IJSEM Papers in Press. Published May 8, 2015 as doi:10.1099/ijs.0.000312
International Journal of Systematic and Evolutionary Microbiology Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal sediment --Manuscript Draft-Manuscript Number:
IJS-D-15-00226R1
Full Title:
Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal sediment
Short Title:
Rheinheimera aestuarii sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Che Ok Jeon Chung-Ang University Seoul, KOREA, REPUBLIC OF
First Author:
Kyunghwa Baek
Order of Authors:
Kyunghwa Baek Che Ok Jeon
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-staining-negative, strictly aerobic, non-pigmented and motile bacterium with a single polar flagellum, designated H29T, was isolated from coastal sediment of Jeju Island, South Korea. Cells were non-spore forming rods showing catalase- and oxidase-positive reactions. Growth of strain H29T was observed at 10-40 C (optimum, 20-25 C) and pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 1-4 % (w/v) NaCl (optimum, 2-3 %). Strain H29T contained C16:0, iso-C15:0 3-OH and summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c) as the major fatty acids and ubiquinone-8 (Q-8) as the sole isoprenoid quinone. Phosphatidylethanolamine and phosphatidylglycerol were identified as the major polar lipids. The G+C content of the genomic DNA was 46.5 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain H29T formed a phyletic lineage with Rheinheimera hassiensis E48T within the genus Rheinheimera of the family Chromatiaceae. Strain H29T was most closely related to Rheinheimera pacifica KMM 1406T, Rheinheimera muenzenbergensis E49T, Rheinheimera hassiensis E48T and Rheinheimera baltica OSBAC1T with 97.8 %, 97.6 %, 97.4 % and 97.2 % of 16S rRNA gene sequence similarities, respectively, but DNA-DNA hybridization values of strain H29T with these type species were lower than 70 %. On the basis of the phenotypic, chemotaxonomic and molecular properties, strain H29T represents a novel species of the genus Rheinheimera, for which the name Rheinheimera aestuarii sp. nov is proposed. The type strain is H29T (= KACC 18251T = JCM 30404T).
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1
Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal
2
sediment
3
Kyunghwa Baek1,2 and Che Ok Jeon1,*
4 5
1
Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea 2
Marine Microorganisms Team, National Marine Biodiversity Institute of Korea,
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Chungcheongnamdo 325-902, Republic of Korea
7 8
*Author for correspondence: Che Ok Jeon.
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Tel: +82-2-820-5864, Fax: +82-2-825-5206.
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E-mail: [email protected]
11 12
Running title: Rheinheimera aestuarii sp. nov.
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Subject category: New taxa (Gammaproteobacteria)
14 15
The GenBank accession number for the 16S rRNA gene sequence of strain H29T is
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KM588222.
17
Transmission electron micrographs, a picture of polar lipid analysis and the results of Biolog
18
GN2 MicroPlate assay are available as supplementary data in the online version of this paper.
19 1
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A Gram-staining-negative, strictly aerobic, non-pigmented and motile bacterium with a
21
single polar flagellum, designated H29T, was isolated from coastal sediment of Jeju
22
Island, South Korea. Cells were non-spore forming rods showing catalase- and oxidase-
23
positive reactions. Growth of strain H29T was observed at 10–40 C (optimum, 20–25
24
C) and pH 6.0–9.0 (optimum, pH 7.0–8.0) and in the presence of 1–4 % (w/v) NaCl
25
(optimum, 2–3 %). Strain H29T contained C16:0, iso-C15:0 3-OH and summed feature 3
26
(comprising C16:1 ω7c/C16:1 ω6c) as the major fatty acids and ubiquinone-8 (Q-8) as the
27
sole isoprenoid quinone. Phosphatidylethanolamine and phosphatidylglycerol were
28
identified as the major polar lipids. The G+C content of the genomic DNA was 46.5
29
mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain
30
H29T formed a phyletic lineage with Rheinheimera hassiensis E48T within the genus
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Rheinheimera of the family Chromatiaceae. Strain H29T was most closely related to
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Rheinheimera pacifica KMM 1406T, Rheinheimera muenzenbergensis E49T,
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Rheinheimera hassiensis E48T and Rheinheimera baltica OSBAC1T with 97.8 %, 97.6 %,
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97.4 % and 97.2 % of 16S rRNA gene sequence similarities, respectively, but DNA-DNA
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hybridization values of strain H29T with these type species were lower than 70 %. On
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the basis of the phenotypic, chemotaxonomic and molecular properties, strain H29T
37
represents a novel species of the genus Rheinheimera, for which the name Rheinheimera
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aestuarii sp. nov is proposed. The type strain is H29T (= KACC 18251T = JCM 30404T).
