Rubrobacter Aplysinae Sp. Nov., Isolated from the Marine Sponge Aplysina Aerophoba

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International Journal of Systematic and Evolutionary Microbiology (2014), 64, 705–709 DOI 10.1099/ijs.0.055152-0

Rubrobacter aplysinae sp. nov., isolated from the marine sponge Aplysina aerophoba

Peter Ka¨mpfer,1 Stefanie P. Glaeser,1 Hans-Ju¨rgen Busse,2 Usama Ramadan Abdelmohsen33 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, A-1210 Wien, giessen.de Austria 3Department of Botany II, Julius-von-Sachs-Institute for Biological Sciences, University of Wuerzburg, D-97082 Wuerzburg, Germany

A Gram-stain-positive, non-spore-forming bacterium (strain RV113T) was isolated from the marine sponge Aplysina aerophoba. 16S rRNA gene sequence analysis showed that strain RV113T belongs to the genus Rubrobacter, and is related most closely to Rubrobacter bracarensis VF70612_S1T (96.9 % similarity) and more distantly related (,93 %) to all other species of the genus Rubrobacter. The peptidoglycan diamino acid was lysine. Strain RV113T exhibited a quinone system with menaquinone MK-8 as the predominant compound. The polar lipid profile of strain RV113T consisted of the major compounds phosphatidylglycerol and two

unidentified phosphoglycolipids. The major fatty acid was anteiso-C17 : 0v9c. These chemotaxonomic traits are in agreement with those of other species of the genus Rubrobacter. The results of physiological and biochemical tests allowed the clear phenotypic differentiation of strain RV113T from all recognized Rubrobacter species. Strain RV113T is thus considered to represent a novel species, for which the name Rubrobacter aplysinae sp. nov. is proposed. The type strain is RV113T (5DSM 27440T5CECT 8425T).

The genus Rubrobacter was established by Suzuki et al. phylogenetically related to the genus Rubrobacter have been (1988), with the reclassification of Arthrobacter radiotolerans detected in numerous environmental samples (Jurado et al., (Yoshinaka et al., 1973) to the genus Rubrobacter.Suzukiet al. 2012), among them different soils (Holmes et al., 2000) and (1988) characterized the type species as a highly gamma- biodeteriorated monuments (Schabereiter-Gurtner et al., radiation-resistant bacterium, which was isolated from a 2001; Imperi et al., 2007). During the characterization of radioactive hot spring in Japan. Later, Carreto et al. (1996) actinobacteria from marine sponges, strain RV113T was described Rubrobacter xylanophilus as a thermophilic, slightly recovered from the Mediterranean sponge Aplysina aero- halotolerant bacterium isolated from a thermally polluted phoba (order Verongida) collected from Rovinj, Croatia industrial run-off in the UK. An additional thermophilic (Abdelmohsen et al., 2010), showing similarities to repre- species, Rubrobacter taiwanensis, was described by Chen et al. sentatives of the genus Rubrobacter. Because members of this (2004), which was also halotolerant and highly resistant to genus have not been described so far from marine habitats, gamma radiation. This species was isolated from a hot spring this strain was studied in more detail. in Taiwan. The mesophilic species Rubrobacter bracarensis The strain was maintained (although growing slowly) on was described by Jurado et al. (2012), strains of which were tryptone soy agar (TSA; Oxoid) at 28 uC and produced isolated from a green biofilm covering the biodeteriorated salmon-coloured colonies on this medium. The strain interior walls of Vilar de Frades Church in Portugal. At the stained Gram-positive using the procedure of Gerhardt time of writing, these four species are the only recognized et al. (1994). Cell morphology was observed with a Zeiss members of the genus, although many 16S rRNA gene clones light microscope at 10006 magnification using cells grown on TSA for 14 days at 25 uC. 3Present address: Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt. Detailed phylogenetic analysis of strain RV113T was Abbreviation: LTP, ‘All-Species Living Tree’ Project. performed using ARB release 5.2 (Ludwig et al., 2004) and The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene the 16S rRNA-based ‘All-Species Living Tree’ Project (LTP) sequence of strain RV113T is GU318365. database (Yarza et al., 2008) release 108 (July 2012).

