v). Growth was measured after 2 days incubation and utilization was assessed by comparing growth in the presence and absence of an added compound. To compare cellular protein profiles, cells were grown for 48 h on NB at 27 °C. Whole-cell lysates were prepared essentially as described by Ohta et al. (1993) and one-dimensional analytical SDS-PAGE was performed by the method of Laemmli (1970) with a 12.5 % separating gel and 4.5 % stacking gel. Proteins were visualized by silver staining with a commercial kit (Daiichi Pure Chemicals). The similarity between all pairs of electrophoresis patterns was calculated by the simple matching coefficient, expressed as a percentage. Cluster analysis was performed by the unweighted pair group method using arithmetic averages (UPGMA) clustering technique. The complete 16S rRNA gene sequences determined previously (Sato et al., 2004) were compared with those of the type strains of Cupriavidus species. The CLUSTAL W algorithm (Thompson et al., 1994) was used to align sequences and to construct a neighbour-joining tree with 1000 bootstrap iterations. For determination of DNA base composition, DNA was extracted, purified by phenol treatment (Saito & Miura, 1963) and enzymically degraded into nucleosides. The nucleoside mixture was separated by reversed-phase HPLC as described by Tamaoka & Komagata (1984). DNA–DNA hybridization experiments were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989). For enzymic development, alkaline phosphatase–streptavidin conjugate (Vector) was used with CDP-Star (Tropix) as substrate and chemiluminescence was determined with a Wallac 1420 ARVOsx multilabel counter as described previously (Ushiba et al., 2003).
The ten bacteria isolated from the volcanic mudflow deposits were strictly aerobic, Gram-negative, non-sporulating, catalase-positive rods (0.3–0.6x0.8–1.6 µm). Their phenotypic characteristics determined by the API 20NE kit test were very similar. All of them reduced nitrate to nitrite but not further to N2, possessed urease and oxidase activities and assimilated gluconate, caprate, adipate, malate, citrate and phenylacetate. None of the strains produced indole, acids from glucose, 
-glucosidase, protease or -galactosidase. None of the strains assimilated arabinose, mannose, mannitol, N-acetyl-D-glucosamine or maltose. Strain 1247 utilized glucose, but the other strains did not. Six strains (1243, 1244a, 1245T, 1247, 1250 and 1278a) produced arginine dihydrolase.
When the almost-complete 16S rRNA gene sequences of the ten strains comprising 1523–1525 nt were compared to each other, nine strains (1243, 1244a, 1245T, 1246a, 1247, 1249, 1250, 1266 and 1278a) formed a homogeneous group with very high similarities (99.7–100 %): the group was named group P. The similarities of the group P strains to the other strain (1263aT) were 98.6–98.7 %. Phylogenetic analysis of the sequences of the ten strains under study, five Ralstonia species, Ralstonia sp. LMG 1197 and nine Cupriavidus species confirmed that the ten strains belonged to the genus Cupriavidus and formed a separate phylogenetic cluster in the tree (Fig. 1). Within the cluster, strain 1263aT formed a distinct branch with group P (a bootstrap resampling value of 94 %). The closest relatives of the group P strains and 1263aT among the type strains of established Cupriavidus species were C. basilensis, with sequence similarities of 98.1–98.3 % and 97.1 %, respectively, and C. taiwanensis, with sequence similarities of 98.0–98.2 % and 97.8 %, respectively. Ralstonia sp. LMG 1197 clustered together with the group P strains and was clearly separate from the clusters of Ralstonia species. The 16S rRNA gene sequence similarities between Ralstonia sp. LMG 1197 and strains of group P and 1263aT were 99.5–99.7 % and 98.6 %, respectively. The homogeneity of group P was further confirmed by the same overall protein-banding pattern in whole-cell protein electrophoretic profiling (see Supplementary Fig. S1 in IJSEM Online). In the following taxonomic analyses, strain 1245T was used as the representative of group P and tested together with strain 1263aT.
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; Kersters & De Ley, 1975): indeed, in the genera Ralstonia and Cupriavidus, high protein electrophoretic similarity correlates with a high level of DNA–DNA hybridization (Coenye et al., 1999, 2003a; Vandamme et al., 1999; Goris et al., 2001; Vandamme & Coenye, 2004). UPGMA cluster analysis of SDS-PAGE protein profiles showed that strain 1245T and Ralstonia sp. LMG 1197 formed a cluster at the similarity level of 87 % and were distinct from strain 1263aT (67 % similarity) and the other type strains of Cupriavidus species (less than 78 % similarity) (Fig. 2).
