Actinokineospora Spheciospongiae Sp. Nov., Isolated from the Marine Sponge Spheciospongia Vagabunda

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International Journal of Systematic and Evolutionary Microbiology (2015), 65, 879–884 DOI 10.1099/ijs.0.000031

Actinokineospora spheciospongiae sp. nov., isolated from the marine sponge Spheciospongia vagabunda

Peter Ka¨mpfer,1 Stefanie P. Glaeser,1 Hans-Ju¨rgen Busse,2 Usama Ramadan Abdelmohsen,33 Safwat Ahmed4 and Ute Hentschel3

Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Peter Ka¨mpfer Germany [email protected] 2Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, giessen.de A-1210 Wien, Austria 3Department of Botany II, Julius-von-Sachs-Institute for Biological Sciences, University of Wuerzburg, D-97082 Wuerzburg, Germany 4Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt

A Gram-staining-positive, aerobic organism, isolated from the Red Sea sponge Spheciospongia vagabunda was investigated to determine its taxonomic position. On the basis of results of 16S rRNA gene sequence analysis strain EG49T was most closely related to Actinokineospora cibodasensis and Actinokineospora baliensis (both 97.3 % similarity) and Actinokineospora diospyrosa and Actinokineospora auranticolor (both 97.0 % similarity). The 16S rRNA gene sequence similarity to all other species of the genus Actinokineospora was ,97.0 %. The quinone T system of strain EG49 contained the menaquinones MK-9(H4) (47 %), MK-9(H6) (27 %) and

MK-9(H2) (15 %) in major amounts. Minor amounts of MK-7(H4) (2 %), MK-9(H0) (1 %), MK-

9(H8) (3 %) and MK-10(H4) (3 %) were detected as well in addition to MK-8(H4), MK-8(H6), MK-

10(H2) and MK-10(H6) (all ,1 %). The diagnostic diamino acid of the peptidoglycan was meso- diaminopimelic acid. In the polar lipid profile, diphosphatidylglycerol, phosphatidylethanolamine and hydroxyphosphatidylethanolamine were predominant. Phosphatidylinositol-mannoside, two unidentified phospholipids and two glycoglipids as well as one aminoglycolipid, one aminolipid and one unidentified lipid were found in addition. The fatty acid profile was composed of mainly

iso-branched fatty acids: iso-C16 : 0, iso-C14 : 0, iso-C15 : 0 and iso-C16 : 1H. All these findings clearly supported the classification of the strain as representing a member of the genus Actinokineospora. In addition, the results of physiological and biochemical tests also allowed phenotypic differentiation of strain EG49T from the most closely related species of the genus Actinokineospora. Strain EG49T represents a novel species of the genus Actinokineospora, for which we propose the name Actinokineospora spheciospongiae sp. nov., with strain EG49T (5DSM 45935T5CCM 8480T5LMG 27700T) as the type strain.

The genus Actinokineospora was proposed by Hasegawa fastidiosa. The genus contains, at the time of writing, 13 (1988), initially for motile, arthrospore-producing organ- species: Actinokineospora riparia (the type species), Actino- isms of the class Actinomycetes. Labeda et al. (2010) kineospora inagensis, Actinokineospora globicatena, Actinokineo- recently emended the description of the genus, which now spora terrae, Actinokineospora diospyrosa, Actinokineospora harbours also species for which the production of motile auranticolor, Actinokineospora enzanensis, Actinokineospora fasti- spores was not observed, and proposed the transfer of diosa, Actinokineospora baliensis, Actinokineospora bangkokensis, Amycolatopsis fastidiosa to this genus as Actinokineospora Actinokineospora cianjurensis, Actinokineospora cibodasensis and Actinokineospora soli (Hasegawa, 1988; Tamura et al., 1995; 3Permanent address: Department of Pharmacognosy, Faculty of Otoguro et al., 2001; Labeda et al., 2010, Lisdiyanti et al., 2010; Pharmacy, Minia University, Minia 61519, Egypt Tang et al., 2012; Intra et al., 2013). These actinomycetes The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene are characterized by having meso-diaminopimelic acid as a T sequence of strain EG49 is GU318361. cell-wall diamino acid, MK-9(H4) as the predominant

