IJSEM Papers in Press. Published April 10, 2015 as doi:10.1099/ijs.0.000245
International Journal of Systematic and Evolutionary Microbiology Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's Seahorse (Hippocampus barbouri) --Manuscript Draft-Manuscript Number:
IJS-D-15-00164R1
Full Title:
Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's Seahorse (Hippocampus barbouri)
Short Title:
Mesonia hippocampi sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Stefanie P. Glaeser Justus-Liebig-Universität Giessen Giessen, GERMANY
First Author:
Judy Kolberg
Order of Authors:
Judy Kolberg Hans-Jürgen Busse Thomas Wilke Patrick Schubert Peter Kämpfer Stefanie P. Glaeser
Manuscript Region of Origin:
GERMANY
Abstract:
An orange pigmented gram-staining-negative, rod-shaped bacterium, designated 96_Hippo_TS_3/13T was isolated from the brood pouch of a diseased seahorse male of the species Hippocampus barbouri from the animal facility of the University of Giessen, Giessen, Hesse, Germany. Phylogenetic analyses based on the nearly fulllength 16S rRNA gene sequence placed strain 96_Hippo_TS_3/13T into the monophyletic cluster of the genus Mesonia within the Flavobacteriaceae. However, the strain shared only 92.2-93.8% sequence similarity to type strains of Mesonia species, with highest sequence similarity to the type strain of Mesonia aquimarina. The fatty acids profile showed a Mesonia-typical fatty acid profile including several branched and hydroxyl fatty acids with highest amounts of iso-C15:0 (40.9%) followed by iso-C17:0 3-OH (14.8%). In the polyamine pattern sym-homospermidine was predominant. The diagnostic diamino acid of the peptidoglycan was mesodiaminopimelic acid. The quinone system contain exclusively menaquinone MK-6. The only identified compound in the polar lipid profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts of an unidentified aminolipid and two unidentified lipids not containing a phosphate-group, an amino-group or a sugar residue were detected. The genomic G+C content of strain 96_Hippo_TS_3/13T was 30 mol% Based on genotypic, chemotaxonomic and physiological characterizations we proposed a new species of the genus Mesonia, Mesonia hippocampi with strain 96_Hippo_TS_3/13T (= DSM 29615T = CIP-110839T) as type strain.
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1
Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's
2
Seahorse (Hippocampus barbouri)
3 4
Judy Kolberg1, 2, Hans-Jürgen Busse3, Thomas Wilke2, Patrick Schubert2, Peter Kämpfer1,
5
Stefanie P. Glaeser1*
6 7
1
8
Germany
9
2
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen,
Institut für Tierökologie und Spezielle Zoologie, Justus-Liebig-Universität Giessen, D-35392
10
Giessen, Germany
11
3
Institut für Mikrobiologie, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria
12 13
Corresponding author:
14
Stefanie P. Glaeser
15
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen
16
Heinrich-Buff-Ring 26–32; D-35392 Giessen, Germany
17
Tel. +49 641 99 37373 ; Fax. +49 641 99 37359
18
e-mail: [email protected]
19 20
Short title: Mesonia hippocampi sp. nov.
21
Subject category: New Taxa; Subsection: Bacteroidetes
22 23 24
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
25
96_Hippo_TS_3/13T is KP119858.
1
26
Abstract
27
An
28
96_Hippo_TS_3/13T was isolated from the brood pouch of a diseased seahorse male of the
29
species Hippocampus barbouri from the animal facility of the University of Giessen, Giessen,
30
Hesse, Germany. Phylogenetic analyses based on the nearly full-length 16S rRNA gene
31
sequence placed strain 96_Hippo_TS_3/13T into the monophyletic cluster of the genus
32
Mesonia within the family Flavobacteriaceae. However, the strain shared only 92.2-93.8%
33
sequence similarity to type strains of Mesonia species, with highest sequence similarity to the
34
type strain of Mesonia aquimarina.
35
The fatty acids profile showed a Mesonia-typical fatty acid profile including several branched
36
and hydroxyl fatty acids with highest amounts of iso-C15:0 (40.9%) followed by iso-C17:0 3-
37
OH (14.8%). In the polyamine pattern sym-homospermidine was predominant. The diagnostic
38
diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system
39
contained exclusively menaquinone MK-6. The only identified compound in the polar lipid
40
profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts
41
of an unidentified aminolipid and two unidentified lipids not containing a phosphate-group,
42
an amino-group or a sugar residue were detected. The genomic G+C content of strain
43
96_Hippo_TS_3/13T was 30 mol%
44
Based on genotypic, chemotaxonomic and physiological characterizations we proposed a new
45
species of the genus Mesonia, Mesonia hippocampi with strain 96_Hippo_TS_3/13T (= CIP-
46
110839T = LMG 28572T = CCM 8557T) as the type strain.
