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Isoptericola Hypogeus Sp. Nov., Isolated from the Roman Catacomb of Domitilla

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Isoptericola Hypogeus Sp. Nov., Isolated from the Roman Catacomb of Domitilla View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Digital.CSIC International Journal of Systematic and Evolutionary Microbiology (2005), 55, 1715–1719 DOI 10.1099/ijs.0.63632-0 Isoptericola hypogeus sp. nov., isolated from the Roman catacomb of Domitilla Ingrid Groth,1 Peter Schumann,2 Barbara Schu¨tze,1 Juan M. Gonzalez,3 Leonila Laiz,3 Cesareo Saiz-Jimenez3 and Erko Stackebrandt2 Correspondence 1Leibniz-Institut fu¨r Naturstoff-Forschung und Infektionsbiologie e. V., Hans-Kno¨ll-Institut, Ingrid Groth Beutenbergstrasse 11a, 07745 Jena, Germany [email protected] 2DSMZ–Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany 3Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Sevilla, Spain In order to clarify the taxonomic position of an actinobacterium from the Roman catacomb of Domitilla, a combination of phenotypic characterization, phylogenetic analysis based on the 16S rRNA gene sequence and DNA–DNA relatedness studies was used. The results from the polyphasic taxonomic study of this organism showed that strain HKI 0342T (=DSM 16849T=NCIMB 14033T) should be considered as the type strain of a novel species of the genus Isoptericola, for which the name Isoptericola hypogeus sp. nov. is proposed. The genus Isoptericola has been proposed by Stackebrandt oxygen requirements) and antibiotic-susceptibility tests et al. (2004) for the misclassified species Cellulosimicro- were performed, using solid or liquid organic medium 79 bium variabile Bakalidou et al. 2002 and is currently based (Prauser & Falta, 1968) and an incubation temperature of on this single species. The type strain, which is the only 28 uC. Cell morphology and cell dimensions were examined representative of Isoptericola variabilis at the time of writing, by using phase-contrast microscopy with a Zeiss Axioscope was isolated from the hindgut of the Australian termite 2 microscope equipped with image-analysing software Mastotermes darwiniensis (Frogatt). I. variabilis is phylo- (Axio Vision 2.05). The colony morphology of 2–10-day- genetically closely related to members of the genera Xyl- old cultures was studied using a stereo microscope animonas (Rivas et al., 2003), Xylanibacterium (Rivas et al., (Olympus). Standard physiological tests were carried out 2004) and Xylanimicrobium (Stackebrandt & Schumann, according to the methods described by Cowan & Steel 2004). The latter genus has been recently established to (1965), Gordon et al. (1974), Lanyi (1987) and Smibert & harbour the misclassified species Promicromonospora Krieg (1994). Acid production from carbon sources and pachnodae (Cazemier et al., 2003, 2004), the type strain of enzyme activities were studied using the API 50 CHB/E kit which was isolated from the hindgut of larvae of the scarab (incubation times of up to 7 days) and API ZYM galleries beetle Pachnoda marginata. Together, these four genera (bioMe´rieux). Additionally, the utilization of carbon constitute a phylogenetically distinct cluster within the sources was tested using Biolog GP2 MicroPlates and suborder Micrococcineae, and have cellulolytic and xylano- MicroLog computer software (Biolog Identification Sys- lytic activities in common. tem). Xylanolytic activity was determined on medium II, described by Cazemier et al. (2003), and incubation times of T Strain HKI 0342 was isolated from a sample of tufa up to 28 days. Susceptibility to antibiotics was examined by collected in the burial chamber of the first arcosolium placing antibiotic discs (Difco) on agar plates that were behind the entrance to the Roman catacomb of Domitilla seeded with suspensions of the test strains grown in a soft (Rome, Italy); PY-BHI agar (Yokota et al., 1993) and a agar layer for 24 h at 28 uC. Oxygen requirements were standard dilution-plate procedure were used. General studied with the GENbag microaer and GENbag anaer laboratory cultivation, morphological studies, determina- incubation systems (bioMe´rieux). The pH range for growth tion of optimal growth parameters (temperature, pH, was established by using liquid medium adjusted to pH values between 4 and 11 with either 1 M HCl or 20 % (w/v) Published online ahead of print on 15 April 2005 as DOI 10.1099/ Na2CO3 solution and incubated at 28 uC for up to 10 days. ijs.0.63632-0. The reference strains used for comparisons in physiological The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene tests and DNA–DNA pairing studies were I. variabilis sequence of strain HKI 0342T is AJ854061. DSM 10177T, Xylanimicrobium pachnodae DSM 12657T, T Detailed physiological characteristics of strain HKI 0342T are available Xylanimonas cellulosilytica DSM 15894 and Myceligene- T as supplementary material in IJSEM Online. rans xiligouense DSM 15700 . Biomass for chemotaxonomic 63632 G 2005 IUMS Printed in