000031 G 2015 IUMS Printed in Great Britain 879 P. Ka¨mpfer and others

menaquinone, phospholipid type II, iso-C16 : 0 fatty acid as the the SILVA Incremental Aligner (SINA; v1.2.11; Pruesse et al., predominant fatty acid and DNA G+C contents of 69– 2012) according to the SILVA seed alignment [http://www. 74 mol%. arb-silva.de; Pruesse et al. (2007)]. The aligned sequence was implemented into the LTP database and added to the Strain EG49T was isolated by Abdelmohsen et al. (2010) database tree using the ARB Parsimony (Quick add marked) from the marine sponge Spheciospongia vagabunda col- tool. The alignment, including all members of the family lected from the Red Sea (Ras Mohamed, Sinai, Egypt; GPS Pseudonocardiaceae and some outgroup species of the u 9 u 9 coordinates 27 47.655 N34 12.904 W). The metabolo- family Micromonosporaceae, was checked manually before mic and the genomic analyses of the strain showed its richness reconstruction of phylogenetic trees. A maximum-like- with diverse bioactive natural products. (Abdelmohsen et al., lihood tree was reconstructed using RAxML v7.04 2014; Harjes et al., 2014; Grkovic et al., 2014). (Stamatakis, 2006) with GTR-GAMMA and rapid boot- The strain showed the presence of aerial mycelium with strap analysis. The tree was based on 16S rRNA gene spore chains. The spores were rod-shaped and were formed sequence termini 73–1443 (Escherichia coli numbering; by fragmentation of the hyphae (arthrospores). Cultural Brosius et al., 1978). The phylogenetic tree showed a clear T characteristics were recorded after 14 days of incubation at allocation of strain EG49 , clustering among species of the 28 uC on International Streptomyces Project (ISP) 2 medium, genus Actinokineospora. Based on these results, further tryptone soy agar (TSA; Oxoid)) and nutrient agar (Oxoid). phylogenetic analyses were performed including all species Light yellow to brown colonies were produced on these of the genus Actinokineospora and species of the genus media. The isolate exhibited good growth on all of the media. Kutzneria as the outgroup. Again, a maximum-likelihood tree, a neighbour-joining tree using ARB neighbour-joining Cells displayed Gram-positive staining (analysed as and the Jukes–Cantor correction (Jukes & Cantor, 1969) described by Gerhardt et al., 1994) and were negative for and a maximum-parsimony tree using DNAPARS v 3.6 cytochrome oxidase, determined by using an oxidase test (Felsenstein, 2005) were generated. The phylogenetic (Merck). Endospores could not be detected. Temperature- trees were reconstructed with 100 resamplings (bootstrap dependent growth was determined on ISP2 agar at 4, 15, analysis; Felsenstein, 1985) and based on 16S rRNA gene 25, 28, 32, 37 and 42 uC. Salinity- and pH-dependent sequence termini 62–1459 (E. coli numbering, Brosius et al., growth were analysed in ISP2 broth either supplemented 1978). Pairwise sequence similarities among the type strains with 1–10 % (w/v) NaCl or adjusted to pH values between of species of the genus Actinokineospora were calculated pH 4 and 12 (at intervals of 0.5 pH units, adjusted by the using the ARB neighbour-joining tool without the use of an addition of HCl or NaOH) and cultured at 28 uC. evolutionary substitution model. Detailed phylogenetic analysis based on 16S rRNA gene The sequenced 16S rRNA gene of strain EG49T represents sequences was performed in ARB release 5.2 (Ludwig et al., a continuous stretch of 1481 nt spanning E. coli positions 2004) using the ‘All-Species Living Tree’ Project (LTP) 9–1514 (E. coli numbering; Brosius et al., 1978). Strain (Yarza et al., 2008) database LTPs115 (March, 2014). The EG49T shared highest 16S rRNA gene sequence similarity 16S rRNA gene sequence of strain EG49T was aligned with with the type strains of Actinokineospora cibodasensis and

