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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
4 VTT Biotechnology, PO Box 1500, FI-02044 VTT, Finland
Correspondence
Ingrid Groth
Ingrid.Groth{at}hki-jena.de
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, L-lys–L-thr–D-Glu; whole-cell sugars (glucose, mannose and galactose); octa-, hexa- and tetrahydrogenated menaquinones with nine isoprene units; phosphatidylglycerol and diphosphatidylglycerol as the major phospholipids; anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0 as the predominant fatty acids; and a DNA G+C content of 72 mol%. These features are consistent with affiliation of these isolates to the genus Myceligenerans. The three isolates shared a 16S rRNA gene similarity of 99·9 % and were most closely related to Myceligenerans xiligouense DSM 15700T (97·9 % sequence similarity). The low level of DNA–DNA relatedness (about 14 %) and the differences in phenotypic characteristics between the novel strains and M. xiligouense DSM 15700T justify the proposal of a novel species of the genus Myceligenerans, Myceligenerans crystallogenes sp. nov., with CD12E2-27T (=HKI 0369T=DSM 17134T=NCIMB 14061T=VTT E-032285T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CD12E2-27T (=HKI 0369T) is AY928181.
Tables showing the physiological test results for the strains in this study are available as supplementary material in IJSEM Online.
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for a single strain that was isolated from an alkaline salt marsh soil in China. The type strain of the species Myceligenerans xiligouense, strain XLG9A10.2T (=DSM 15700T), forms a distinct phylogenetic line within the family Promicromonosporaceae, suborder Micrococcineae, order Actinomycetales, and shares 94·8–95·1 % 16S rRNA gene sequence similarity with representatives of the genus Promicromonospora Krasil'nikov et al. 1961 and 94·4–95·7 % similarity with strains of the genera Xylanimonas Rivas et al. 2003, Xylanibacterium Rivas et al. 2004 and Isoptericola Stackebrandt et al. 2004. M. xiligouense shares the ability to hydrolyse xylan with members of the last three genera and with Xylanimicrobium pachnodae (Stackebrandt & Schumann, 2004). From different sampling sites in the Roman catacombs of Domitilla and San Callisto, three morphologically similar actinobacteria were isolated that were able to degrade xylan and to produce crystals of iodinin (1,6-phenazinediol 5,10-dioxide). Preliminary phylogenetic data obtained from 16S rRNA gene sequence comparison indicated a close relationship with M. xiligouense (DSM 15700T). Therefore a polyphasic taxonomic study was carried out to establish the taxonomic position of these isolates.
Strain CD12E2-27T (=HKI 0369T) was isolated from a sample of tufa collected in the first arcosolium behind the entrance of the Roman catacomb of Domitilla using peptone/yeast extract/brain heart infusion agar (Yokota et al., 1993) and a standard dilution plate procedure. Strain CD12Tz-28 (=HKI 0371=VTT E-032288) was obtained from the stone surface of the burial chamber at the same place by touching the stone with a sterile cotton swab and suspending the adherent bacteria in 1 : 10-diluted organic medium 79 (Prauser & Falta, 1968). Aliquots of this suspension were spread on peptone/yeast extract/brain heart infusion agar plates and incubated at 28 °C for 10 days. Strain CSC13Tb-79 (=HKI 0372=VTT E-032289) was isolated from a fresco in the cubiculum of Oceano, catacomb of San Callisto, Rome, using the same procedure and humic acid agar (Hayakawa & Nonomura, 1987) for growth. (In the following text and in the tables and figures, only the HKI numbers are used for the isolates.)
