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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, Inhoffenstraße 7b, 38124 Braunschweig, Germany
Correspondence
Ingrid Groth
Ingrid.Groth{at}hki-jena.de
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ABSTRACT |
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Present address: CLONDIAG Chip Technologies GmbH, Löbstedter Straße 103–105, 07749 Jena, Germany. 
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains HKI 0478T, HKI 0479 and HKI 0480 are EF126967, EF126968 and EF126969, respectively.
Cellular fatty acid compositions of strains HKI 0478T, HKI 0479, HKI 0480 and type strains of closely related Kribbella species are presented in a supplementary table available with the online version of this paper.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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emend. Sohn et al. 2003 is a member of the family Nocardioidaceae, which was proposed by Nesterenko et al. (1985, 1990) and the description of which was emended by Rainey et al. (in Stackebrandt et al., 1997). The genus Kribbella is represented by Gram-positive or Gram-variable, non-motile actinomycetes that form an extensively branched vegetative mycelium and aerial hyphae that fragment into short to elongated rod-like or coccoid elements. Strains of the 11 Kribbella species with validly published names share the following chemotaxonomic characteristics (Park et al., 1999; Sohn et al., 2003): the presence of LL-diaminopimelic acid in the peptidoglycan (wall chemotype I sensu Lechevalier & Lechevalier, 1970), fatty acids that consist mainly of anteiso- and iso- branched components, MK-9(H4) as the main menaquinone and polar lipids comprising phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol (phospholipid pattern III sensu Lechevalier et al., 1977). A combination of these chemotaxonomic markers serves to separate the strains of the genus Kribbella from members of the other genera within the family Nocardioidaceae.
Three filamentously growing actinomycetes, strains HKI 0478T, HKI 0479 and HKI 0480, were isolated from acidic and heavy-metal-containing rock surfaces in a small mining area behind the ‘Märchendom’, the third level of the Feengrotten medieval alum slate mine (Saalfeld, Thuringia, Germany). The rock surface was touched with a sterile cotton swab and the adhering bacteria were suspended in about 1 ml sterile distilled water. Aliquots of this suspension were spread over agar plates containing water agar supplemented with Bacto peptone (0.1 %) and yeast extract (0.02 %), mineral agar Gauze 1 (Gauze et al., 1983) or starch-casein agar (Küster & Williams, 1964). All of the media were supplemented with cycloheximide (50 µg ml–1). The agar plates were incubated at 28 °C for about 4 weeks. Pure cultures of the isolates were maintained on organic medium 79 (Prauser & Falta, 1968) and preserved at –80 °C as a mixture of well-growing cultures in organic medium 79 broth and 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 %). Stock cultures of the isolates in liquid organic medium 79 supplemented with 5 % DMSO were also maintained in the vapour phase of liquid nitrogen.
Bacterial growth for chemotaxonomic and molecular systematic investigations of the isolates was prepared by cultivating cells at 28 °C for 24–48 h in liquid organic medium 79 or Bacto tryptic soy broth (Sigma-Aldrich). For morphological and cultural studies, the strains were cultivated on ISP media 2, 3, 4 and 5 (Difco; Shirling & Gottlieb, 1966) and on minimal agar (Amoroso et al., 2000) at 28 °C for up to 21 days. Growth parameters were determined using organic medium 79. The pH range for growth was established in shake flasks of liquid medium that was adjusted to pH values between 4.5 and 10.0 with either 1 M HCl or 20 % (w/v) Na2CO3 solution after sterilization. The cultures were incubated at 28 °C for up to 6 days. Physiological tests, including the determination of enzyme activities and susceptibility to antibiotics, were carried out as described by Groth et al. (2003). The reference strains used for comparison were Kribbella jejuensis JCM 12204T, Kribbella solani DSM 17294T and Kribbella swartbergensis DSM 17345T.