95 Actinokineospora enzanensis IFO 16517T (AB058395) ** Actinokineospora bangkokensis 44EHWT (JQ922512) Actinokineospora riparia NRRL B–16432T (AF114802) 100 Actinokineospora soli YIM 75948T (JN005785) ** Actinokineospora fastidiosa NRRL B–16697T (GQ200601) Actinokineospora cibodasensis NBRC 104212T (AB447489) * Actinokineospora spheciospongiae EG49T (GU318361) Actinokineospora diospyrosa NRRL B–24047T (AF114797) Actinokineospora auranticolor IFO 16518T (AB058396) Actinokineospora baliensis NBRC 104211T (AB447488) Actinokineospora cianjurensis BTCC B–558T (AB473945) Actinokineospora globicatena NRRL B-24048T (AF114798) 100 Actinokineospora terrae IFO 15668T (AB058394) Actinokineospora inagensis NRRL B–24050T (AF114799) 100 3 Kutzneria 0.10

Fig. 1. Maximum-likelihood tree showing the phylogenetic position of strain EG49T among type strains of species of the genus Actinokineospora. The tree was generated in ARB using RAxML (GTR-GAMMA, Rapid Bootstrap analysis) and based on nucleotide sequences at 16S rRNA gene sequence positions 62–1459 (E. coli numbering). Type strains of species of the genus Kutzneria were used as the outgroup. Bootstrap values ¢70 % are given in the tree. Nodes marked with asterisks also occurred in the maximum-parsimony and neighbour-joining trees; those nodes marked with two asterisks also showed bootstrap values .70 %. Bar, 0.1 substitutions per nucleotide position.

880 International Journal of Systematic and Evolutionary Microbiology 65 Actinokineospora spheciospongiae sp. nov.

Actinokineospora baliensis (both 97.3 %) and Actinokineospora from casein, 0.3 % yeast extract, pH 7.2) at 28 uC. diospyrosa and Actinokineospora auranticolor (both 97.1 %). Biomasses used for extraction of diamino acids, quinones The 16S rRNA gene sequence similarities to all other type and polar lipids were harvested at the stationary growth strains of species of the genus Actinokineospora were phase. Diamino acid extraction was carried out according below 97.0 %. Results of phylogenetic analysis showed that to the protocol of Schumann (2011). Quinones and polar strain EG49T clustered within the genus Actinokineospora lipids were extracted and analysed as described by Tindall with the type strain of Actinokineospora cibodasensis. The (1990a, b) and Altenburger et al. (1996). The HPLC clustering was obtained with all applied treeing methods, but apparatus used was described by Stolz et al. (2007). The was not supported by high bootstrap values, indicating that diagnostic diamino acid of the peptidoglycan was meso- T the direct phylogenetic relationship of the novel strain and diaminopimelic acid. Strain EG49 showed a complex the species of the genus Actinokineospora cannot be resolved quinone system which contained 47 % MK-9(H4), 27 % at the species level solely on the basis of 16S rRNA gene MK-9(H6) and 15 % MK-9(H2). Minor amounts of MK- sequence analysis (Fig. 1). Because of the low 16S rRNA 7(H4) (2 %), MK-9(H0) (1 %), MK-9(H8) (3 %) and MK- gene sequence similarities to all species of the genus 10(H4) (3 %) were detected as well in addition to MK-8(H4), Actinokineospora (,97.3 %), DNA–DNA hybridizations MK-8(H6), MK-10(H2) and MK-10(H6) (all ,1 %). were not performed. In none of the previously published The polar lipid profile (Fig. 2) consisted of the major studies, DNA–DNA pairing values .50 % could be found lipids diphosphatidylglycerol, phosphatidylethanolamine for pairs of strains showing ,98 % 16S rRNA gene sequence and hydroxyphosphatidylethanolamine. Phosphatidylinosi- similarity (Tamura et al., 1995; Lisdiyanti et al., 2010; Tang tol mannoside, two unidentified phospholipids and two et al., 2012; Intra et al., 2013). Furthermore, Meier-Kolthoff glycoglipids as well as one aminoglycolipid, one aminolipid et al. (2013) showed clearly for the class Actinobacteria that and one unidentified lipid were found in addition. at a threshold of 97.6 % 16S rRNA gene sequence similarity between a pair of strains, the maximum probability of an The meso-diaminopimpelic acid detected in the peptido- error that these strains represent the same species is only glycan and the quinone system consisting predominantly of 0.01 %. menaquinone MK-9(H4) is in agreement with the description of the genus Actinokineospora. The polar lipid profile Biomass subjected to analyses of diamino acids, quinones and polar lipids was grown in PYE broth (0.3 % peptone Table 1. Major fatty acid composition of strain EG49T and the type strains of the most closely related species

