IJSEM Papers in Press. Published October 29, 2013 as doi:10.1099/ijs.0.055152-0
1
Rubrobacter aplysinae sp. nov. isolated from the marine sponge Aplysina aerophoba
2 3
Peter Kämpfer,1 Stefanie P. Glaeser,1 Hans-Jürgen Busse2, Usama Ramadan
4
Abdelmohsen3,† , Ute Hentschel3
5 6
1
7
Giessen, Germany
8
2
9
1210 Wien, Austria
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392
Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-
10
3
11
Wuerzburg, D-97082 Wuerzburg, Germany
Dept. of Botany II, Julius-von-Sachs-Institute for Biological Sciences, University of
12 13
Corresponding author:
14
Professor Dr Peter Kämpfer
15
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen
16
Heinrich-Buff-Ring 26–32; D-35392 Giessen, Germany
17
Tel. +49 641 99 37352 ; Fax. +49 641 99 37359
18
e-mail: [email protected]
19 20
Subject category: New Taxa; Subsection: Gram-positive bacteria
21 22
The EMBL accession number for the 16S rRNA gene sequence of strain RV113T is
23
GU318365.
24
†
Permanent address: Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
1
25
A Gram-positive, non-spore-forming bacterium (RV113T) was isolated from
26
the marine sponge Aplysina aerophoba. The 16S rRNA gene sequence analysis
27
and similarity studies showed that strain RV113T belongs to the genus
28
Rubrobacter, and is most closely related to Rubrobacter bracariensis VF70612 S1T
29
(96.9%) and more distantly related (< 93%) to all other Rubrobacter species. The
30
peptidoglycan diaminoacid was lysine. Strain RV113T exhibited a quinone system
31
with the predominant compound menaquinone MK-8.
32
The polar lipid profile of RV113T consisted of the major compounds
33
phosphatidylglycerol and two unidentified phosphoglycolipids.
34
The major fatty acid was anteiso C17:0ω9. These chemotaxonomic traits are in
35
excellent agreement with those of other Rubrobacter species. The results of
36
physiological and biochemical tests allowed a clear phenotypic differentiation of
37
strain RV113T from all other Rubrobacter species. RV113T merits species status,
38
and we propose the name Rubrobacter aplysinae sp. nov. with the type strain
39
RV113T (= DSM 27440T = CECT 8425T).
40 41 42 43 44 45 46 47 48 49
2
50
The genus Rubrobacter was already established in 1988 by Suzuki et al. (1988), who
51
reclassified Arthrobacter radiotolerans (Yoshinaka et al., 1973) into the genus
52
Rubrobacter and characterised the type species as a highly gamma-radiation
53
resistant bacterium, isolated from a radioactive hot spring in Japan. Later, Carreto et
54
al. (1996) described Rubrobacter xylanophilus as a thermophilic, slightly halotolerant
55
bacterium isolated from a thermally polluted industrial run-off in the United Kingdom.
56
An additional thermophilic species, Rubrobacter taiwanensis was decribed by Chen
57
et al. (2004), which was in addition also halotolerant, highly resistant to gamma-
58
radiation. This species was isolated from a hot spring in Taiwan. Very recently the
59
mesophilic species Rubrobacter bracarensis was described by Jurado et al. (2012).
60
The stains belonging to this species were isolated from a green biofilm covering the
61
biodeteriorated interior walls of Vilar de Frades Church in Portugal. To date, only
62
these four species have been described, although in the past, many 16S rRNA gene
63
clones phylogenetically related to the genus Rubrobacter have been detected in
64
many environmental samples (Jurado et al., 2012), among them different soils
65
(Holmes et al., 2000) and biodeteriorated monuments (Schabereiter-Gurtner et al.,
66
2001; Imperi et al., 2007). During the characterization of actinobacteria from marine
67
sponges, interestingly strain RV113T was recovered from the Mediterranean sponge
68
Aplysina aerophoba (order Verongida) collected from Rovinj, Croatia (Abdelmohsen
69
et al., 2010), showing similarities to representatives of the genus Rubrobacter.
70
Because members of this genus have not been described so far from marine
71
habitats, this strain was studied in more detail.