orange
pigmented,
Gram-staining-negative,
47 48
2
rod-shaped
bacterium,
designated
49
The genus Mesonia forms a monophyletic clade within the Flavobacteriaceae
50
(Bacteriodestes). It was first proposed in 2003 by Nedashkovskaya et al. (2003). At the time
51
of writing the genus contained five species, Mesonia algae (Nedashkovskaya et al. 2003),
52
Mesonia mobilis (Nedashkovskaya et al., 2006), Mesonia phycicola (Kang & Lee 2010),
53
Mesonia ostreae (Lee et al., 2012) and Mesonia aquimarina (Choi et al., 2014). Type strains
54
of all species were isolated from marine ecosystem, including from green algae of the species
55
Acrosiphonia sonderi in the Sea of Japan (Nedashkovskaya et al., 2003), seawater of the Sea
56
of Japan (Nedashkovskaya et al., 2006), of an oyster farm in the South Sea of Korea (Lee et
57
al., 2012) and off the east sea of Korea (Choi et al., 2014), and from seaweeds near the island
58
of Maryland in Korea (Kang & Lee, 2010).
59
Mesonia are described as thin-rod shaped strictly aerobic chemoheterotrophic bacteria
60
forming yellow colonies with a non-diffusible yellow pigments but not producing flexirubin-
61
type pigments. Cells are oxidase-, catalase- and alkaline phosphatase positive and negative for
62
ß-galactosidase activity. Seawater or Na+ ions are reported to be a prerequisite for growth.
63
The major respiratory quinone is MK-6, phosphatidylethanolamine is the major respiratory
64
polar lipid, and straight- and branched-chain unsaturated fatty acids are the predominant fatty
65
acids (Nedashkovskaya & Kim, 2011). The genomic DNA G+C content varies between 30-37
66
mol% (Lee et al., 2012).
67 68
Strain 96_Hippo_TS_3/13T was isolated in March 2013 from a ditch sample taken
69
from a brood pouch of a male seahorse of the species Hippocampus barbouri kept at the
70
animal facility of the University of Giessen, Germany. At the time of sampling the seahorse
71
was diseased and later died on the so-called „air bubble“ disease. The ditch sample was
72
serially diluted in 0.9 %(v/w) autoclaved NaCl up to a dilution of 10-5 and 100 µL of each
73
dilution were plated on Tryptic soy agar (TSA, Oxoid) supplemented with 1.5 (w/v)% NaCl.
74
After three days of incubation at 28°C in the dark, strain 96_Hippo_TS_3/13T was isolated 3
75
from the 10-3 dilution of the ditch sample. The original colony had a diameter of
76
approximately 1 cm, with smooth edges and an orange pigmentation. The strain was purified
77
and sub-cultured on TSA supplemented with 1.5% (w/v) NaCl at 28°C. For long-term
78
storage, the strain was cultured in Tryptic soy broth (TSB, Oxoid) supplement with 1.5%
79
(v/w%) NaCl at 28°C, mixed after growth with a final concentration of 20 % (v/v) glycerol,
80
and stored at -80°C after the culture was shock-frozen in liquid nitrogen.
81 82
For phylogenetic analysis, genomic DNA of strain 96_Hippo_TS_3/13T was released
83
from a loop of cell biomass suspended in pure water (Carl Roth GmbH, Germany) and
84
subjected to three cycles of freezing (-20°C) and heating (100°C, 1 min). The nearly full-
85
length 16S rRNA gene of strain 96_Hippo_TS_3/13T was PCR-amplified with the primer
86
system
87
ACGGCTACCTTGTTACGACTT-3´, Lane et al., 1991) with 1 µL cell lysate as template in
88
a 25 µL PCR reaction containing 1x DreamTaq PCR buffer, 0.2 mM of each dNTPs, 0.2 µM
89
of each primer, 0.04 mg L-1 BSA, and 0.5 U DreamTaq DNA polymerase. All chemicals
90
except for the primers were obtained from Thermo Scientific (formerly Fermentas). PCR
91
conditions were as follows: 95°C, 3 min, 28 cycle of 95°C, 30 sec, 57.3°C, 30 sec, and 72°C,
92
1.5 min, and finally 72°C, 15 min. PCR purification and Sanger sequencing with primer 27F
93
(5´-GAGTTTGATCMTGGCTCAG-3´) and E786F (5´-GATTAGATACCCTGGTAG-3´)
94
(Coloqhoun, 1997) were performed by LGC Genomics, Berlin. The DNA sequence was
95
manually processed in MEGA 5 (Tamura et al., 2011) based on the electropherograms. After
96
removing ambiguous positions at the 5´and 3´ends, the final sequence was 1447 nt spanning
97
16S rRNA gene positions 46 to 1505 (according to the E. coli numbering, Brosius et al.,
98
1978). A first phylogenetic placement of the strain was obtained using the EzTaxon type
99
strain database (Kim et al., 2012).