PL1 Fatty acid 1234 5 6

PE iso-C14 : 0 8.7 5.5 11.3 12.2 12.6 1.6 iso-C14 : 0 3-OH 3.4 3.9 C15 : 1v6c 3.1 2.0 OH-PE iso-C15 : 0 7.8 25.2 13.8 13.7 19.7 18.2 PL2 anteiso-C15 : 0 4.9 2.2 2.0 2.7 1.4 C15 : 0 4.5 4.4 3.3 3.3 iso-C H 10.7 8.3 4.2 3.1 5.8 2.7 AL1 16 : 1 C16 : 1 2-OH 1.4 GL1 iso-C16 : 0 59.3 39.7 47.5 48.8 35.8 24.9 GL2 C16 : 0 1.5 1.7 3.1 AGL1 PIM Summed feature 3* 6.9 3.5 1.7 4.9 7.2

iso-C17 : 1v9c 1.2 C17 : 1v6c 6.4 2.9 2.1 4.5 T Fig. 2. Polar lipid profile of strain EG49 after two-dimensional C17 : 1v8c 3.3 10.2 TLC and detection with molybdatophosphoric acid. DPG, iso-C17 : 0 8.1 1.7 2.0 6.2 diphosphatidylglycerol; OH-PE, hydroxyphosphatidylathanolamine; anteiso-C17 : 0 8.3 1.8 2.2 9.7 PE, phosphatidylethanolamine; PIM, phosphatidylinositol mannoside; C17 : 0 cyclo 6.6 GL1, GL2, unidentified glycolipids; AL1, unidentified aminolipid; C17 : 0 1.7 3.0 3.7 AGL1, unidentified amnioglycolipid; PL1, PL2, unidentified phospholipids; L1, unidentified polar lipid. *Summed feature 3 comprises C16 : 1v7c and/or iso-C15 : 0 2-OH. http://ijs.sgmjournals.org 881 P. Ka¨mpfer and others

Table 2. Differential phenotypic characteristics of EG49T and the type strains of the closest related species

Strains: 1, EG49T;2,Actinokineospora diospyrosa NRRL B-24047T;3,Actinokineospora riparia NRRL B-16432T;4,Actinokineospora auranticolor CIP 107896T;5,Actinokineospora baliensis NBRC 104211T;6,Actinokineospora cibodasensis NBRC 104212T;7,Alloactinosynnema album CCM 7461T. All data are from this study. +, Positive; 2, negative; (+), weakly positive. All strains were negative for acid production from glucose, lactose, sucrose, D-mannitol, dulcitol, salicin, adonitol, myo-inositol, L-arabinose, raffinose, rhamnose, maltose, D-xylose, trehalose, cellobiose, methyl D-glucoside, erythritol, melibiose, D-arabitol and D-mannose. None of the strains hydrolysed para-nitrophenyl (pNP)-b-D-glucuronide and pNP-b-D- xylopyranoside and none of the strains assimilated L-arabinose, p-arbutin, a-melibiose, salicin, adonitol, myo-inositol, D-sorbitol, putrescine, trans- aconitate, 4-aminobutyrate, citrate, itaconate, mesaconate, 3-hydroxybenzoate or 4-hydroxybenzoate as a sole source of carbon. All strains were positive for hydrolysis of L-alanine-pNA and assimilation of N-acetyl-D-glucosamine, D-glucose and oxoglutarate as sole sources of carbon.