39 40
The genus Rheinheimera, a member of the family Chromatiaceae of the phylum
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Gammaproteobacteria, was first described by Brettar et al. (2002) with Rheinheimera baltica
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isolated from the central Baltic Sea. The genus Rheinheimera features Gram-staining2
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negative, oxidase-positive, catalase-variable, aerobic and flagellated rod-shaped to coccoid
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(Brettar et al., 2002). Cells of the genus Rheinheimera contain straight- and branched-
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saturated fatty acids as the major cellular fatty acids, ubiquinone-8 (Q-8) as the major
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isoprenoid quinone and phosphatidylethanolamine and phosphoglycerol as the major polar
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lipids and have a range of 47–52 mol% DNA G+C content (Yoon et al., 2007; Zhang et al.,
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2008; Chen et al. 2010; Liu et al., 2012; Suarez et al., 2014). At the time of writing, the genus
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Rheinheimera includes 16 type species with validly published names (Parte, 2014), which
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have been isolated from various environmental habitats such as seawater (Brettar et al., 2002,
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2006; Yoon et al., 2007; Romanenko et al., 2003), marine sediment (Li et al., 2011; Park et
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al., 2014), soil (Ryu et al., 2008), rhizosphere (Zhang et al., 2008; Suarez et al., 2014),
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chiromomid egg mass (Halpern et al., 2007) and freshwater (Merchant et al., 2007; Chen et
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al., 2010, 2013; Liu et al., 2012; Zhong et al., 2014), which suggests that members of the
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genus Rheinheimera may have diverse metabolic functions in environments. In this study, we
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isolated a presumably new Rheinheimera strain, designated H29T, from coastal sediment of
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Jeju Island in Korea and determined its taxonomic position using a polyphasic approach.
58 59
Strain H29T was isolated from coastal sea sediment (33° 30' 46.24" N 126° 53' 54.49" E),
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located in Jeju Island, Republic of Korea. A coastal sediment sample was obtained and
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serially diluted in 2 % (w/v) NaCl solution. The diluted samples were spread on marine agar
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2216 (MA, BD) and incubated at 25 °C for 3 days under an aerobic condition. The 16S rRNA
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genes of colonies grown on MA were PCR-amplified using the universal primers, F1 and
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R13, as described previously (Jeong et al., 2013). The amplicons were double-digested using
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a mixture of HaeIII and HhaI and their fragment patterns were analyzed on 2.5 % (w/v) 3
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agarose gels. The representative PCR products showing unique fragment patterns were
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partially sequenced using the F1 primer and the resulting 16S rRNA gene sequences were
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compared with those of validly reported all type strains using the Nucleotide Similarity
69
Search program in the EzTaxon-e server (Kim et al., 2012). A putative novel species
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belonging to the genus Rheinheimera, designated strain H29T, was selected for further
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analysis of the phenotypic and phylogenetic properties. Rheinheimera pacifica CCUG 46544T
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and Rheinheimera baltica DSM 14885T were purchased from their culture collection centers
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and Rheinheimera muenzenbergensis E49T and Rheinheimera hassiensis E48T were provided
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by Dr. Ratering in Justus-Liebig University Giessen, Germany as gifts. All four strains were
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used as reference strains for the comparisons of phenotypic properties and fatty acid
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composition and DNA-DNA hybridization.