8F
(5´-AGAGTTTGATCCTGGCTCAG-3´)
4
and
1492R
(5´-
100
Detailed phylogenetic analyses were performed in ARB release 5.2 (Ludwig et al.,
101
2004) using the “All-Species Living Tree" Project (LTP) (Yarza et al., 2008) database
102
LTPs115 (March, 2014). Sequences not included in the database were aligned using the
103
SILVA Incremental Aligner (SINA; v1.2.11; Pruesse et al., 2012) based on the SILVA seed
104
alignment [http://www.arb-silva.de; Pruesse et al. (2007)] and implemented into the LTP
105
database. The alignment of all sequences included in the analysis was checked manually.
106
Sequence similarities were calculated with the ARB Neighbor-joining tool, without an
107
evolutionary model. Maximum-likelihood trees were calculated with RAxML version 7.04
108
(Stamatakis, 2006) using GTR-GAMMA and a rapid bootstrap analysis and PhyML with the
109
HKM nucleotide substitution model, a neighbor-joining tree was calculated using ARB
110
Neighbor joining with the Jukes-Cantor correction (Jukes & Cantor, 1969), and a maximum-
111
parsimony tree using DNAPARS v 3.6 (Felsenstein, 2005). Tree calculations were based on
112
100 resamplings (bootstrap analysis; Felsenstein, 1985) and sequences spanning gene termini
113
62 to 1401 (E. coli numbering, Brosius et al., 1978).
114
The initial EzTaxon analysis showed that strain 96_Hippo_TS_3/13T shared 94.0%
115
16S rRNA gene sequence similarity with Mesonia mobilis KMM 6059T (Acc. number:
116
DQ367409). Sequence similarities to all other type strains were lower. A detailed
117
phylogenetic analysis in ARB showed that strain 96_Hippo_TS_3/13T was placed within the
118
monophyletic cluster formed by the type strain of the fife Mesonia species, M. mobilis, M.
119
phycicola, M. algae, M. ostreae, and M. aquimarina (Fig. 1). A distinct clustering (supported
120
by high bootstrap values) to any of the Mesonia type strains was not obtained. In the
121
maximum-parsimony and neighbor-joining trees, strain 96_Hippo_TS_3/13T formed an
122
outlying branch next to the type strains of the five Mesonia species, but, in the maximum-
123
likelihood tree, strain 96_Hippo_TS_3/13T clustered among the Mesonia type strains (Fig. 1).
124
Strain 96_Hippo_TS_3/13T shared only 92.2-93.7% sequence similarity to the type strains of
125
the fife Mesonia species, with highest sequence similarity to Mesonia mobilis KMM 6059T as 5
126
already obtained in the EzTaxon analysis. Sequence similarities to other genera placed in the
127
phylogenetic tree next to the monophyletic cluster of the genus Mesonia were below 92%,
128
Salegentibacter (90.0-91.9%), Salinimicrobium (90.6-91.4%), Psychroflexus (90.2-91.4%),
129
Zunongwangia (91.1-91.3%), Aquimarina (89.5-91.3%), Gramella (89.4-90.6%) and Gillisia
130
(88.5-89.9%).
131 132
For further genotypic analyses, high molecular weight genomic DNA was extracted as
133
described by Pitcher et al. (1989). The genomic DNA G+C content was determined by the
134
DNA melting temperature method established by Gonzales and Saiz-Jimenez (2002) as
135
described previously (Glaeser et al., 2013). The genomic DNA G+C content determined for
136
96_Hippo_TS_3/13T was 30 mol%, which was slightly lower than the range of G+C content
137
reported for Mesonia type strains (30 to 42.1%; Lee et al., 2012).
138 139
For phenotypic tests, strain 96_Hippo_TS_3/13T was cultured on TSA at 28°C for 3
140
days. Gram-staining was performed by the modified Hucker method according to Gerhardt et
141
al. (1994). Morphology analyses were performed by light microscopy at 1000 ×
142
magnification (Axiophot 2, Carl Zeiss AG, Germany) with glass slides covered with three
143
times washed 2% (w/v) agar (Becton Dickinson GmbH, Germany). The AxioVision Rel. 4.7.