Characteristic 1 2 3 4 5 6 7

Hydrolysis of: Aesculin + 2 (+)(+)(+) ++ oNP-b-D-galactopyranoside 222222+ pNP-a-D-glucopyranoside +++2 +++ pNP-b-D-glucopyranoside + 2 (+) 22+ (+) Bis-pNP-phosphate 2 ++++++ pNP-phenylphosphonate 2 (+) +++++ pNP-phosphorylcholine 22+ 222+ 2-Deoxythymidine-59-thymidine-pNP-phosphate +++2 +++ L-Glutamate-c-3-carboxy-para-nitroanilide + (+)(+) 2 +++ L-Proline-pNA ++2 ++++ Assimilation of: N-Acetyl-D-galactosamine 222222+ Cellobiose + 22222+ D-Fructose + (+) 222++ D-Galactose 222222+ Gluconate ++++++2 D-Glucose (+) ++++++ Maltose + (+) 2222+ D-Mannose + (+) 222++ L-Rhamnose (+) 222222 D-Ribose 22222+ 2 Sucrose ++222(+) + Salicin 2222222 Trehalose ++(+) + 22+ D-Xylose + 222222 Maltitol (+) 222222 D-Mannitol + 22222+ Acetate + (+)(+) 2 +++ Propionate + 222++(+) cis-Aconitate 222222+ Adipate (+) 222222 Azelate (+) 22222+ Fumarate (+) 2 (+) + 2 ++ Glutarate (+) 2 (+) 2222 DL-3-Hydroxybutyrate 2222(+) 22 DL-Lactate 22222(+) 2 L-Malate (+) 22+ 2 ++ Pyruvate (+) 22+ 2 ++ Suberate + (+) 2222+ L-Alanine ++2 + (+) ++ b-Alanine 222(+)(+) 22 L-Aspartate (+) + (+)(+) + 2 + L-Histidine ++2 (+) +++ L-Leucine (+) 222(+) ++ L-Ornithine + (+) 22(+) ++ L-Phenylalanine (+) 22(+) +++

882 International Journal of Systematic and Evolutionary Microbiology 65 Actinokineospora spheciospongiae sp. nov.

Table 2. cont.

Characteristic 1 2 34567

L-Proline (+) + 2 ++++ L-Serine (+) + 2 + (+) ++ L-Tryptophan (+)(+) 222+ (+) Phenylacetate (+) 222222