72
The strain was maintained (although growing slowly) on tryptone soy agar (TSA,
73
Oxoid) at 28°C and showed on this agar salmon coloured colonies. The strain
74
stained Gram-positive using the procedure of Gerhardt et al. (1994) and cell
75
morphology was observed with a Zeiss light microscope at 1000 x magnification 3
76
using cells grown on TSA for 14 days at 25 °C.
77
Detailed phylogenetic analysis of strain RV113T was performed in ARB release 5.2
78
(Ludwig et al., 2004) using the 16S rRNA-based “All-Species Living Tree" Project
79
(LTP) database (Yarza et al., 2008) release 108 (July 2012). Sequences not included
80
in the LTP database were aligned with the SINA alignment tool version 1.2.11
81
(Pruesse et al., 2012) and were implemented in the LTP database. The alignment of
82
sequences selected for tree construction was controlled manually based on
83
secondary structure information. Sequence similarities were calculated in ARB using
84
the ARB Neighbour-joining tool without the use of an evolutionary substitution model.
85
A phylogenetic trees was constructed with the Maximum-likelihood method using the
86
RAxML algorithm version 7.04 (Stamatakis, 2006) with GTR-GAMMA and rapid
87
bootstrap analysis based on 16S rRNA gene sequences between sequence termini
88
79 and 1407 (numbering according to the E. coli rRNA sequence published by
89
Brosius et al., 1978).
90
The sequenced nearly full-length 16S rRNA gene fragment of strain RV113T was a
91
continuous stretch of 1512 unambiguous nucleotides between E. coli positions 9 to
92
1514.
93
Strain RV113T shares 89.6 to 96.9% 16S rRNA gene sequence similarities to type
94
strains of the genus Rubrobacter with R. bracarensis VF70612_S1T (96.9%) as the
95
closest relative. Phylogenetic tree construction showed a clear positioning of strain
96
RV113T within the monophyletic cluster of the genus Rubrobacter clustering closest
97
to the type strain of R. bracarensis (Fig. 1).
98 99
Biomass of RV113T for analysis of polar lipids and menaquinones was grown on PYE medium (0.3% peptone from casein, 0.3% yeast extract, pH 7.2)
100
supplemented with 4% sea salts (Sigma). Analyses of quinones and polar lipids were
101
done as described by Tindall (1990a, b) and Altenburger et al. (1996). The quinone 4
102
system of RV113T showed the presence of the major menaquinone MK-8 (88%) and
103
less amounts of MK-7 (11%) and MK-6 (1%). This menaquinone system with the
104
predominant MK-8 is in accordance with the quinone systems reported for R.
105
radiotolerans (Suzuki et al., 1988), R. xylanophilus (Carreto et al., 1996), and R.
106
bracarensis (Jurado et al., 2012). The polar lipid profile (Fig. 2) was composed of the
107
major lipids phosphatidylglycerol and two unidentified phosphoglycolipids.
108
Furthermore, less amounts of diphosphatidylglycerol, an unidentified glycolipid GL1
109
with medium chromatographic motility, an unidentified glycolipid GL2 showing a
110
highly hydrophilic chromatographic motility, two unidentified phospholipids, an
111
unidentified aminoglycolipid (AGL1) with highly hydrophobic chromatrographic
112
motility, an unidentified aminolipid (AL1) and two polar lipids (L1, L2) negative for
113
phosphate, an amino group and a sugar moiety. This polar lipid profile was similar to
114
those of R. radiotolerans and R. xylanophilus in respect to the major compounds
115
phosphatidylglycerol, an unidentified phosphoglycolipid and minor amounts of
116
diphosphatidylglycerol. On the other hand the presence of major amounts of a
117
second phosphoglycolipid and minor amounts of an unidentified aminoglycolipid
118
clearly distinguished strain RV113T from R. radiotolerans and R. xylanophilus (no
119
lipid data available for the other two established species of the genus R. bracarensis
120
and R. taiwanensis).
121
Fatty acid analysis was performed according to Kämpfer & Kroppenstedt (1996)
122
using the MIDI Sherlock Version 2.11 (TSBA 4.1). The fatty acid profile of strain
123
RV113T was obtained after growth on TS agar at 25°C for 14 days. The results are
124
given in Table 1. The profile was similar to those from the R. bracarensis strains but
125
slightly different to those obtained for the other Rubrobacter species.
