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1 Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V., Hans-Knöll-Institut, Beutenbergstrasse 11a, 07745 Jena, Germany
2 DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany
3 Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Sevilla, Spain
Correspondence
Ingrid Groth
Ingrid.Groth{at}hki-jena.de
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain HKI 0342T is AJ854061.
Detailed physiological characteristics of strain HKI 0342T are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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for the misclassified species Cellulosimicrobium variabile Bakalidou et al. 2002 and is currently based on this single species. The type strain, which is the only representative of Isoptericola variabilis at the time of writing, was isolated from the hindgut of the Australian termite Mastotermes darwiniensis (Frogatt). I. variabilis is phylogenetically closely related to members of the genera Xylanimonas (Rivas et al., 2003), Xylanibacterium (Rivas et al., 2004) and Xylanimicrobium (Stackebrandt & Schumann, 2004). The latter genus has been recently established to harbour the misclassified species Promicromonospora pachnodae (Cazemier et al., 2003, 2004), the type strain of which was isolated from the hindgut of larvae of the scarab beetle Pachnoda marginata. Together, these four genera constitute a phylogenetically distinct cluster within the suborder Micrococcineae, and have cellulolytic and xylanolytic activities in common.
Strain HKI 0342T was isolated from a sample of tufa collected in the burial chamber of the first arcosolium behind the entrance to the Roman catacomb of Domitilla (Rome, Italy); PY-BHI agar (Yokota et al., 1993) and a standard dilution-plate procedure were used. General laboratory cultivation, morphological studies, determination of optimal growth parameters (temperature, pH, oxygen requirements) and antibiotic-susceptibility tests were performed, using solid or liquid organic medium 79 (Prauser & Falta, 1968) and an incubation temperature of 28 °C. Cell morphology and cell dimensions were examined by using phase-contrast microscopy with a Zeiss Axioscope 2 microscope equipped with image-analysing software (Axio Vision 2.05). The colony morphology of 2–10-day-old cultures was studied using a stereo microscope (Olympus). Standard physiological tests were carried out according to the methods described by Cowan & Steel (1965), Gordon et al. (1974), Lanyi (1987) and Smibert & Krieg (1994). Acid production from carbon sources and enzyme activities were studied using the API 50 CHB/E kit (incubation times of up to 7 days) and API ZYM galleries (bioMérieux). Additionally, the utilization of carbon sources was tested using Biolog GP2 MicroPlates and MicroLog computer software (Biolog Identification System). Xylanolytic activity was determined on medium II, described by Cazemier et al. (2003), and incubation times of up to 28 days. Susceptibility to antibiotics was examined by placing antibiotic discs (Difco) on agar plates that were seeded with suspensions of the test strains grown in a soft agar layer for 24 h at 28 °C. Oxygen requirements were studied with the GENbag microaer and GENbag anaer incubation systems (bioMérieux). The pH range for growth was established by using liquid medium adjusted to pH values between 4 and 11 with either 1 M HCl or 20 % (w/v) Na2CO3 solution and incubated at 28 °C for up to 10 days. The reference strains used for comparisons in physiological tests and DNA–DNA pairing studies were I. variabilis DSM 10177T, Xylanimicrobium pachnodae DSM 12657T, Xylanimonas cellulosilytica DSM 15894T and Myceligenerans xiligouense DSM 15700T. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strain in shake flasks containing liquid organic medium 79 or Bacto tryptic soy broth (Sigma–Aldrich) for 24–48 h. Stock cultures of strain HKI 0342T in liquid organic medium 79 supplemented with 5 % DMSO were maintained in either the vapour phase of liquid nitrogen or at –80 °C by adding a 1 : 1 mixture of glycerol and a medium that consisted of K2HPO4 (1·26 %), KH2PO4 (0·36 %), MgSO4 (0·01 %), sodium citrate (0·09 %), (NH4)2SO4 (0·18 %) and glycerol (8·8 %).
On medium 79, strain HKI 0342T formed wrinkled, circular, smooth, pale-yellow colonies about 1–4 mm in diameter. In young cultures (8–24 h), a well-developed primary mycelium (width 0·6–0·7 µm) was observed which fragmented, in the stationary growth phase (48–72 h), into irregular, non-motile, short rods and cocci (diameter 0·8–1·0 µm). In contrast to stationary-phase cultures of I. variabilis, both cell types were arranged mainly as short flexible chains or as clusters. Furthermore, strain HKI 0342T and I. variabilis DSM 10177T exhibited different growth characteristics (Table 1). The catacomb isolate was an aerobic to microaerophilic organism and did not grow in an anaerobic atmosphere, unlike I. variabilis DSM 10177T, which is a facultatively anaerobic organism (Bakalidou et al., 2002). I. variabilis grew at temperatures up to 45 °C, but not at 10 °C, while strain HKI 0342T did not grow above 40 °C, but grew well at 10 °C. Both strains had in common the ability to hydrolyse xylan. However, they could be readily distinguished from one another by means of a broad spectrum of physiological properties. Clearly differential characteristics of the two strains under study are listed in Table 1. Full detailed results (classical physiological tests, API kits and Biolog test) are provided as supplementary tables in IJSEM Online.
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. The 16S rRNA gene was amplified by a PCR using the conserved primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522R (5'-AAGGAGGTGATCCAGCCGCA-3'). PCR thermal conditions were as follows: 95 °C for 1 min; 35 cycles of 95 °C for 15 s, 55 °C for 15 s and 72 °C for 2 min; and a final extension cycle at 72 °C for 10 min. Forward and reverse strands of the amplified DNA fragment were sequenced in an ABI 3700 sequencer (Applied Biosystems). The 16S rRNA gene sequences of the strains studied were aligned manually with nucleotide sequences obtained from the GenBank and EMBL databases. The algorithm of Jukes & Cantor (1969) was applied in order to transform sequence dissimilarities into evolutionary distances. Phylogenetic dendrograms were reconstructed by using the method of De Soete (1983) and the neighbour-joining method (Felsenstein, 1993).