General laboratory cultivation, morphological studies, determination of the optimal growth parameters (temperature, pH, oxygen requirements) and analysis of the susceptibility to antibiotics were performed using solid or liquid organic medium 79 and an incubation temperature of 28 °C. Cell morphology and cell dimensions were examined by phase-contrast microscopy using a Zeiss Axioscope 2 microscope equipped with image-analysing software (Axio Vision 2.05; Zeiss). The colony morphology of 2–10-day-old cultures was studied using an Olympus stereo microscope. 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 was studied using the API 50 CH system and API 50 CHB/E medium (bioMérieux) according to the manufacturer's instructions (incubation times of up to 7 days). API ZYM galleries (bioMérieux) were used to study enzymic activities. Additionally, the utilization of carbon sources was tested using Biolog GP2 MicroPlates and MicroLog computer software (Biolog Identification System). The cell density of the inoculum for the GP2 MicroPlates was adjusted to the range specified by the Biolog turbidity standard GP-COC/GP-ROD/GN-FAS (20 %T). Xylanolytic activity was determined on medium II, described by Cazemier et al. (2003), and using 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 then incubating the samples at 28 °C for up to 10 days. The reference strains used for comparisons in physiological tests and DNA–DNA pairing studies were Isoptericola variabilis DSM 10177T, Xylanimicrobium pachnodae DSM 12657T, Xylanimonas cellulosilytica DSM 15894T and M. xiligouense DSM 15700T. Biomass for chemotaxonomic and molecular systematic studies was prepared by growing the strains in shake flasks containing liquid organic medium 79 or Bacto tryptic soy broth (Sigma-Aldrich) for 24–48 h. Stock cultures of the three isolates, HKI 0369T, HKI 0371 and HKI 0372, in liquid organic medium 79 supplemented with 5 % DMSO were maintained either in the vapour phase of liquid nitrogen or at –80 °C as a 1 : 1 mixture of the liquid culture and a glycerol 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 %).
The colonies of the three isolates under study were white to cream in colour, circular, convex and smooth (with diameters of about 1 mm). Larger wrinkled colonies with diameters of 2–4 mm were also observed. The colonies were distinguished from those of M. xiligouense DSM 15700T by their white to cream colour, which never changed to yellow. Aerial mycelium was absent. The three organisms produced spore-like cells in the substrate mycelium after growth on solid organic medium 79: these cells were similar to those described for M. xiligouense by Cui et al. (2004). In liquid cultures, a well-developed primary mycelium was produced within 8–24 h (width 0·5–0·7 µm); this underwent fragmentation into irregular non-motile rods and cocci after about 48 h cultivation.
The morphological and physiological similarities of the three strains under study underline the genomic coherence of these isolates (see Supplementary Tables S1 and S2 available in IJSEM Online). Although the novel strains share numerous physiological characteristics with M. xiligouense DSM 15700T, they could be readily distinguished from the latter by means of the physiological characteristics listed in Table 1. Full details of the results of the physiological tests are available as Supplementary Tables S1 and S2 in IJSEM Online.
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for M. xiligouense DSM 15700T. However, in this study growth at 6 and 42 °C, at pH 5 and 10 and at a NaCl concentration of 10 % (w/v) was not observed for strain DSM 15700T or for the three isolates, as shown in Supplementary Table S1. Furthermore, it should be mentioned that differentiation between the isolates and M. xiligouense DSM 15700T using the API 50 CHB/E kit was not possible because of the generally weakly expressed metabolic activity in all strains. The results (data not shown) obtained at different incubation temperatures (28 and 35 °C) did not allow an unambiguous interpretation (exceptions were the utilization of aesculin and arbutin). Weak activities were also observed in the utilization of carbon sources provided in the Biolog GP2 MicroPlates. The low colour intensities in the wells and the mycelial growth of the strains under study contributed to the fact that numerous reactions had to be considered as variable.
For sequence analysis of the 16S rRNA gene, the genomic DNA was extracted by following the method described by Marmur (1961). PCR-mediated amplification of the 16S rRNA gene, sequencing of the amplified DNA fragment and phylogenetic analysis were carried out as described previously (Groth et al., 2005).
Phylogenetic analysis of the almost-complete 16S rRNA gene (1437 bases) revealed that strain HKI 0369T is a member of the family Promicromonosporaceae, showing between 93·0 and 97·9 % similarity with its members. The highest level of relatedness (97·9 %) was obtained with M. xiligouense DSM 15700T. A bootstrap value of 100 confirmed the isolated position of the two strains within the radiation of the Promicromonosporaceae (Fig. 1).
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. DNA–DNA hybridization was carried out as described by De Ley et al. (1970), incorporating 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).
Ribotyping was performed using the standard method of the automated ribotyping device RiboPrinter System (DuPont Qualicon) according to the manufacturer's instructions, as described by Bruce (1996). The restriction enzyme used was PvuII (DuPont Qualicon).
The three catacomb isolates shared a 16S rRNA gene similarity of 99·9 %. This close phylogenetic relationship was underlined by the results of the riboprint analysis (Fig. 2). Strains HKI 0369T and HKI 0372 showed identical riboprint patterns and were affiliated to the same ribogroup. In strain HKI 0371, an additional band occurred that was unique to this strain and which did not occur in the pattern of M. xiligouense DSM 15700T.