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Sequences not included in the LTP database were aligned Biomass of strain RV113T for analysis of polar lipids and with the SINA alignment tool version 1.2.11 (Pruesse et al., menaquinones was grown on PYE medium (0.3 % peptone 2012) and were implemented in the LTP database. The from casein, 0.3 % yeast extract, pH 7.2) supplemented alignment of sequences selected for tree construction was with 4 % sea salts (Sigma). Analyses of quinones and polar controlled manually based on secondary structure informa- lipids were done as described by Tindall (1990a, b) and tion. Sequence similarities were calculated in ARB using Altenburger et al. (1996). The quinone system of strain T the ARB neighbour-joining tool without the use of an RV113 showed the presence of the major menaquinone evolutionary substitution model. A phylogenetic tree was MK-8 (88 %) and lesser amounts of MK-7 (11 %) and MK- constructed with the maximum-likelihood method using 6 (1 %). This menaquinone system with MK-8 as the the RAxML algorithm version 7.04 (Stamatakis, 2006) with predominant component is in accordance with the GTR-GAMMA and rapid bootstrap analysis based on 16S quinone systems reported for Rubrobacter radiotolerans rRNA gene sequences between sequence termini 79 and (Suzuki et al., 1988), R. xylanophilus (Carreto et al., 1996) 1407 (numbering according to the Escherichia coli 16S and R. bracarensis (Jurado et al., 2012). The polar lipid rRNA gene sequence published by Brosius et al., 1978). profile (Fig. 2) was composed of the major lipids phosphatidylglycerol and two unidentified phosphoglyco- The sequenced nearly full-length 16S rRNA gene fragment lipids. The following compounds were further identified as of strain RV113T was a continuous stretch of 1512 minor components: diphosphatidylglycerol, an uniden- unambiguous nucleotides between E. coli positions 9 and tified glycolipid (GL1) with medium chromatographic 1514. motility, an unidentified glycolipid (GL2) showing highly Strain RV113T shared 89.6–96.9 % 16S rRNA gene hydrophilic chromatographic motility, two unidentified sequence similarity with the type strains of species of the phospholipids, an unidentified aminoglycolipid (AGL1) genus Rubrobacter, with R. bracarensis VF70612_S1T with highly hydrophobic chromatrographic motility, an (96.9 %) as its closest relative. Phylogenetic tree construc- unidentified aminolipid (AL1) and two polar lipids (L1, tion showed a clear positioning of strain RV113T within the L2) negative for phosphate, an amino group and a sugar monophyletic cluster of the genus Rubrobacter, clustering moiety. This polar lipid profile was similar to those of R. closest to the type strain of R. bracarensis (Fig. 1). radiotolerans and R. xylanophilus with respect to the major