Great Britain 1715 I. Groth and others Table 1. Differential physiological characteristics of strain Table 1. cont. HKI 0342T and I. variabilis DSM 10177T Characteristic HKI DSM Characteristic HKI DSM 0342T 10177T 0342T 10177T 29-Deoxyadenosine 2 + Decomposition of: Inosine 2 + Urea 2 + Thymidine 2 + Utilization of: Adenosine 59-monophosphate 2 + Aconitate 2 + Citrate 2 + *Delayed reaction. Malate +* 2 DData from Cui et al. (2004). Growth characteristics GENbag anaer 2 +D Growth at 10 uC + 2 and molecular systematic studies was prepared by growing Growth at 42 uC 2 + the strain in shake flasks containing liquid organic medium Growth in the presence of 10?0 % NaCl 2 + 79 or Bacto tryptic soy broth (Sigma–Aldrich) for 24–48 h. + Susceptibility to streptomycin (10 mg) 2 Stock cultures of strain HKI 0342T in liquid organic medium Enzyme assay (API ZYM) 79 supplemented with 5 % DMSO were maintained in either + a-Galactosidase 2 the vapour phase of liquid nitrogen or at 280 uC by adding API 50 CH/BE a 1 : 1 mixture of glycerol and a medium that consisted of + Methyl b-xyloside 2 K HPO (1?26 %), KH PO (0?36 %), MgSO (0?01 %), + 2 4 2 4 4 Methyl a-D-glucoside 2 sodium citrate (0?09 %), (NH ) SO (0?18 %) and glycerol + 4 2 4 Melibiose 2 (8?8 %). Melezitose 2 + T D-Raffinose 2 + On medium 79, strain HKI 0342 formed wrinkled, circular, Biolog GP2 MicroPlate (24 h incubation) smooth, pale-yellow colonies about 1–4 mm in diameter. In Glycogen 2 + young cultures (8–24 h), a well-developed primary myce- N-Acetyl-D-mannosamine 2 + lium (width 0?6–0?7 mm) was observed which fragmented, Amygdalin 2 + in the stationary growth phase (48–72 h), into irregular, L-Fucose 2 + non-motile, short rods and cocci (diameter 0?8–1?0 mm). In Gentiobiose 2 + contrast to stationary-phase cultures of I. variabilis, both D-Gluconic acid 2 + cell types were arranged mainly as short flexible chains or as a-D-Lactose 2 + clusters. Furthermore, strain HKI 0342T and I. variabilis Lactulose 2 + DSM 10177T exhibited different growth characteristics D-Mannitol 2 + (Table 1). The catacomb isolate was an aerobic to micro- D-Melezitose 2 + aerophilic organism and did not grow in an anaerobic D-Melibiose 2 + atmosphere, unlike I. variabilis DSM 10177T, which is a Methyl a-D-galactoside 2 + facultatively anaerobic organism (Bakalidou et al., 2002). Methyl b-D-galactoside 2 + I. variabilis grew at temperatures up to 45 uC, but not at Methyl a-D-glucoside 2 + 10 uC, while strain HKI 0342T did not grow above 40 uC, but Methyl b-D-glucoside 2 + grew well at 10 uC. Both strains had in common the ability Methyl a-D-mannoside 2 + to hydrolyse xylan. However, they could be readily distin- Palatinose 2 + guished from one another by means of a broad spectrum D-Raffinose 2 + of physiological properties. Clearly differential characteris- L-Rhamnose 2 + tics of the two strains under study are listed in Table 1. D-Sorbitol 2 + Full detailed results (classical physiological tests, API kits Stachyose 2 + and Biolog test) are provided as supplementary tables in Sucrose 2 + IJSEM Online. D-Trehalose 2 + Turanose 2 + For sequence analysis of the 16S rRNA gene, bacterial Acetic acid 2 + DNA was extracted according to the method described by c-Hydroxybutyric acid + 2 Marmur (1961). The 16S rRNA gene was amplified by a PCR L-Lactic acid 2 + using the conserved primers 27F (59-AGAGTTTGATCC- Monomethyl succinate 2 + TGGCTCAG-39) and 1522R (59-AAGGAGGTGATCCAG- Propionic acid 2 + CCGCA-39). PCR thermal conditions were as follows: 95 uC L-Glutamic acid 2 + for 1 min; 35 cycles of 95 uC for 15 s, 55 uC for 15 s and 72 uC L-Serine 2 + for 2 min; and a final extension cycle at 72 uC for 10 min. Forward and reverse strands of the amplified DNA fragment 1716 International Journal of Systematic and Evolutionary Microbiology 55 Isoptericola hypogeus sp. nov. were sequenced in an ABI 3700 sequencer (Applied (1987), Groth et al. (1996) and Frank et al. (1980); the acyl Biosystems). The 16S rRNA gene sequences of the strains type of the muramic acid, as described by Uchida et al. studied were aligned manually with nucleotide sequences (1999); the whole-cell sugars present, as described by Becker obtained from the GenBank and EMBL databases. The et al. (1965) and Saddler et al. (1991); the menaquinones algorithm of Jukes & Cantor (1969) was applied in order to present, as described by Groth et al. (1996); the polar lipids transform sequence dissimilarities into evolutionary dis- present, as described by Minnikin et al. (1979) and Collins & tances. Phylogenetic dendrograms were reconstructed by Jones (1980); the mycolic acids present, as described by using the method of De Soete (1983) and the neighbour- Minnikin et al. (1975); and the fatty acid profile (MIDI joining method (Felsenstein, 1993). system; Agilent). The binary 16S rRNA gene sequence similarity values The chemotaxonomic characteristics of strain HKI 0342T between strain HKI 0342T and its nearest phylogenetic were most similar to those of I.
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