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). Utilization of ten aromatic compounds and six organic acids by strain 1245T, strain 1263aT, Ralstonia sp. LMG 1197 and C. basilensis DSM 11853T was tested and substrate utilization profiles were compared. Seven out of ten aromatic compounds were used by strain 1245T, Ralstonia sp. LMG 1197 and C. basilensis DSM 11853T. The utilization profile of strain 1245T was the same as that of Ralstonia sp. LMG 1197: benzoate, caffeate, catechol, phenol, 2-hydroxybenzoate, 3,4-dihydroxybenzoate and 4-hydroxybenzoate were used, but ferulate, guaiacol and vanillate were not used. C. basilensis DSM 11853T also used a wide range of aromatic compounds: benzoate, caffeate, catechol, phenol, 2-hydroxybenzoate, 4-hydroxybenzoate and vanillate. In contrast, strain 1263aT utilized a narrower range of aromatic compounds (caffeate, catechol, phenol and vanillate). Other phenotypic characteristics of strains 1245T and 1263aT are shown in Table 1 or are given in the species description.
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.
Description of Cupriavidus pinatubonensis sp. nov.
Cupriavidus pinatubonensis (pin.a.tu.bo.nen'sis. N.L. masc. adj. pinatubonensis pertaining to Mt. Pinatubo, the volcano on Luzon Island, the Philippines, from which first strains were isolated).
Cells are aerobic, Gram-negative, non-sporulating, peritrichously flagellated rods (0.3–0.6x0.8–1.6 µm). Colonies on 100-fold diluted NB agar are circular, entire, convex, opaque and white. Growth is observed in NB and 10-fold and 100-fold diluted NB at 27, 30 and 41 °C. Oxidase, catalase and urease are present; protease, -galactosidase and -glucosidase are not present. No indole production or production of acid from glucose occurs. Nitrate is reduced to nitrite, but not further to N2. Assimilates gluconate, caprate, adipate, malate, citrate, phenylacetate, D-fructose, acetate, glutamate, lactate, benzoate, caffeate, catechol, phenol, 2-hydroxybenzoate, 3,4-dihydroxybenzoate and 4-hydroxybenzoate, but not glucose, arabinose, mannose, mannitol, N-acetyl-D-glucosamine, maltose, sucrose, formate, ferulate, guaiacol or vanillate. Chemolithoautotrophic growth occurs in the presence of hydrogen, oxygen and carbon dioxide. The major isoprenoid quinone is ubiquinone Q-8.
The type strain is 1245T (=CIP 108725T=PNCM 10346T), isolated from volcanic mudflow deposits sampled around Mt. Pinatubo, the Philippines. The DNA G+C content of strain 1245T is 65.9 mol%.
Description of Cupriavidus laharis sp. nov.
Cupriavidus laharis (la.har'is. N.L. gen. n. laharis of a lahar, a volcanic mudflow).
Cells are Gram-negative, aerobic, non-sporulating, peritrichously flagellated rods (0.3–0.6x0.8–1.6 µm). Colonies on 100-fold diluted NB agar are circular, entire, convex, opaque and white. Growth is observed in NB and 10-fold and 100-fold diluted NB at 27 and 30 °C, but not at 41 °C. Oxidase, catalase and urease are present; protease, -galactosidase and -glucosidase are not present. No indole production or production of acid from glucose occurs. Nitrate is reduced to nitrite, but not further to N2. Assimilates gluconate, caprate, adipate, malate, citrate, phenylacetate, D-fructose, acetate, caffeate, catechol, phenol and vanillate, but not sucrose, formate, glutamate, lactate, benzoate, ferulate, guaiacol, 2-hydroxybenzoate, 3,4-dihydroxybenzoate or 4-hydroxybenzoate. Chemolithoautotrophic growth occurs in the presence of hydrogen, oxygen and carbon dioxide. The major isoprenoid quinone is ubiquinone Q-8.
The type strain is 1263aT (=CIP 108726T=PNCM 10347T), isolated from volcanic mudflow deposits sampled around Mt. Pinatubo, the Philippines. The DNA G+C content of strain 1263aT is 65.2 mol%.
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ACKNOWLEDGEMENTS |
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