126 127
Detailed results of the physiological characterization are given in Table 2 and the species description and were carried out in a microliter plate panel including 87 5
128
physiological tests as described by Kämpfer et al. (1991). In addition, some tests on
129
the degradation of polymeric substances were performed using the standard
130
procedures according to Williams et al. (1983). The results of these tests were read
131
after 7 days incubation at 28 °C.
132
On the basis of the results reported here, it is justified to propose a novel Rubrobacter
133
species.
134 135 136
Description of Rubrobacter aplysinae sp. nov. Rubrobacter aplysinae (a.ply´si.ae N.L. gen. n. aplysina of/from
137
Aplysina the zoological name of a genus of sponge, referring to the isolation of the
138
type strain from the sponge Aplysina aerophoba)
139
Cells are irregular rods, 0.9–1.5 µm in diameter and occur singly or in
140
agglomerates. Forms pink salmon coloured colonies after 7 to 14 days of incubation.
141
Gram-positive, oxidase-negative, showing an oxidative metabolism. Slow growth
142
occurs on tryptone soy agar, nutrient agar, R2A agar and marine agar at 28°C,
143
Growth was observed at pH values 6.5, 7.5, 8.5, and 9.5, but not at pH 4.5 and 5.5
144
and pH 10.5 and 11.5 ranging in tryptone soy broth at 28°C . The temperature range
145
of growth was 20°C to 30°C. No growth (withing 3 weeks of incubation) was
146
observed at 15°C and below and at 36°C and above.
147
The characteristic diaminoacid of the peptidoglycan is meso-diaminopimelic acid.
148
The polar lipid profile contains major lipids phosphatidylglycerol, two unidentified
149
phosphoglycolipids, and less amounts of diphosphatidylglycerol, two unidentified
150
glycolipid (GL1, GL2), two unidentified phospholipids, an unidentified aminoglycolipid
151
(AGL1), an unidentified aminolipid (AL1) and two polar lipids (L1, L2). The quinone
152
system is characterized by the major compound menaquinone MK-8.
6
153
The major fatty acid is anteiso C17:0ω9. No acid is produced from L-arabinose,
154
arbutin, D-cellobiose, dulcitol, D-fructose, D-galactose, D-glucose, glycerol, glycogen,
155
inositol, D-lactose, D-melezitose, methyl-α-D-glucopyranoside, D-maltose, D-
156
mannitol, D-mannose, D-melibiose, D-raffinose, rhamnose, D-ribose, D-sucrose,
157
salicin, L-sorbose, starch, D-trehalose, D-turanose and D-xylose.
158
The substrates D-fructose, D-gluconate, D-ribose, D-mannitol, sorbitol, acetate, fumarate,
159
glutarate, DL-lactate, L-malate, 2-oxoglutarate, pyruvate, L-aspartate, L-ornithine and L-
160
proline are utilized as sole source of carbon after 14 days of incubation. Does not utilize
161
N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, p-arbutin, D-cellobiose,
162
D-galactose, D-gluconate, D-mannose, D-maltose, alpha-D-melibiose, L-rhamnose,
163
salicin, sucrose, D-xylose, D-adonitol, i-inositol, maltitol, putrescine, cis-aconitate, trans-
164
aconitate, citrate, DL-lactate, L-malate, itaconate, mesaconate, L-alanine, ß-alanine, D-
165
histidine, L-leucine, L-phenylalanine, L-serine, L-tryptophane, 3-hydroxybenzoate, 4-
166
hydroxybenzate, and phenylacetate.
167
The type strain was isolated from the sponge Aplysina aerophoba collected close to
168
Rovinj, Croatia. Type strain is RV113T (= DSM 27440T = CECT 8425T).
169 170
Acknowledgements
171 172
Financial support was provided by the Deutsche Forschungsgemeinschaft , SFB630-TP
173
A5 to U. H.
174 175
REFERENCES
176
Abdelmohsen, U. R., Pimentel-Elardo, S. M., Hanora, A., Radwan, M., Abou-El-Ela,
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Classification of bacteria isolated from a medieval wall painting. J Biotechnol 47, 39–52.
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Altenburger, P., Kämpfer, P., Akimov, V. N., Lubitz, W. & Busse, H.-J. (1997).