The three isolates showed lichenous growth on all media tested. Colonies on organic medium 79 were wrinkled, pasty and cream to pale yellow in colour. The substrate mycelium was extensively branched. White aerial mycelium was produced in abundance on ISP medium 5 and less abundantly on ISP media 2, 3 and 4. No aerial mycelium was produced on organic medium 79. Substrate and aerial hyphae fragmented into irregular, elongated rod-shaped to coccoid elements. Diffusible pigments were not observed.
The morphological and physiological characteristics of the three strains under study were almost identical (see Table 1 and the species description). Although the novel strains shared numerous physiological properties with their most closely related phylogenetic neighbours K. jejuensis JCM 12204T, K. solani DSM 17294T and K. swartbergensis DSM 17345T, they could be readily distinguished from these organisms on the basis of the characteristics listed in Table 1.
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), the predominant whole-organism sugars (Becker et al., 1965; Schön & Groth, 2006), the fatty acids present (MIDI system; http://www.midi-inc.com/), the predominant menaquinones (Collins et al., 1977; Groth et al., 1996) and the polar lipids present (Minnikin et al., 1979; Collins & Jones, 1980).
The chemotaxonomic characteristics of the strains were consistent with an affiliation with the genus Kribbella. Whole-organism hydrolysates contained LL-diaminopimelic acid as the diagnostic diamino acid of the peptidoglycan and the sugars comprised glucose together with minor amounts of mannose and ribose (wall chemotype I sensu Lechevalier & Lechevalier, 1970). The predominant menaquinone was MK-9(H4) (91–93 %). The phospholipids were composed of phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and unknown phospho- and glycolipids, corresponding to phospholipid pattern III sensu Lechevalier et al. (1977). The fatty acid profiles of the three isolates were characterized by the presence of significant amounts of anteiso-C15 : 0 (35.9, 44.6 and 37.2 %), iso-C16 : 0 (17.6, 7.5 and 15.1 %), C16 : 0 9-methyl (8.7, 7.0 and 8.2 %) and iso-C15 : 0 (6.1, 9.7 and 7.4 %), together with minor amounts of iso-C14 : 0 (5.7, 2.7 and 6.1 %) and anteiso-C17 : 0 (5.4, 5.1 and 4.6 %). These profiles also served to distinguish the novel strains from their phylogenetic neighbours (see Supplementary Table S1 available with the online version of this paper).
Chromosomal DNA was extracted from the three isolates by using a slightly modified version of the method of Pospiech & Neumann (1995). Amplification of the 16S rRNA genes and subsequent purification and direct sequencing of the respective PCR products were performed as described by Carlsohn et al. (2007).
The 16S rRNA gene sequences were aligned with actinobacterial sequences from the database of DSMZ by using the ae2 editor (Maidak et al., 1997). Evolutionary distances were calculated by using the Jukes–Cantor method (Jukes & Cantor, 1969). Dendrograms were constructed by using the neighbour-joining and maximum-likelihood algorithms (Felsenstein, 1993) and by using the algorithm of De Soete (1983). Bootstrap analysis (500 resamplings) was used to evaluate the tree topology (Felsenstein, 1985).
Almost-complete 16S rRNA gene sequences (1429–1463 nt) were obtained from the three novel strains. These sequences were virtually identical (99.9–100 %) and showed between 98.8 and 98.9 % similarity to the corresponding genes of the closest relatives, namely the type strains of K. solani, K. swartbergensis and K. jejuensis. The levels of similarity with the 16S rRNA genes of the other Kribbella species were lower, ranging between 97.7 and 98.7 %. The topologies of both the maximum-likelihood and distance-matrix dendrograms (Fig. 1) were identical in that the three isolates and the three closest relatives in the genus Kribbella formed an individual clade. The topologies differed in that the positions of the type strains of K. jejuensis and K. swartbergensis were transposed. Most of the bootstrap percentages were below 70 %, indicating the low statistical significance of the branching points of the majority of the type strains.
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.