144
(Carl Zeiss) software was used for cell size measurements. Gliding motility was tested as
145
described by Bernardet et al. (2002) using the hanging trop methods and phase contrast
146
microcopy. Cytochrome oxidase activity was tested with Microbiology Bactident oxidase test
147
strip (Merck), catalase activity by texting gas bubble formation after dropping HCl onto a
148
fresh culture grown on TSA. The presence of flexirubin type pigments was tested with the
149
KOH method according to Reichenbach (1992). Growth was tested at 28°C on following
150
growth media, Tryptic Soy Agar (TSA, Becton Dickinson GmbH, Germany), M92 (according
151
to DSMZ, Germany), CASO agar (Carl Roth GmbH + Co. KG, Germany), Luria Bertani (LB, 6
152
Sigma-Aldrich Chemie GmbH, Germany), Marine agar 2216 (MA, Becton Dickinson GmbH,
153
Germany), R2A (Oxoid, Germany), Nutrient (Oxoid, Germany), Malt agar (Merck KAaG,
154
Darmstadt, Germany), Medium 65 (according to DSMZ), DEV agar (DEV, Merck KAaG,
155
Darmstadt, Germany), K7 [0.1% (w/v) of yeast extract, peptone, and glucose, 15 g L-1 agar,
156
pH 7.2], and Nutrient agar (NA, Becton Dickinson GmbH, Germany). Temperature-
157
dependent growth was tested on M92 agar at 4, 10, 15, 20, 25, 28, 30, 36, 45, 50 and 55°C;
158
salinity and pH-dependent growth at 28°C in TSB supplemented with 1 to 12% (w/v) NaCl
159
(in 1% intervals) or adjusted to pH values of pH 4.5 to 11.5 (in 1 pH units intervals) adjusted
160
with 6 M HCl and 1 M NaOH after autoclaving. Anaerobic growth was tested on MA at 25°C
161
in an anaerobic jar with an oxygen depleted CO2 enriched atmosphere generated by the
162
addition of Anaerocult C (Merck, Germany). Further physiological tests were performed with
163
API20NE, APIZYM and API50CH test strips (bioMérieux) as described by the manufacturer.
164
For API20NE and APIZYM bacterial biomass was suspended in 0.9% (w/v) NaCl, for the
165
assimilation testing using API50CH, bacterial biomass was suspended in 0.2% (w/v) sea salts
166
(Sigma-Aldrich Chemie GmbH, Germany) as performed by Choi et al. (2014) and for acid
167
production (API 50CH) with CHB/E media (bioMérieux).
168
Strain 96_Hippo_TS_3/13T was rod-shaped with a cell size of 0.5 (± 0.1) x 1.5 (± 0.1)
169
µm (Supplementary Fig. 1). It shows optimal growth on 28°C on TSA with 1.5% (w/v) NaCl
170
and pH 7.5 to 9.5 similar to other Mesonia type strains. In contrast to other Mesonia species,
171
strain 96_Hippo_TS_3/13T showed a positive reaction for flexirubin-type pigments.
172
Comparison of the physiological properties of strain 96_Hippo_TS_3/13T with other Mesonia
173
type strains showed 18 to 23 differentiating out of 79 tested reactions to other Mesonia type
174
strains (Nedashkovskaya et al., 2003; 2006; Kang et al., 2010; Lee et al., 2012; Choi et al.,
175
2014). Differentiating characteristic compared to other Mesonia type strains are listed in
176
Table 1. All physiological properties are provided in the species description.
177 7
178
Biomass for fatty acid analyses was harvested after growth on MA at 25°C for 3 days
179
(late exponential growth phase) as reported for all other Mesonia type strains. The analysis
180
was performed as described by Kämpfer & Kroppenstedt (1996) by fatty acid separation with
181
a 5898A gas chromatograph (Hewlett Packard). Peaks were automatically integrated and fatty
182
acid names and percentages were determined with the Sherlock MIDI version 2.1 (TSBA
183
version 4.1).
184
The fatty acids profile showed the characteristic profile described for the genus Mesonia with
185
several branched and hydroxyl fatty acids and highest amounts of iso-C15:0 (40.9%) followed
186
by iso-C17:0 3-OH (14.8%). Differences in the concentration of several fatty acids
187
differentiates the new strain form other Mesonia type strains (Table 2).
188 189
For production of biomass subjected to analyses of the peptidoglycan diamino acid,
190
polyamines, quinones and polar lipids cells were grown in 3.3xPYE broth (1.0% peptone
191
from casein, 1.0% yeast extract, pH 7.2), supplemented with 3% Marine sea salts (Tetra
192
GmbH, Melle, Germany). The diamino acid meso-diaminopimelic acid was identified from
193
extract of whole cells as described by Schumann (2011). Though this is for the first time that
194
the presence of meso-diaminopimelic acid is reported in a strain of Mesonia this result is not
195
surprising because so far no species of the Flavobacteriaceae has been reported not to contain
196
the diagnostic diamino acid meso-diaminopimelic acid in the peptidoglycan.
197
Polyamines were extracted from biomass that was harvested at the late exponential growth
198
phase (Busse & Auling, 1988). Analyses of polyamines by HPLC was carried out as
199
described by Busse et al. (1997). Quinones and polar lipids were extracted from biomass that
200
had been harvested at the stationary growth phase, applying an integrated procedure and
201
analyzed as reported previously (Tindall, 1990a, b; Altenburger et al., 1996). The HPLC
202
apparatus used to analyze polyamines and quinones was described by Stolz et al. (2007). In
203
the polyamine pattern, 3.0 µmol (g dry weight)-1 sym-homospermidine, 0.1 µmol (g dry 8
204
weight)-1 cadaverine and trace amounts [
International Journal of Systematic and Evolutionary Microbiology Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's Seahorse (Hippocampus barbouri) --Manuscript Draft-Manuscript Number:
IJS-D-15-00164R1
Full Title:
Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's Seahorse (Hippocampus barbouri)
Short Title:
Mesonia hippocampi sp. nov.