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genera Brevibacterium, Corynebacterium and Tsukamurella. Int J Syst Bacteriol 47, 270-
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Busse, H.-J. & Auling, G. (1988). Polyamine pattern as a chemotaxonomic marker
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Carreto, L., Moore, E., Nobre, M.F., Wait, R., Riley, P.W., Sharp, R.K. & da Costa,
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M.S. (1996). Rubrobacter xylanophilus sp. nov., a new thermophilic species isolated
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from a thermally polluted effluent. Int J Syst Bacteriol 46, 460-465.
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Chen, M.Y., Wu, S.H., Lin, G.H., Lu, C.P., Lin, Y.T., Chang, W.C. & Tsay, S.S.
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(2004). Rubrobacter taiwanensis sp. nov., a novel thermophilic radiation-resistant
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species isolated from hot springs. Int J Syst Bacteriol 54, 1849-1855.
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Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods
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for General and Molecular Bacteriology. Washington, DC: American Society for
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Holmes, A.J. Bowyer, J., Holley, M.P., O’Donoghue, M. & Gillings, M.R. (2000).
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Imperi, F., Caneva, G., Cancellieri, L., Ricci, M.A., Soldo, A. & Visca, P. (2007). 8
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The bacterial aetiology of rosy discoloration of ancient wall paintings. Environ
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Microbiol 9, 2894-2902.
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Jurado, V., Miller, A.Z., Alias-Villegas, C., Laiz, L. & Saiz-Jimenez, C. (2012).
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Rubrobacter bracarensis sp. nov., a novel member of the genus Rubrobacter isolated
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from a biodeteriorated monument. Syst Appl Microbiol 35, 306-309.
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Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of
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coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
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Kämpfer, P., Steiof, M. & Dott, W. (1991). Microbiological characterisation of a fuel-oil
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Pruesse, E., Peplies J. & Glöckner, F. O. (2012) SINA: accurate high throughput
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multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28(14): 1823-
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Schabereiter-Gurtner, C., Piñar, G., Vybiral, D., Lubitz, W. & Rolloke, S. (2001).
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Rubrobacter-related bacteria associated with rosy discoloration of masonry and lime
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Schleifer, K. H. (1985). Analysis of the chemical composition and primary structure
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Suzuki, K., Collins, M.D., Iljima, E. & Komagata, K. (1988) Chemotaxonomic
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characterization of a radiotolerant bacterium Arthrobacter radiotolerans: description
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Tindall, B. J. (1990a). A comparative study of the lipid composition of Halobacterium 9
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Williams, S.T., Goodfellow, M., Alderson, G., Wellington, E.M.H., Sneath, P.H.A., and
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Yarza, P., Richter, M., Peplies, J., Euzeby, J., Amann, R., Schleifer, K. H. et al.
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(2008). The all-species living tree project: A 16S rRNA-based phylogenetic tree of all
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Yoshinaka, T., Yano, K. & Yamaguchi, H. (1973). Isolation of highly radioresistant
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242 243 244 245 246 247 248 249 250 251
Table 1. Comparison of the major fatty acids found in strain RV113T and the Rubrobacter bracarensis strains VF70612_S1T, VF70612_S4 and VF70612_S5. Taxa: 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°C, for R. bracarensis strains on TSANa-Mg at 28 °C; 37 °C for R. radiotolerans and 60 °C for R. xylanophilus and R. taiwanensis. Values represent percentages of total fatty acid content and standard deviations are showed in parentheses. *, 70% (100 replicates). Bar, 0.10 substitutions per side.
13
281
Fig. 2: Two-dimensional TLC of polar lipid profile of strain RV113T, stained with
282
molybdatophosphoric acid.
283
PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PL1, PL2, unidentified
284 285 286 287 288
phospholipids; AL1, unidentified aminolipid; PGL1, PGL2, unidentified phosphoglycolipids; GL1, GL2, unidentified glycolipids; AGL1, unidentified aminoglycolipid; L1, 2, unidentified polar lipids
289 290 291 292 293
14
1
Rubrobacter aplysinae sp. nov. isolated from the marine sponge Aplysina aerophoba
2 3
Peter Kämpfer,1 Stefanie P. Glaeser,1 Hans-Jürgen Busse2, Usama Ramadan
4
Abdelmohsen3,† , Ute Hentschel3
5 6
1
7
Giessen, Germany
8
2
9
1210 Wien, Austria
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392
Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-
10
3
11
Wuerzburg, D-97082 Wuerzburg, Germany
Dept. of Botany II, Julius-von-Sachs-Institute for Biological Sciences, University of
12 13
Corresponding author:
14
Professor Dr Peter Kämpfer
15
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen
16
Heinrich-Buff-Ring 26–32; D-35392 Giessen, Germany
17
Tel. +49 641 99 37352 ; Fax. +49 641 99 37359
18
e-mail: [email protected]
19 20
Subject category: New Taxa; Subsection: Gram-positive bacteria
21 22
The EMBL accession number for the 16S rRNA gene sequence of strain RV113T is
23
GU318365.