DNA–DNA hybridization was carried out between strain HKI 0478T and its closest relatives as described by De Ley et al. (1970) taking into account the modifications described by Huß et al. (1983), using a Cary 100 Bio UV/Vis spectrophotometer (Varian) equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with an in situ temperature probe. The low levels of DNA–DNA relatedness (in 2x SSC plus 12 % formamide at 70 °C) between strain HKI 0478T and K. jejuensis JCM 12204T (46.9 and 35.7 %), K. solani DSM 17294T (13.0 and 15.3 %) and K. swartbergensis DSM 17345T (20.7 and 16.4 %) clearly indicate that the novel organism cannot be affiliated with any of these species.
The genome structure of the three isolates and their closest phylogenetic neighbours was examined using universally primed PCRs with primers AS4/AA2M2 (Bulat et al., 2000). The set-up and protocol were as described previously (Carlsohn et al., 2007). The resultant fingerprints were analysed using GelCompar II software (version 4.5; Applied Maths). The banding patterns were aligned using the pGEM DNA marker (Promega) as the reference system. The corresponding bands were automatically matched (optimization value, 0.3 %; position tolerance value, 0.18 %); the resultant matching was examined and then refined manually. Phylogenetic clustering was obtained using the built-in maximum-parsimony tree-making algorithm (Fitch, 1971). The resulting most parsimonious tree (Fig. 2) was artificially rooted; the topology was evaluated by using bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. It is clearly shown in Fig. 2 that the isolates shared almost identical fingerprint patterns from universally primed PCRs, distinguishing them from the corresponding patterns of their phylogenetic neighbours.
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Description of Kribbella aluminosa sp. nov.
Kribbella aluminosa (a.lu.mi.no'sa. L. fem. adj. aluminosa aluminous, full of alum, alum-containing, referring to the source of isolation of the first strains).
Gram-positive, aerobic, non-motile actinomycete that produces an extensively branched, beige to pale yellow substrate mycelium and a white aerial mycelium. Substrate and aerial hyphae fragment into irregular rod-shaped elements. Colonies on organic medium 79 are wrinkled and of a pasty consistency. Diffusible pigments are not produced. Grows between 20 and 37 °C and between pH 5 and 8. Tolerates 2 % NaCl in the culture medium. Grows on minimal medium in the presence of NiCl2 (5 mM) and CuSO4 (0.5 mM). No growth occurs at 6 or 42 °C, at pH 9.5 or in the presence of 4 % NaCl. Catalase-positive and oxidase-negative. Aesculin and hippurate are hydrolysed. H2S is produced, but indole is not. Acetate, aconitate (delayed), citrate, malate, succinate, L-arabinose, D-fructose, D-glucose, D-raffinose, L-rhamnose, sucrose and D-xylose are utilized as sole carbon sources, but benzoate, DL-tartrate and cellulose are not. Produces leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), 
-galactosidase, 
-galactosidase, N-acetyl--glucosaminidase, -glucosidase, -glucosidase, -mannosidase, naphthol-AS-BI-phosphohydrolase, acid phosphatase and alkaline phosphatase, but not -chymotrypsin (type strain, weak) or -glucuronidase (API ZYM tests). Susceptible to the following antibiotics (amounts per disc shown in parentheses): chloramphenicol (30 µg), ciprofloxacin (5 µg, weakly), novobiocin (5 µg, weakly), ofloxacin (10 µg, weakly), oxytetracycline hydrochloride (30 µg) and polymyxin B (300 IU, weakly). Resistant to the following antibiotics (amounts per disc shown in parentheses): lincomycin hydrochloride (2 µg), meticillin (5 µg), nalidixic acid (30 µg), norfloxacin (10 µg) and penicillin G (10 IU). Additional phenotypic properties are shown in Table 1
. Chemotaxonomic characteristics are typical of those for Kribbella species.
The type strain, HKI 0478T (=DSM 18824T =JCM 14599T), was isolated from a medieval alum slate mine in Thuringia, Germany. Strains HKI 0479 and HKI 0480 are also members of this species.
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ACKNOWLEDGEMENTS |
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