0.10 Actinoalloteichus cyanogriseus IFO 14455T (AB006178) Kutzneria viridogrisea JCM 3298T (U58530) Crossiella cryophila NRRL B-16238T (AF114806) Allokutzneria albata DSM 44149T (AJ512462) Alloactinosynnema album 03-9939T (EU438907) Actinosynnema mirum IMSNU 20048T (AF328679) Saccharothrix australiensis NRRL 11239T (AF114803) 82 Amycolatopsis orientalis IMSNU 20058T (AJ400711) Saccharopolyspora hirsuta subsp. hirsuta ATCC 27875T (DQ381814) Actinokineospora riparia NRRL B-16432T (AF114802) Lechevalieria aerocolonigenes NRRL B-3298T (AF114804) Lentzea albidocapillata IMSNU 21253T (AF328680) 10096 Lentzea californiensis NRRL B-16137T (AF174435) 100 Actinoplanes cyaneus IFO 14990T (AB036997) Actinoplanes philippinensis ATCC 12427T (U58525) 95 Rubrobacter aplysinae RV113T (GU318365) 100 Rubrobacter bracarensis VF70612_S1T (EU512991) 76 Rubrobacter radiotolerans JCM 2153T (U65647) 100 Rubrobacter taiwanensis LS-293T (AF465803) 100 Rubrobacter xylanophilus DSM 9941T (CP000386) 100 Conexibacter woesei DSM 14684T (AJ440237) Conexibacter arvalis KV-962T (AB597950) 97 Solirubrobacter pauli B33D1T (AY039806) Solirubrobacter soli Gsoil 355T (AB245334) 10098 Solirubrobacter ginsenosidimutans BXN5-15T (EU332825) 76 Patulibacter minatonensis KV-614T (AB193261) 100 Patulibacter ginsengiterrae P4-5T (EU710748) 91 Patulibacter americanus CP177-2T (AJ87130) Thermoleophilum album ATCC 35263T (AJ458462) Gaiella occulta F2-233T (JF423906) 100 Chloroflexus aurantiacus J-10-flT (D38365) Roseiflexus castenholzii DSM 13941T (CP00080)

Fig. 1. Maximum-likelihood tree showing the phylogenetic position of strain RV113T within the genus Rubrobacter. The tree was generated in ARB using RAxML (GTR-GAMMA, rapid bootstrap analysis, 100 bootstraps) and based on 16S rRNA gene sequences between positions 79 and 1407 (E. coli numbering, Brosius et al., 1978). Representatives of closely related genera were included in the analysis. GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values .70 % (100 replicates). Bar, 0.10 substitutions per site.

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compounds phosphatidylglycerol, an unidentified phosphoglycolipid and minor amounts of diphosphatidylglycerol. On the other hand, the presence of major amounts of DPG L1 a second phosphoglycolipid and minor amounts of an AGL1 unidentified aminoglycolipid clearly distinguished strain RV113T from R. radiotolerans and R. xylanophilus (no lipid data were available for R. bracarensis and R. taiwanensis). AL1 Fatty acid analysis was performed according to Ka¨mpfer & PG Kroppenstedt (1996) using MIDI Sherlock Version 2.11 T PGL1 (TSBA 4.1). The fatty acid profile of strain RV113 was GL1 obtained after growth on TSA at 28 uC for 14 days. The results are given in Table 1. The profile was similar to those PL1 from strains of R. bracarensis but slightly different from PGL2 those obtained for other species of the genus Rubrobacter. PL2 Detailed results of the physiological characterization of T L2 strain RV113 are given in Table 2 and the species description and were carried out in a microtitre plate panel GL2 including 87 physiological tests as described by Ka¨mpfer et al. (1991). In addition, some tests on the degradation of polymeric substances were performed using standard procedures according to Williams et al. (1983). The results Fig. 2. Two-dimensional TLC image of the polar lipid profile of strain of these tests were read after 7 days of incubation at 28 uC. RV113T, stained with molybdatophosphoric acid. PG, phosphati- T dylglycerol; DPG, diphosphatidylglycerol; PL1, PL2, unidentified On the basis of the results reported here, strain RV113 is phospholipids; AL1, unidentified aminolipid; PGL1, PGL2, unidentified considered to represent a novel species of the genus phosphoglycolipids; GL1, GL2, unidentified glycolipids; AGL1, Rubrobacter, for which the name Rubrobacter aplysinae sp. unidentified aminoglycolipid; L1, L2, unidentified polar lipids. nov. is proposed.