24
†
Permanent address: Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
1
25
A Gram-positive, non-spore-forming bacterium (RV113T) was isolated from
26
the marine sponge Aplysina aerophoba. The 16S rRNA gene sequence analysis
27
and similarity studies showed that strain RV113T belongs to the genus
28
Rubrobacter, and is most closely related to Rubrobacter bracariensis VF70612 S1T
29
(96.9%) and more distantly related (< 93%) to all other Rubrobacter species. The
30
peptidoglycan diaminoacid was lysine. Strain RV113T exhibited a quinone system
31
with the predominant compound menaquinone MK-8.
32
The polar lipid profile of RV113T consisted of the major compounds
33
phosphatidylglycerol and two unidentified phosphoglycolipids.
34
The major fatty acid was anteiso C17:0ω9. These chemotaxonomic traits are in
35
excellent agreement with those of other Rubrobacter species. The results of
36
physiological and biochemical tests allowed a clear phenotypic differentiation of
37
strain RV113T from all other Rubrobacter species. RV113T merits species status,
38
and we propose the name Rubrobacter aplysinae sp. nov. with the type strain
39
RV113T (= DSM 27440T = CECT 8425T).
40 41 42 43 44 45 46 47 48 49
2
50
The genus Rubrobacter was already established in 1988 by Suzuki et al. (1988), who
51
reclassified Arthrobacter radiotolerans (Yoshinaka et al., 1973) into the genus
52
Rubrobacter and characterised the type species as a highly gamma-radiation
53
resistant bacterium, isolated from a radioactive hot spring in Japan. Later, Carreto et
54
al. (1996) described Rubrobacter xylanophilus as a thermophilic, slightly halotolerant
55
bacterium isolated from a thermally polluted industrial run-off in the United Kingdom.
56
An additional thermophilic species, Rubrobacter taiwanensis was decribed by Chen
57
et al. (2004), which was in addition also halotolerant, highly resistant to gamma-
58
radiation. This species was isolated from a hot spring in Taiwan. Very recently the
59
mesophilic species Rubrobacter bracarensis was described by Jurado et al. (2012).
60
The stains belonging to this species were isolated from a green biofilm covering the
61
biodeteriorated interior walls of Vilar de Frades Church in Portugal. To date, only
62
these four species have been described, although in the past, many 16S rRNA gene
63
clones phylogenetically related to the genus Rubrobacter have been detected in
64
many environmental samples (Jurado et al., 2012), among them different soils
65
(Holmes et al., 2000) and biodeteriorated monuments (Schabereiter-Gurtner et al.,
66
2001; Imperi et al., 2007). During the characterization of actinobacteria from marine
67
sponges, interestingly strain RV113T was recovered from the Mediterranean sponge
68
Aplysina aerophoba (order Verongida) collected from Rovinj, Croatia (Abdelmohsen
69
et al., 2010), showing similarities to representatives of the genus Rubrobacter.
70
Because members of this genus have not been described so far from marine
71
habitats, this strain was studied in more detail.
72
The strain was maintained (although growing slowly) on tryptone soy agar (TSA,
73
Oxoid) at 28°C and showed on this agar salmon coloured colonies. The strain
74
stained Gram-positive using the procedure of Gerhardt et al. (1994) and cell
75
morphology was observed with a Zeiss light microscope at 1000 x magnification 3
76
using cells grown on TSA for 14 days at 25 °C.