Article Type:
Note
Section/Category:
New taxa - Bacteroidetes
Corresponding Author:
Stefanie P. Glaeser Justus-Liebig-Universität Giessen Giessen, GERMANY
First Author:
Judy Kolberg
Order of Authors:
Judy Kolberg Hans-Jürgen Busse Thomas Wilke Patrick Schubert Peter Kämpfer Stefanie P. Glaeser
Manuscript Region of Origin:
GERMANY
Abstract:
An orange pigmented gram-staining-negative, rod-shaped bacterium, designated 96_Hippo_TS_3/13T was isolated from the brood pouch of a diseased seahorse male of the species Hippocampus barbouri from the animal facility of the University of Giessen, Giessen, Hesse, Germany. Phylogenetic analyses based on the nearly fulllength 16S rRNA gene sequence placed strain 96_Hippo_TS_3/13T into the monophyletic cluster of the genus Mesonia within the Flavobacteriaceae. However, the strain shared only 92.2-93.8% sequence similarity to type strains of Mesonia species, with highest sequence similarity to the type strain of Mesonia aquimarina. The fatty acids profile showed a Mesonia-typical fatty acid profile including several branched and hydroxyl fatty acids with highest amounts of iso-C15:0 (40.9%) followed by iso-C17:0 3-OH (14.8%). In the polyamine pattern sym-homospermidine was predominant. The diagnostic diamino acid of the peptidoglycan was mesodiaminopimelic acid. The quinone system contain exclusively menaquinone MK-6. The only identified compound in the polar lipid profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts of an unidentified aminolipid and two unidentified lipids not containing a phosphate-group, an amino-group or a sugar residue were detected. The genomic G+C content of strain 96_Hippo_TS_3/13T was 30 mol% Based on genotypic, chemotaxonomic and physiological characterizations we proposed a new species of the genus Mesonia, Mesonia hippocampi with strain 96_Hippo_TS_3/13T (= DSM 29615T = CIP-110839T) as type strain.
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Manuscript Including References (Word document) Click here to download Manuscript Including References (Word document): Mesonia_hippocampi_manu_resubmission_ang2.docx
1
Mesonia hippocampi sp. nov. isolated from the brood pouch of a diseased Barbour's
2
Seahorse (Hippocampus barbouri)
3 4
Judy Kolberg1, 2, Hans-Jürgen Busse3, Thomas Wilke2, Patrick Schubert2, Peter Kämpfer1,
5
Stefanie P. Glaeser1*
6 7
1
8
Germany
9
2
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen,
Institut für Tierökologie und Spezielle Zoologie, Justus-Liebig-Universität Giessen, D-35392
10
Giessen, Germany
11
3
Institut für Mikrobiologie, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria
12 13
Corresponding author:
14
Stefanie P. Glaeser
15
Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen
16
Heinrich-Buff-Ring 26–32; D-35392 Giessen, Germany
17
Tel. +49 641 99 37373 ; Fax. +49 641 99 37359
18
e-mail: [email protected]
19 20
Short title: Mesonia hippocampi sp. nov.
21
Subject category: New Taxa; Subsection: Bacteroidetes
22 23 24
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
25
96_Hippo_TS_3/13T is KP119858.
1
26
Abstract
27
An
28
96_Hippo_TS_3/13T was isolated from the brood pouch of a diseased seahorse male of the
29
species Hippocampus barbouri from the animal facility of the University of Giessen, Giessen,
30
Hesse, Germany. Phylogenetic analyses based on the nearly full-length 16S rRNA gene
31
sequence placed strain 96_Hippo_TS_3/13T into the monophyletic cluster of the genus
32
Mesonia within the family Flavobacteriaceae. However, the strain shared only 92.2-93.8%
33
sequence similarity to type strains of Mesonia species, with highest sequence similarity to the
34
type strain of Mesonia aquimarina.
35
The fatty acids profile showed a Mesonia-typical fatty acid profile including several branched
36
and hydroxyl fatty acids with highest amounts of iso-C15:0 (40.9%) followed by iso-C17:0 3-
37
OH (14.8%). In the polyamine pattern sym-homospermidine was predominant. The diagnostic
38
diamino acid of the peptidoglycan was meso-diaminopimelic acid. The quinone system
39
contained exclusively menaquinone MK-6. The only identified compound in the polar lipid
40
profile was phosphatidylethanolamine present in major amounts. Additionally, major amounts
41
of an unidentified aminolipid and two unidentified lipids not containing a phosphate-group,
42
an amino-group or a sugar residue were detected. The genomic G+C content of strain
43
96_Hippo_TS_3/13T was 30 mol%
44
Based on genotypic, chemotaxonomic and physiological characterizations we proposed a new
45
species of the genus Mesonia, Mesonia hippocampi with strain 96_Hippo_TS_3/13T (= CIP-
46
110839T = LMG 28572T = CCM 8557T) as the type strain.