The binary 16S rRNA gene sequence similarity values between strain HKI 0342T and its nearest phylogenetic neighbours range between 95·0 and 98·8 %. A dendrogram of relationships based on the Jukes & Cantor (1969) corrections of similarity values (Fig. 1) shows strain HKI 0342T as branching next to I. variabilis DSM 10177T (98·8 %), whereas members of Xylanimonas and relatives, Cellulosimicrobium and Promicromonospora are less closely related. To determine the genomic relatedness between strains DSM 10177T and HKI 0342T, DNA–DNA similarity studies were performed.
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. DNA–DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huß et al. (1983), using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in-situ temperature probe (Varian). The DNA G+C content was determined as recommended by Mesbah et al. (1989). Strains HKI 0342T and DSM 10177T shared a mean DNA–DNA relatedness value of 31·7 % (individual values of 33·4 and 30·0 %), a value which clearly indicates that the two strains belong to distinct genomic species.
The following chemotaxonomic characteristics were determined: the structure of the peptidoglycan, as described by Schleifer & Kandler (1972), Schleifer (1985), MacKenzie (1987), Groth et al. (1996) and Frank et al. (1980); the acyl type of the muramic acid, as described by Uchida et al. (1999); the whole-cell sugars present, as described by Becker et al. (1965) and Saddler et al. (1991); the menaquinones present, as described by Groth et al. (1996); the polar lipids present, as described by Minnikin et al. (1979) and Collins & Jones (1980); the mycolic acids present, as described by Minnikin et al. (1975); and the fatty acid profile (MIDI system; Agilent).
The chemotaxonomic characteristics of strain HKI 0342T were most similar to those of I. variabilis and supported the affiliation of this organism to the genus Isoptericola. The peptidoglycan contained N-acetylated muramic acid and corresponded to type A4
, L-lys–D-Glu (A11.33 according to http://www.dsmz.de/species/murein.htm). Strain HKI 0342T differs from I. variabilis in having D-glu instead of D-Asp as the interpeptide bridge. Glucose, rhamnose, galactose and a minor amount of mannose were found in whole-cell hydrolysates. Strain HKI 0342T was characterized by the presence of menaquinones with nine isoprene units, which differs from I. variabilis in that the major components were MK-9(H2) and MK-9 (peak areas 42 : 29, respectively) instead of MK-9(H4) (peak area 10). The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unknown phospholipids and an unknown glycolipid. In addition to the polar lipid components reported by Stackebrandt et al. (2004) for I. variabilis, phosphatidylinositol mannoside was also detected in strain DSM 10177T in this study. The fatty acid profile consisted of anteiso-C15 : 0 (44·0 mol%), iso-C16 : 0 (22·6 mol%), iso-C15 : 0 (18·1 mol%), iso-C14 : 0 (8·4 mol%) and anteiso-C17 : 0 (3·1 mol%). Mycolic acids were not detected.
It is evident from Fig. 1
that strain HKI 0342T is most closely related to the type strain of I. variabilis. The low level of DNA–DNA relatedness (about 30 %) between the two strains, together with the numerous differences in chemotaxonomic, morphological and a broad range of physiological characteristics (Table 1), clearly indicates that strain HKI 0342T represents a novel species of the genus Isoptericola, for which the name Isoptericola hypogeus is proposed.
Description of Isoptericola hypogeus sp. nov.
Isoptericola hypogeus (hy.po.ge'us. L. masc. adj. hypogeus underground, referring to the site of isolation).
Gram-positive, aerobic to microaerophilic actinomycete with a well-developed primary mycelium (diameter of hyphae 0·6–0·7 µm) that undergoes fragmentation into short, irregular, non-motile rods and cocci (diameter 0·8–1·0 µm) in the stationary growth phase. Aerial mycelium is absent. Colonies on organic medium 79 are wrinkled, circular, smooth and pale yellow (diameter about 1–4 mm). Growth occurs between 10 and 40 °C (optimal growth at 28 °C) and at pH values in the range of 6–9. NaCl in the culture medium is tolerated at concentrations up to 10 %. Physiological characteristics relating to the utilization of carbohydrates, enzyme activities and susceptibility to antibiotics are listed in Table 1 and in the supplementary data available in IJSEM Online. The peptidoglycan type is A4, L-lys–D-Glu. The whole-cell sugars comprise glucose, rhamnose, galactose and a minor amount of mannose. The acyl type is acetyl. The menaquinones comprise MK-9(H2), MK-9 and MK-9(H4) (peak areas 42 : 29 : 10) and the major fatty acids are anteiso-C15 : 0 (44·0 mol%), iso-C16 : 0 (22·6 mol%), iso-C15 : 0 (18·1 mol%), iso-C14 : 0 (8·4 mol%) and anteiso-C17 : 0 (3·1 mol%). The phospholipids comprise diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, two unknown phospholipids and an unknown glycolipid. Mycolic acids are absent. The G+C content of the DNA of the type strain is 73·8 mol%.
The type strain, HKI 0342T (=DSM 16849T=NCIMB 14033T), was isolated from a tufa sample from a burial chamber in the catacomb of Domitilla, Rome, Italy.
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ACKNOWLEDGEMENTS |
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