77
Detailed phylogenetic analysis of strain RV113T was performed in ARB release 5.2
78
(Ludwig et al., 2004) using the 16S rRNA-based “All-Species Living Tree" Project
79
(LTP) database (Yarza et al., 2008) release 108 (July 2012). Sequences not included
80
in the LTP database were aligned with the SINA alignment tool version 1.2.11
81
(Pruesse et al., 2012) and were implemented in the LTP database. The alignment of
82
sequences selected for tree construction was controlled manually based on
83
secondary structure information. Sequence similarities were calculated in ARB using
84
the ARB Neighbour-joining tool without the use of an evolutionary substitution model.
85
A phylogenetic trees was constructed with the Maximum-likelihood method using the
86
RAxML algorithm version 7.04 (Stamatakis, 2006) with GTR-GAMMA and rapid
87
bootstrap analysis based on 16S rRNA gene sequences between sequence termini
88
79 and 1407 (numbering according to the E. coli rRNA sequence published by
89
Brosius et al., 1978).
90
The sequenced nearly full-length 16S rRNA gene fragment of strain RV113T was a
91
continuous stretch of 1512 unambiguous nucleotides between E. coli positions 9 to
92
1514.
93
Strain RV113T shares 89.6 to 96.9% 16S rRNA gene sequence similarities to type
94
strains of the genus Rubrobacter with R. bracarensis VF70612_S1T (96.9%) as the
95
closest relative. Phylogenetic tree construction showed a clear positioning of strain
96
RV113T within the monophyletic cluster of the genus Rubrobacter clustering closest
97
to the type strain of R. bracarensis (Fig. 1).
98 99
Biomass of RV113T for analysis of polar lipids and menaquinones was grown on PYE medium (0.3% peptone from casein, 0.3% yeast extract, pH 7.2)
100
supplemented with 4% sea salts (Sigma). Analyses of quinones and polar lipids were
101
done as described by Tindall (1990a, b) and Altenburger et al. (1996). The quinone 4
102
system of RV113T showed the presence of the major menaquinone MK-8 (88%) and
103
less amounts of MK-7 (11%) and MK-6 (1%). This menaquinone system with the
104
predominant MK-8 is in accordance with the quinone systems reported for R.
105
radiotolerans (Suzuki et al., 1988), R. xylanophilus (Carreto et al., 1996), and R.
106
bracarensis (Jurado et al., 2012). The polar lipid profile (Fig. 2) was composed of the
107
major lipids phosphatidylglycerol and two unidentified phosphoglycolipids.
108
Furthermore, less amounts of diphosphatidylglycerol, an unidentified glycolipid GL1
109
with medium chromatographic motility, an unidentified glycolipid GL2 showing a
110
highly hydrophilic chromatographic motility, two unidentified phospholipids, an
111
unidentified aminoglycolipid (AGL1) with highly hydrophobic chromatrographic
112
motility, an unidentified aminolipid (AL1) and two polar lipids (L1, L2) negative for
113
phosphate, an amino group and a sugar moiety. This polar lipid profile was similar to
114
those of R. radiotolerans and R. xylanophilus in respect to the major compounds
115
phosphatidylglycerol, an unidentified phosphoglycolipid and minor amounts of
116
diphosphatidylglycerol. On the other hand the presence of major amounts of a
117
second phosphoglycolipid and minor amounts of an unidentified aminoglycolipid
118
clearly distinguished strain RV113T from R. radiotolerans and R. xylanophilus (no
119
lipid data available for the other two established species of the genus R. bracarensis
120
and R. taiwanensis).
121
Fatty acid analysis was performed according to Kämpfer & Kroppenstedt (1996)
122
using the MIDI Sherlock Version 2.11 (TSBA 4.1). The fatty acid profile of strain
123
RV113T was obtained after growth on TS agar at 25°C for 14 days. The results are
124
given in Table 1. The profile was similar to those from the R. bracarensis strains but
125
slightly different to those obtained for the other Rubrobacter species.
126 127
Detailed results of the physiological characterization are given in Table 2 and the species description and were carried out in a microliter plate panel including 87 5
128
physiological tests as described by Kämpfer et al. (1991). In addition, some tests on
129
the degradation of polymeric substances were performed using the standard
130
procedures according to Williams et al. (1983). The results of these tests were read
131
after 7 days incubation at 28 °C.
132
On the basis of the results reported here, it is justified to propose a novel Rubrobacter
133
species.