orange
pigmented,
Gram-staining-negative,
47 48
2
rod-shaped
bacterium,
designated
49
The genus Mesonia forms a monophyletic clade within the Flavobacteriaceae
50
(Bacteriodestes). It was first proposed in 2003 by Nedashkovskaya et al. (2003). At the time
51
of writing the genus contained five species, Mesonia algae (Nedashkovskaya et al. 2003),
52
Mesonia mobilis (Nedashkovskaya et al., 2006), Mesonia phycicola (Kang & Lee 2010),
53
Mesonia ostreae (Lee et al., 2012) and Mesonia aquimarina (Choi et al., 2014). Type strains
54
of all species were isolated from marine ecosystem, including from green algae of the species
55
Acrosiphonia sonderi in the Sea of Japan (Nedashkovskaya et al., 2003), seawater of the Sea
56
of Japan (Nedashkovskaya et al., 2006), of an oyster farm in the South Sea of Korea (Lee et
57
al., 2012) and off the east sea of Korea (Choi et al., 2014), and from seaweeds near the island
58
of Maryland in Korea (Kang & Lee, 2010).
59
Mesonia are described as thin-rod shaped strictly aerobic chemoheterotrophic bacteria
60
forming yellow colonies with a non-diffusible yellow pigments but not producing flexirubin-
61
type pigments. Cells are oxidase-, catalase- and alkaline phosphatase positive and negative for
62
ß-galactosidase activity. Seawater or Na+ ions are reported to be a prerequisite for growth.
63
The major respiratory quinone is MK-6, phosphatidylethanolamine is the major respiratory
64
polar lipid, and straight- and branched-chain unsaturated fatty acids are the predominant fatty
65
acids (Nedashkovskaya & Kim, 2011). The genomic DNA G+C content varies between 30-37
66
mol% (Lee et al., 2012).
67 68
Strain 96_Hippo_TS_3/13T was isolated in March 2013 from a ditch sample taken
69
from a brood pouch of a male seahorse of the species Hippocampus barbouri kept at the
70
animal facility of the University of Giessen, Germany. At the time of sampling the seahorse
71
was diseased and later died on the so-called „air bubble“ disease. The ditch sample was
72
serially diluted in 0.9 %(v/w) autoclaved NaCl up to a dilution of 10-5 and 100 µL of each
73
dilution were plated on Tryptic soy agar (TSA, Oxoid) supplemented with 1.5 (w/v)% NaCl.
74
After three days of incubation at 28°C in the dark, strain 96_Hippo_TS_3/13T was isolated 3
75
from the 10-3 dilution of the ditch sample. The original colony had a diameter of
76
approximately 1 cm, with smooth edges and an orange pigmentation. The strain was purified
77
and sub-cultured on TSA supplemented with 1.5% (w/v) NaCl at 28°C. For long-term
78
storage, the strain was cultured in Tryptic soy broth (TSB, Oxoid) supplement with 1.5%
79
(v/w%) NaCl at 28°C, mixed after growth with a final concentration of 20 % (v/v) glycerol,
80
and stored at -80°C after the culture was shock-frozen in liquid nitrogen.
81 82
For phylogenetic analysis, genomic DNA of strain 96_Hippo_TS_3/13T was released
83
from a loop of cell biomass suspended in pure water (Carl Roth GmbH, Germany) and
84
subjected to three cycles of freezing (-20°C) and heating (100°C, 1 min). The nearly full-
85
length 16S rRNA gene of strain 96_Hippo_TS_3/13T was PCR-amplified with the primer
86
system
87
ACGGCTACCTTGTTACGACTT-3´, Lane et al., 1991) with 1 µL cell lysate as template in
88
a 25 µL PCR reaction containing 1x DreamTaq PCR buffer, 0.2 mM of each dNTPs, 0.2 µM
89
of each primer, 0.04 mg L-1 BSA, and 0.5 U DreamTaq DNA polymerase. All chemicals
90
except for the primers were obtained from Thermo Scientific (formerly Fermentas). PCR
91
conditions were as follows: 95°C, 3 min, 28 cycle of 95°C, 30 sec, 57.3°C, 30 sec, and 72°C,
92
1.5 min, and finally 72°C, 15 min. PCR purification and Sanger sequencing with primer 27F
93
(5´-GAGTTTGATCMTGGCTCAG-3´) and E786F (5´-GATTAGATACCCTGGTAG-3´)
94
(Coloqhoun, 1997) were performed by LGC Genomics, Berlin. The DNA sequence was
95
manually processed in MEGA 5 (Tamura et al., 2011) based on the electropherograms. After
96
removing ambiguous positions at the 5´and 3´ends, the final sequence was 1447 nt spanning
97
16S rRNA gene positions 46 to 1505 (according to the E. coli numbering, Brosius et al.,
98
1978). A first phylogenetic placement of the strain was obtained using the EzTaxon type
99
strain database (Kim et al., 2012).