Table 1. Comparison of the major fatty acids found in strain RV113T and members of the genus Rubrobacter

Strains: 1, RV113T;2,R. bracarensis VF70612_S1T;3,R. bracarensis VF70612_S4; 4, R. bracarensis VF70612_S5; 5, R. radiotolerans DSM 5868T;6,R. xylanophilus DSM 9941T;7,R. taiwanensis JCM 12932T. Results from this study for strain RV113T were obtained from cells grown on TSA at 28 uC, for R. bracarensis strains on TSANa-Mg at 28 uC, at 37 uC for R. radiotolerans, and 60 uC for R. xylanophilus and R. taiwanensis. Values represent percentages of total fatty acid content and standard deviations are shown in parentheses. *,1%; ND, not detected. Data for all reference strains are from Jurado et al. (2012). Data for the major components are in bold type.

Fatty acid 1 2 3 4 5 6 7

C16 : 0 1.0 1.9 (0.7) 1.5 (0.0) 1.7 (0.1) 5.9 (0.5) 6.4 (0.6) 3.1 (0.4) C16 : 0 10 methyl ND ND ND ND ND ND 1.9 (0.2) C15 : 0 3-OH ND ND ND ND ND ND 21.6 (2.5) anteiso-C17 : 1v9c 89.9 91.6 (0.7) 76.6 (1.0) 91.2 (0.4) 79.3 (0.6) 1.7 (2.5) ND C17 : 0 cyclo 3.7 1.5 (2.2) 11.1 (0.4) 3.2 (0.1) 7.6 (1.6) ND ND iso-C17 : 0 ND ND ND ND ND ND 1.8 (0.1) C17 : 1v6c ND ND ND ND ND ND 6.7 (0.2) C17 : 0 ND ND ND ND ND ND 7.0 (0.4) C17 : 0 10-methyl ND ND ND ND ND ND 1.6 (0.1) iso-H C18 : 1 4.0 2. (0.4) 6.4 (0.6) 2.0 (0.1) 1.56 (0.4) ND 25.1 (0.1) C18 : 3v6c (6,9,12) ND ND 1.1 (0.8) ND 1.4 (1.1) 2.5 (1.3) 1.3 (0.0) iso-C18 : 0 ND ND ND ND ND ND 3.8 (0.2) C18 : 0 1.4 1.9 (0.7) 1.1 (0.3) 1.5 (0.2) 3.43 (1.0) 84.0 (3.7) 12.0 (0.1) C19 : 0 ND ND ND ND ND ND 1.4 (0.0) C20 : 0 ND ND ND ND ND 1.0 (0.1) ND iso-I C17 : 1/anteiso B ND ND ND ND ND 1.7 (0.8) 2.4 (2.4) C19 : 1v11c/C19 : 1v9c ND ND ND ND ND ND 1.7 (0.0) Unknown 18.846/C19 : 1v6c ND ND ND ND ND ND 8.3 (0.5)

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Table 2. Characteristics that differentiate strain RV113T from members of the genus Rubrobacter

Strains: 1, RV113T;2,R. bracarensis VF70612_S1T;3,R. bracarensis VF70612_S4; 4, R. bracarensis VF70612_S5; 5, R. radiotolerans DSM 5868T;6,R. xylanophilus DSM 9941T;7,R. taiwanensis JCM 12932T. Data for the reference strains are from Jurado et al. (2012). Strains of R. bracarensis were reported not to be able to grow on TSA (Jurado et al., 2012), and hence a direct comparison of fatty acid profiles from strains grown on the same medium was not possible. 2, Negative; +, positive; (+), weakly positive; ±, variable.

Characteristic 1 2 3 4 5 6 7

Oxidase 2222+++ Growth on/with/at: TSA + 222+++ 10 % (w/v) NaCl 2 +++222 50 uC 2222+++ Utilization of: D-Galactose 22(+) + 2 ++ D-Mannitol +±(+) ++22 myo-Inositol 2 +++2 ++ D-Xylose (+) +++2 ++ Acid production from: D-Fructose 2222+++ D-Glucose 2222+++ Melibiose 2222+++ D-Tagatose 2222(+) 2 + Turanose 2222(+) ++ Alkaline phosphatase ++++2 ++

Major fatty acid(s) anteiso- anteiso- anteiso- anteiso- anteiso- C18 : 0 iso-H C18 : 1, C17 : 0v9c C17 : 0v9c C17 : 0v9c C17 : 0v9c C17 : 0v9c C15 : 0 3-OH

T Acid production for strain RV113 was negative for all tested substrates (see species description). The strain utilized D-fructose, D-gluconate, D- ribose, D-mannitol, sorbitol, acetate, fumarate, glutarate, DL-lactate, L-malate, 2-oxoglutarate, pyruvate, L-aspartate, L-ornithine and L-proline.