134 135 136
Description of Rubrobacter aplysinae sp. nov. Rubrobacter aplysinae (a.ply´si.ae N.L. gen. n. aplysina of/from
137
Aplysina the zoological name of a genus of sponge, referring to the isolation of the
138
type strain from the sponge Aplysina aerophoba)
139
Cells are irregular rods, 0.9–1.5 µm in diameter and occur singly or in
140
agglomerates. Forms pink salmon coloured colonies after 7 to 14 days of incubation.
141
Gram-positive, oxidase-negative, showing an oxidative metabolism. Slow growth
142
occurs on tryptone soy agar, nutrient agar, R2A agar and marine agar at 28°C,
143
Growth was observed at pH values 6.5, 7.5, 8.5, and 9.5, but not at pH 4.5 and 5.5
144
and pH 10.5 and 11.5 ranging in tryptone soy broth at 28°C . The temperature range
145
of growth was 20°C to 30°C. No growth (withing 3 weeks of incubation) was
146
observed at 15°C and below and at 36°C and above.
147
The characteristic diaminoacid of the peptidoglycan is meso-diaminopimelic acid.
148
The polar lipid profile contains major lipids phosphatidylglycerol, two unidentified
149
phosphoglycolipids, and less amounts of diphosphatidylglycerol, two unidentified
150
glycolipid (GL1, GL2), two unidentified phospholipids, an unidentified aminoglycolipid
151
(AGL1), an unidentified aminolipid (AL1) and two polar lipids (L1, L2). The quinone
152
system is characterized by the major compound menaquinone MK-8.
6
153
The major fatty acid is anteiso C17:0ω9. No acid is produced from L-arabinose,
154
arbutin, D-cellobiose, dulcitol, D-fructose, D-galactose, D-glucose, glycerol, glycogen,
155
inositol, D-lactose, D-melezitose, methyl-α-D-glucopyranoside, D-maltose, D-
156
mannitol, D-mannose, D-melibiose, D-raffinose, rhamnose, D-ribose, D-sucrose,
157
salicin, L-sorbose, starch, D-trehalose, D-turanose and D-xylose.
158
The substrates D-fructose, D-gluconate, D-ribose, D-mannitol, sorbitol, acetate, fumarate,
159
glutarate, DL-lactate, L-malate, 2-oxoglutarate, pyruvate, L-aspartate, L-ornithine and L-
160
proline are utilized as sole source of carbon after 14 days of incubation. Does not utilize
161
N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, L-arabinose, p-arbutin, D-cellobiose,
162
D-galactose, D-gluconate, D-mannose, D-maltose, alpha-D-melibiose, L-rhamnose,
163
salicin, sucrose, D-xylose, D-adonitol, i-inositol, maltitol, putrescine, cis-aconitate, trans-
164
aconitate, citrate, DL-lactate, L-malate, itaconate, mesaconate, L-alanine, ß-alanine, D-
165
histidine, L-leucine, L-phenylalanine, L-serine, L-tryptophane, 3-hydroxybenzoate, 4-
166
hydroxybenzate, and phenylacetate.
167
The type strain was isolated from the sponge Aplysina aerophoba collected close to
168
Rovinj, Croatia. Type strain is RV113T (= DSM 27440T = CECT 8425T).
169 170
Acknowledgements
171 172
Financial support was provided by the Deutsche Forschungsgemeinschaft , SFB630-TP
173
A5 to U. H.
174 175
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Table 1. Comparison of the major fatty acids found in strain RV113T and the Rubrobacter bracarensis strains VF70612_S1T, VF70612_S4 and VF70612_S5. Taxa: 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°C, for R. bracarensis strains on TSANa-Mg at 28 °C; 37 °C for R. radiotolerans and 60 °C for R. xylanophilus and R. taiwanensis. Values represent percentages of total fatty acid content and standard deviations are showed in parentheses. *, 70% (100 replicates). Bar, 0.10 substitutions per side.
13
281
Fig. 2: Two-dimensional TLC of polar lipid profile of strain RV113T, stained with
282
molybdatophosphoric acid.
283
PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PL1, PL2, unidentified
284 285 286 287 288
phospholipids; AL1, unidentified aminolipid; PGL1, PGL2, unidentified phosphoglycolipids; GL1, GL2, unidentified glycolipids; AGL1, unidentified aminoglycolipid; L1, 2, unidentified polar lipids
289 290 291 292 293
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