8F
(5´-AGAGTTTGATCCTGGCTCAG-3´)
4
and
1492R
(5´-
100
Detailed phylogenetic analyses were performed in ARB release 5.2 (Ludwig et al.,
101
2004) using the “All-Species Living Tree" Project (LTP) (Yarza et al., 2008) database
102
LTPs115 (March, 2014). Sequences not included in the database were aligned using the
103
SILVA Incremental Aligner (SINA; v1.2.11; Pruesse et al., 2012) based on the SILVA seed
104
alignment [http://www.arb-silva.de; Pruesse et al. (2007)] and implemented into the LTP
105
database. The alignment of all sequences included in the analysis was checked manually.
106
Sequence similarities were calculated with the ARB Neighbor-joining tool, without an
107
evolutionary model. Maximum-likelihood trees were calculated with RAxML version 7.04
108
(Stamatakis, 2006) using GTR-GAMMA and a rapid bootstrap analysis and PhyML with the
109
HKM nucleotide substitution model, a neighbor-joining tree was calculated using ARB
110
Neighbor joining with the Jukes-Cantor correction (Jukes & Cantor, 1969), and a maximum-
111
parsimony tree using DNAPARS v 3.6 (Felsenstein, 2005). Tree calculations were based on
112
100 resamplings (bootstrap analysis; Felsenstein, 1985) and sequences spanning gene termini
113
62 to 1401 (E. coli numbering, Brosius et al., 1978).
114
The initial EzTaxon analysis showed that strain 96_Hippo_TS_3/13T shared 94.0%
115
16S rRNA gene sequence similarity with Mesonia mobilis KMM 6059T (Acc. number:
116
DQ367409). Sequence similarities to all other type strains were lower. A detailed
117
phylogenetic analysis in ARB showed that strain 96_Hippo_TS_3/13T was placed within the
118
monophyletic cluster formed by the type strain of the fife Mesonia species, M. mobilis, M.
119
phycicola, M. algae, M. ostreae, and M. aquimarina (Fig. 1). A distinct clustering (supported
120
by high bootstrap values) to any of the Mesonia type strains was not obtained. In the
121
maximum-parsimony and neighbor-joining trees, strain 96_Hippo_TS_3/13T formed an
122
outlying branch next to the type strains of the five Mesonia species, but, in the maximum-
123
likelihood tree, strain 96_Hippo_TS_3/13T clustered among the Mesonia type strains (Fig. 1).
124
Strain 96_Hippo_TS_3/13T shared only 92.2-93.7% sequence similarity to the type strains of
125
the fife Mesonia species, with highest sequence similarity to Mesonia mobilis KMM 6059T as 5
126
already obtained in the EzTaxon analysis. Sequence similarities to other genera placed in the
127
phylogenetic tree next to the monophyletic cluster of the genus Mesonia were below 92%,
128
Salegentibacter (90.0-91.9%), Salinimicrobium (90.6-91.4%), Psychroflexus (90.2-91.4%),
129
Zunongwangia (91.1-91.3%), Aquimarina (89.5-91.3%), Gramella (89.4-90.6%) and Gillisia
130
(88.5-89.9%).
131 132
For further genotypic analyses, high molecular weight genomic DNA was extracted as
133
described by Pitcher et al. (1989). The genomic DNA G+C content was determined by the
134
DNA melting temperature method established by Gonzales and Saiz-Jimenez (2002) as
135
described previously (Glaeser et al., 2013). The genomic DNA G+C content determined for
136
96_Hippo_TS_3/13T was 30 mol%, which was slightly lower than the range of G+C content
137
reported for Mesonia type strains (30 to 42.1%; Lee et al., 2012).
138 139
For phenotypic tests, strain 96_Hippo_TS_3/13T was cultured on TSA at 28°C for 3
140
days. Gram-staining was performed by the modified Hucker method according to Gerhardt et
141
al. (1994). Morphology analyses were performed by light microscopy at 1000 ×
142
magnification (Axiophot 2, Carl Zeiss AG, Germany) with glass slides covered with three
143
times washed 2% (w/v) agar (Becton Dickinson GmbH, Germany). The AxioVision Rel. 4.7.