T of strain EG49 showed some similarities to those of other salicin, D-adonitol, myo-inositol, D-sorbitol, L-arabinose, species of the genus Actinokineospora with respect to the raffinose, L-rhamnose, maltose, trehalose, cellobiose, ery- presence of phospholipids, but showed also some differences. thritol, melibiose or D-arabitol. Several sugar compounds are utilized including N-acetyl-D-glucosamine, D-fructose, Fatty acids analysis of cells, grown in tryptone soy broth cellobiose, D-gluconate, D-glucose, D-mannose, maltose, D- (TSB) at 28 uC, was done as described by Ka¨mpfer & maltitol (weakly), D-mannitol, L-rhamnose (weakly), sucrose, Kroppenstedt (1996) using the Sherlock Microbial Identi- trehalose and D-xylose. L-Arabinose, arbutin, D-galactose, D- fication System (Sherlock software version 2.11 and a TSBA adonitol, myo-inositol, melibiose, ribose, D-sorbitol and peak naming table version 4.1; MIDI). salicin are not utilized. Major fatty acids are iso-C16 : 0,iso- The fatty acid profile comprised mainly iso-branched fatty C14 : 0,iso-C15 : 0 and iso-C16 : 1 H. The diagnostic diamino acid acids and was similar to those of the most closely related of the peptidoglycan is meso-diaminopimelic acid. Major species (Table 1). polar lipids are diphosphatidylglycerol, phosphatidylethanolamine and hydroxyphosphatidylethanolamine. Phosphatidy- The results of the physiological characterization, performed linositol mannoside, two unidentified phospholipids and two using methods described previously (Ka¨mpfer et al., 1991), glycoglipids as well as one aminoglycolipid, one aminolipid are given in Table 2 and in the species description. Strain and one unidentified lipid are detected in addition. Major EG49T was able to utilize several sugars or sugar-related quinones are MK-9(H4), MK-9(H6)andMK-9(H2). Minor compounds. A distinct physiological and biochemical amounts of MK-7(H ), MK-9(H ), MK-9(H ) and MK- profile allowed differentiation of the strain from the 4 0 8 10(H4) are detected as well in addition to MK-8(H4), MK- type strains of the most closely related species of the 8(H ), MK-10(H ) and MK-10(H ). genus Actinokineospora. Based on the low 16S rRNA gene 6 2 6 sequence similarities (,97.3 %) to all other species of the The type strain, EG49T (5DSM 45935T5CCM 8480T5 genus with validly published names, DNA–DNA hybridi- LMG 27700T), was isolated on ISP 2 medium from the Red zations were not performed. From the results of the Sea sponge Spheciospongia vagabunda. phylogenetic and chemotaxonomic analyses, it is obvious that strain EG49T represents a novel species, which is allocated to the genus Actinokineospora. For this species we References propose the name Actinokineospora spheciospongiae sp. nov. Abdelmohsen, U. R., Cheng, C., Viegelmann, C., Zhang, T., Grkovic, T., Quinn, R. J., Safwat A., Hentschel, U. & Edrada-Ebel, R. (2014). Description of Actinokineospora Dereplication strategies for targeted isolation of new anti-trypanosomal spheciospongiae sp. nov. actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49. Mar Drugs 12, 1220–1244. Actinokineospora spheciospongiae (sphe.ci.o.spon9gi.ae N.L. Abdelmohsen, U. R., Pimentel-Elardo, S. M., Hanora, A., Radwan, M., gen. n. spheciospongia of/from Spheciospongia the zoological Abou-El-Ela, S. H., Ahmed, S. & Hentschel, U. (2010). Isolation, name of a genus of sponge, referring to the isolation of the phylogenetic analysis and anti-infective activity screening of marine type strain from the sponge Spheciospongia vagabunda). sponge-associated actinomycetes. Mar Drugs 8, 399–412. Vegetative mycelium is yellow to tan. When formed, aerial Altenburger, P., Ka¨ mpfer, P., Makristathis, A., Lubitz, W. & Busse, H.-J. (1996). Classification of bacteria isolated from a medieval wall mycelium is white. Aerial mycelium produces rod-shaped painting. J Biotechnol 47, 39–52. arthrospores (diameter 1.5–1.8 mm). Motility of spores is Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978). u not observed. Good growth is observed at 25–28 C. Grows Complete nucleotide sequence of a 16S ribosomal RNA gene from well on ISP media 2 and 3 and on tryptone soy agar and Escherichia coli. Proc Natl Acad Sci U S A 75, 4801–4805. u nutrient agar. Optimal temperature for growth is 28 C; Felsenstein, J. (1985). Confidence limits of phylogenies: an approach growth occurs at 20–36 uC but not at 15 uC and below or using the bootstrap. Evolution 39, 783–791. 40 uC and above on ISP2 agar. Optimal pH for growth is Felsenstein, J. (2005). PHYLIP (Phylogeny Inference Package) version pH 7.0; growth occurs at pH 5.5–10.5. Growth is observed 3.6. Distributed by the author. Department of Genome Sciences, with a NaCl concentration from 1 % (w/v) up to 5 % (w/v), University of Washington, Seattle. but not above. Test for catalase is positive; oxidase activity Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) is weakly positive. No acid formation can be observed from (1994). Methods for General and Molecular Bacteriology. Washington, D-glucose, D-xylose, lactose, sucrose, D-mannitol, dulcitol, DC: American Society for Microbiology. http://ijs.sgmjournals.org 883 P. Ka¨mpfer and others