Description of Rubrobacter aplysinae sp. nov. cellobiose, dulcitol, D-fructose, D-galactose, D-glucose, glycerol, glycogen, inositol, lactose, melezitose, methyl a-D- Rubrobacter aplysinae (a.ply9si.nae. N.L. gen. n. aplysinae glucopyranoside, maltose, D-mannitol, D-mannose, meli- of/from Aplysina the zoological name of a genus of sponge, biose, raffinose, rhamnose, D-ribose, sucrose, salicin, L- referring to the isolation of the type strain from the sponge sorbose, starch, trehalose, turanose or D-xylose. The Aplysina aerophoba). substrates D-fructose, D-gluconate, D-ribose, D-mannitol, Cells are irregular rods, 0.9–1.5 mm in diameter and occur sorbitol, acetate, fumarate, glutarate, DL-lactate, L-malate, 2- singly or in agglomerates. Forms salmon-coloured colonies oxoglutarate, pyruvate, L-aspartate, L-ornithine and L-pro- after 7–14 days of incubation. Gram-stain-positive and line are utilized as sole source of carbon after 14 days of oxidase-negative, showing an oxidative metabolism. Slow incubation. Does not utilize N-acetyl-D-galactosamine, N- growth occurs on TSA, nutrient agar, R2A agar and marine acetyl-D-glucosamine, L-arabinose, p-arbutin, cellobiose, D- agar at 28 uC. Growth occurs at pH 6.5, 7.5, 8.5 and 9.5, but galactose, D-gluconate, D-mannose, maltose, a-melibiose, L- not at pH 4.5 and 5.5 or pH 10.5 and 11.5 in tryptone soy rhamnose, salicin, sucrose, D-xylose, D-adonitol, i-inositol, broth at 28 uC . The temperature range for growth is maltitol, putrescine, cis-aconitate, trans-aconitate, citrate, 20–30 uC. No growth (within 3 weeks of incubation) is DL-lactate, L-malate, itaconate, mesaconate, L-alanine, b- observed at ¡15 uCor¢36 uC. The characteristic diamino alanine, D-histidine, L-leucine, L-phenylalanine, L-serine, L- acid of the peptidoglycan is meso-diaminopimelic acid. The tryptophan, 3-hydroxybenzoate, 4-hydroxybenzate or phe- polar lipid profile contains phosphatidylglycerol and two nylacetate. unidentified phosphoglycolipids as major components, with The type strain, RV113T (5DSM 27440T5CECT 8425T), lesser amounts of diphosphatidylglycerol, two unidentified was isolated from the sponge Aplysina aerophoba collected glycolipids (GL1, GL2), two unidentified phospholipids close to Rovinj, Croatia. (PL1, PL2), an unidentified aminoglycolipid (AGL1), an unidentified aminolipid (AL1) and two polar lipids (L1, L2). The quinone system is characterized by the major com- Acknowledgements pound menaquinone MK-8. The major fatty acid is anteiso- Financial support was provided by the Deutsche Forschungsge- C17 : 0v9c. No acid is produced from L-arabinose, arbutin, meinschaft, grant SFB630-TP A5, to U. H. Downloaded from www.sgmjournals.org by 708 International Journal of Systematic and Evolutionary Microbiology 64 IP: 134.245.215.185 On: Thu, 02 Jul 2015 09:27:41 Rubrobacter aplysinae sp. nov.

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