144
(Carl Zeiss) software was used for cell size measurements. Gliding motility was tested as
145
described by Bernardet et al. (2002) using the hanging trop methods and phase contrast
146
microcopy. Cytochrome oxidase activity was tested with Microbiology Bactident oxidase test
147
strip (Merck), catalase activity by texting gas bubble formation after dropping HCl onto a
148
fresh culture grown on TSA. The presence of flexirubin type pigments was tested with the
149
KOH method according to Reichenbach (1992). Growth was tested at 28°C on following
150
growth media, Tryptic Soy Agar (TSA, Becton Dickinson GmbH, Germany), M92 (according
151
to DSMZ, Germany), CASO agar (Carl Roth GmbH + Co. KG, Germany), Luria Bertani (LB, 6
152
Sigma-Aldrich Chemie GmbH, Germany), Marine agar 2216 (MA, Becton Dickinson GmbH,
153
Germany), R2A (Oxoid, Germany), Nutrient (Oxoid, Germany), Malt agar (Merck KAaG,
154
Darmstadt, Germany), Medium 65 (according to DSMZ), DEV agar (DEV, Merck KAaG,
155
Darmstadt, Germany), K7 [0.1% (w/v) of yeast extract, peptone, and glucose, 15 g L-1 agar,
156
pH 7.2], and Nutrient agar (NA, Becton Dickinson GmbH, Germany). Temperature-
157
dependent growth was tested on M92 agar at 4, 10, 15, 20, 25, 28, 30, 36, 45, 50 and 55°C;
158
salinity and pH-dependent growth at 28°C in TSB supplemented with 1 to 12% (w/v) NaCl
159
(in 1% intervals) or adjusted to pH values of pH 4.5 to 11.5 (in 1 pH units intervals) adjusted
160
with 6 M HCl and 1 M NaOH after autoclaving. Anaerobic growth was tested on MA at 25°C
161
in an anaerobic jar with an oxygen depleted CO2 enriched atmosphere generated by the
162
addition of Anaerocult C (Merck, Germany). Further physiological tests were performed with
163
API20NE, APIZYM and API50CH test strips (bioMérieux) as described by the manufacturer.
164
For API20NE and APIZYM bacterial biomass was suspended in 0.9% (w/v) NaCl, for the
165
assimilation testing using API50CH, bacterial biomass was suspended in 0.2% (w/v) sea salts
166
(Sigma-Aldrich Chemie GmbH, Germany) as performed by Choi et al. (2014) and for acid
167
production (API 50CH) with CHB/E media (bioMérieux).
168
Strain 96_Hippo_TS_3/13T was rod-shaped with a cell size of 0.5 (± 0.1) x 1.5 (± 0.1)
169
µm (Supplementary Fig. 1). It shows optimal growth on 28°C on TSA with 1.5% (w/v) NaCl
170
and pH 7.5 to 9.5 similar to other Mesonia type strains. In contrast to other Mesonia species,
171
strain 96_Hippo_TS_3/13T showed a positive reaction for flexirubin-type pigments.
172
Comparison of the physiological properties of strain 96_Hippo_TS_3/13T with other Mesonia
173
type strains showed 18 to 23 differentiating out of 79 tested reactions to other Mesonia type
174
strains (Nedashkovskaya et al., 2003; 2006; Kang et al., 2010; Lee et al., 2012; Choi et al.,
175
2014). Differentiating characteristic compared to other Mesonia type strains are listed in
176
Table 1. All physiological properties are provided in the species description.
177 7
178
Biomass for fatty acid analyses was harvested after growth on MA at 25°C for 3 days
179
(late exponential growth phase) as reported for all other Mesonia type strains. The analysis
180
was performed as described by Kämpfer & Kroppenstedt (1996) by fatty acid separation with
181
a 5898A gas chromatograph (Hewlett Packard). Peaks were automatically integrated and fatty
182
acid names and percentages were determined with the Sherlock MIDI version 2.1 (TSBA
183
version 4.1).
184
The fatty acids profile showed the characteristic profile described for the genus Mesonia with
185
several branched and hydroxyl fatty acids and highest amounts of iso-C15:0 (40.9%) followed
186
by iso-C17:0 3-OH (14.8%). Differences in the concentration of several fatty acids
187
differentiates the new strain form other Mesonia type strains (Table 2).
188 189
For production of biomass subjected to analyses of the peptidoglycan diamino acid,
190
polyamines, quinones and polar lipids cells were grown in 3.3xPYE broth (1.0% peptone
191
from casein, 1.0% yeast extract, pH 7.2), supplemented with 3% Marine sea salts (Tetra
192
GmbH, Melle, Germany). The diamino acid meso-diaminopimelic acid was identified from
193
extract of whole cells as described by Schumann (2011). Though this is for the first time that
194
the presence of meso-diaminopimelic acid is reported in a strain of Mesonia this result is not
195
surprising because so far no species of the Flavobacteriaceae has been reported not to contain
196
the diagnostic diamino acid meso-diaminopimelic acid in the peptidoglycan.
197
Polyamines were extracted from biomass that was harvested at the late exponential growth
198
phase (Busse & Auling, 1988). Analyses of polyamines by HPLC was carried out as
199
described by Busse et al. (1997). Quinones and polar lipids were extracted from biomass that
200
had been harvested at the stationary growth phase, applying an integrated procedure and
201
analyzed as reported previously (Tindall, 1990a, b; Altenburger et al., 1996). The HPLC
202
apparatus used to analyze polyamines and quinones was described by Stolz et al. (2007). In
203
the polyamine pattern, 3.0 µmol (g dry weight)-1 sym-homospermidine, 0.1 µmol (g dry 8
204
weight)-1 cadaverine and trace amounts [