Grkovic, T., Abdelmohsen, U. R., Othman, E. M., Stopper, H., Edrada- Otoguro, M., Hayakawa, M., Yamazaki, T., Tamura, T., Hatano, K. & Ebel, R., Hentschel, U. & Quinn R. J. (2014). Two new Antioxidant Iimura, Y. (2001). Numerical phenetic and phylogenetic analysis of actinosporin analogues from the calcium alginate beads culture of sponge- Actinokineospora isolates, with a description of Actinokineospora auranti- associated Actinokineospora sp. strain EG49. Bioorg Med Chem Lett 24, color sp. nov. and Actinokineospora enzaensis sp. nov. Actinomycetologica 5089–5092. 15,30–39. Harjes, J., Ryu, T., Abdelmohsen, U. R., Moitinho-Silva, L., Horn, H., Pruesse, E., Quast, C., Knittel, K., Fuchs, B. M., Ludwig, W., Peplies, Ravasi, T. & Hentschel, U. (2014). Draft genome sequence of the J. & Glo¨ ckner, F. O. (2007). SILVA: a comprehensive online resource antitrypanosomally active sponge associated-bacterium Actinokineospora for quality checked and aligned ribosomal RNA sequence data sp. strain EG49. Genome Announcments. Doi:10.1128/genomeA.00160-14. compatible with ARB. Nucleic Acids Res 35, 7188–7196. Hasegawa, T. (1988). Actinokineospora: a new genus of the Pruesse, E., Peplies, J. & Glo¨ ckner, F. O. (2012). SINA: accurate high- actinomycetales. Actinomycetologica 2, 31–45. throughput multiple sequence alignment of ribosomal RNA genes. Intra, B., Matsumoto, A., Inahashi, Y., Omura, S., Takahashi, Y. & Bioinformatics 28, 1823–1829. Panbangred, W. (2013). Actinokineospora bangkokensis sp. nov., isolated Schumann, P. (2011). Peptidoglycan structure. Methods Microbiol 38, from rhizospheric soil. Int J Syst Evol Microbiol 63, 2655–2660. 101–129. Jukes, T. H. & Cantor, C. R. (1969). Evolution of the protein Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by phylogenetic analyses with thousands of taxa and mixed models. H. N. Munro. New York: Academic Press. Bioinformatics 22, 2688–2690. Ka¨ mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of Stolz, A., Busse, H.-J. & Ka¨ mpfer, P. (2007). Pseudomonas fatty acid patterns of coryneform bacteria and related taxa. Can J knackmussii sp. nov. Int J Syst Evol Microbiol 57, 572–576. Microbiol 42, 989–1005. Tamura, T., Hayakawa, M., Nonomura, H., Yokota, A. & Hatano, K. Ka¨ mpfer, P., Steiof, M. & Dott, W. (1991). Microbiological (1995). Four new species of the genus Actinokineospora: Actinokineospora characterization of a fuel-oil contaminated site including numerical inagensis sp. nov., Actinokineospora globicatena sp. nov., Actinokineospora identification of heterotrophic water and soil bacteria. Microb Ecol 21, 227–251. terrae sp. nov. and Actinokineospora diospyrosa sp. nov. Int J Syst Evol Microbiol 45, 371–378. Labeda, D. P., Price, N. P., Tan, G. Y. A., Goodfellow, M. & Klenk, H.-P. (2010). Emended description of the genus Actinokineospora Hasegawa Tang, X., Zhou, Y., Zhang, J., Ming, H., Nie, G.-X., Yang, L.-L., Tang, 1988 and transfer of Amycolatopsis fastidiosa Henssen et al. 1987 as S.-K. & Li, W.-J. (2012). Actinokineospora soli sp. nov., a thermo- Actinokineospora fastidiosa comb. nov. Int J Syst Evol Microbiol 60, tolerant actinomycete isolated from soil, and emended description of 1444–1449. the genus Actinokineospora. Int J Syst Evol Microbiol 62, 1845– 1849. Lisdiyanti, P., Otoguro, M., Ratnakomala, S., Lestari, Y., Hastuti, R. D., Triana, E., Katsuhiko, A. & Widyastuti, Y. (2010). Actinokineospora Tindall, B. J. (1990a). Lipid composition of Halobacterium lacu- baliensis sp. nov., Actinokineospora cibodasensis sp. nov. and sprofundi. FEMS Microbiol Lett 66, 199–202. Actinokineospora cianjurensis sp. nov., isolated from soil and plant Tindall, B. J. (1990b). A comparative study of the lipid composition of litter. Int J Syst Evol Microbiol 60, 2331–2335. Halobacterium saccharovorum from various sources. Syst Appl Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Microbiol 13, 128–130. Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a software Yarza, P., Richter, M., Peplies, J., Euzeby, J., Amann, R., Schleifer, environment for sequence data. Nucleic Acids Res 32, 1363–1371. K. H., Ludwig, W., Glo¨ ckner, F. O. & Rossello´ -Mo´ ra, R. (2008). Meier-Kolthoff, J. P., Go¨ ker, M., Spro¨ er, C. & Klenk, H.-P. (2013). The All-Species Living Tree project: a 16S rRNA-based phylogenetic When should a DDH experiment be mandatory in microbial tree of all sequenced type strains. Syst Appl Microbiol 31, 241– taxonomy? Arch Microbiol 195, 413–418. 250.

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