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The following chemotaxonomic characteristics were determined as described previously: the structure of the peptidoglycan (Schleifer & Kandler, 1972; Schleifer, 1985; MacKenzie, 1987; Groth et al., 1996); the muramic acid type (Uchida & Aida, 1984); the whole-cell sugars (Becker et al., 1965; Saddler et al., 1991); the menaquinones (Groth et al., 1996); the polar lipids (Minnikin et al., 1979; Collins & Jones, 1980); and the mycolic acids (Minnikin et al., 1975). The fatty acid profile was determined using the MIDI system (Agilent).
The chemotaxonomic characteristics of the catacomb isolates were consistent with their affiliation to the genus Myceligenerans. The peptidoglycan of strain HKI 0369T contained N-acetylated muramic acid and corresponded to type A4, L-lys–L-thr–D-Glu (A11·57 according to http://www.dsmz.de/species/murein.htm). This type has been found in members of the family Cellulomonadaceae and from members of the family Promicromonosporaceae only in the genus Myceligenerans. The organisms under study shared with M. xiligouense DSM 15700T the characteristic sugars analysed in whole-cell hydrolysates, the major phospholipids and the predominant fatty acids [anteiso-C15 : 0 (54·4 mol%), iso-C15 : 0 (18·5 mol%) and iso-C16 : 0 (16·5 mol%)]. The minor fatty acids were iso-C14 : 0 (4·8 mol%) and anteiso-C17 : 0 (3·7 mol%). The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, two unknown phospholipids and three unknown glycolipids. In contrast to the data given by Cui et al. (2004), phosphatidylinositol was not detected in preparations of M. xiligouense DSM 15700T or in those of the three isolates. The three strains under study could be differentiated from M. xiligouense by the patterns of their menaquinones. Strain HKI 0369T was characterized by the major menaquinones MK-9(H8), MK-9(H4), MK-9(H6) and MK-9(H2), with peak areas of 36 : 19 : 16 : 13, and the minor amounts of MK-8(H4), MK-9 and MK-8(H2) (peak areas 6 : 3 : 1), while for M. xiligouense DSM 15700T, the major menaquinones were MK-9(H4) and MK-9(H6).
The DNA G+C value for strain HKI 0369T, determined by HPLC according to Groth et al. (1996), was 72·3 mol%.
It is evident from the genotypic and phenotypic data that the three strains from the Roman catacombs merit recognition as a novel species in the genus Myceligenerans. The name proposed for this new taxon is Myceligenerans crystallogenes sp. nov.
Description of Myceligenerans crystallogenes sp. nov.
Myceligenerans crystallogenes [crys.tall.o.ge'nes. Gr. n. krustallos crystal; Gr. v. gennao produce; N.L. neut. adj. crystallogenes producing crystals of iodinin (1,6-phenazinediol 5,10-dioxide)].
Gram-positive, aerobic to microaerophilic actinomycete with a well-developed primary mycelium (diameter of hyphae 0·5–0·7 µm) that undergoes fragmentation into short, irregular, non-motile rods and cocci in the stationary growth phase. Aerial mycelium is absent. Spore-like cells occur in the substrate mycelium. Colonies on organic medium 79 are wrinkled, circular, smooth and white to cream (diameter about 1–4 mm). Grows between 10 and 40 °C (optimal growth is at 28 °C) and at pH values in the range pH 6–9. NaCl in the culture medium is well tolerated up to 5 %. Physiological characteristics (utilization of carbohydrates, enzymic activities and susceptibility to antibiotics) are listed in Supplementary Tables S1 and S2 in IJSEM Online. The peptidoglycan type is A4, L-lys–L-thr–D-Glu. The whole-cell sugars are glucose, mannose and galactose. The acyl type is acetyl. The menaquinones are MK-9(H8), MK-9(H4), MK-9(H6), MK-9(H2) and minor amounts of MK-8(H4), MK-9 and MK-8(H2). The predominant fatty acids are anteiso-C15 : 0, iso-C15 : 0 and iso-C16 : 0. The phospholipids are diphosphatidylglycerol, phosphatidylglycerol, two unknown phospholipids and three unknown glycolipids. Mycolic acids are absent. The DNA G+C content is 72·0–72·3 mol% (type strain, 72·3 mol%).
The type strain is CD12E2-27T (=HKI 0369T=DSM 17134T=NCIMB 14061T=VTT E-032285T), which was isolated from the catacomb of Domitilla in Rome, Italy.
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ACKNOWLEDGEMENTS |
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