77 78
The PCR amplicon from the 16S rRNA gene of strain H29T was ligated into the pCR2.1
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vector using a TOPO cloning kit (Invitrogen) and sequenced with the M13 reverse and T7
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primers of the pCR2.1 vector to obtain almost complete 16S rRNA gene sequence. The
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resulting 16S rRNA gene sequence (1419 nucleotides) of strain H29T was manually quality-
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checked and compared with those of all type strains using the Nucleotide Similarity Search
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program in the EzTaxon-e server. The 16S rRNA gene sequences of strain H29T and closely
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related taxa were aligned using the fast secondary-structure aware infernal aligner available
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in Ribosomal Database Project (RDP) (Nawrocki & Eddy, 2007). Phylogenetic trees based on
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the neighbor-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP)
87
algorithms were built using the MEGA6 software package (Tamura et al., 2013).
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Evolutionary distances for the NJ algorithm were calculated with the Juke-Cantor model and 4
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the nearest-neighbor-interchange (NNI) metric was applied to calculate the distances in the
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ML analysis. MP analysis was conducted using a branch swapping heuristic search option
91
based on Tree-Bisection Reconnection (TBR). The phylogenetic tree topologies were
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evaluated using bootstrap analyses based on 1,000 resampled datasets. DNA-DNA
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hybridization analyses between strain H29T and four reference strains, R. pacifica CCUG
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46544T, R. muenzenbergensis E49T, R. hassiensis E48T and R. baltica DSM 14885T, showing
95
greater than 97 % 16S rRNA gene sequence similarities were performed in triplicate to
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evaluate their DNA-DNA relatedness according to the procedure described previously (Lee et
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al., 2011) using the non-radioactive DIG-High Prime system (Roche). Hybridization signals
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were scanned and analyzed using an Adobe Photoshop CS6 (ver. 13.0). Hybridization signals
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produced by the hybridization of the probes to the homologous target DNA were taken to be
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100 %, and the signal intensities from self-hybridizations of serial dilutions were used for the
101
calculation of the DNA-DNA relatedness. The DNA-DNA relatedness was confirmed by
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reciprocal interchange of the probe and the target DNA.
103 104
Comparative analysis of the 16S rRNA gene sequences showed that strain H29T was most
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closely related to R. pacifica KMM 1406T, R. muenzenbergensis E49T, R. hassiensis E48T
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and R. baltica OSBAC1T with similarities of 97.8 %, 97.6 % 97.4 % and 97.2 %,
107
respectively. The phylogenetic tree using the NJ algorithm showed that strain H29T formed a
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phyletic lineage with R. hassiensis E48T within the genus Rheinheimera of the family
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Chromatiaceae (Fig. 1), which was also recovered by the phylogenetic inferences based on
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the MP and ML algorithms. The DNA-DNA relatedness of strain H29T with R. pacifica
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CCUG 46544T, R. muenzenbergensis E49T, R. hassiensis E48T and R. baltica DSM 14885T 5
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were 17.7 ± 4.5 %, 21.1 ± 6.0 %, 17.7 ± 4.5 % and 15.0 ± 4.1 %, respectively, which were
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clearly lower than the 70 % threshold generally accepted for the new species delineation
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(Rosselló-Mora & Amann, 2001). These results indicated that strain H29T could be
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considered as a novel species of the genus Rheinheimera.
116 117
Growth of strain H29T at different temperatures (5–45 °C at 5 °C intervals) and pH (5.0–10.0
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at 0.5 pH unit intervals) was assessed in marine broth. Marine broth with pH below 8.0 and
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pH 8.0–10.0 was prepared using the Na2HPO4-NaH2PO4 and Tris-HCl buffers, respectively.
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The pH values of the marine broth were adjusted again if necessary after sterilization
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(121 °C, 15 min). Growth of strain H29T at different NaCl concentrations (0, 0.5, 1, 2, 3, 4, 5
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and 6 % (w/v)) was tested in marine broth prepared in the laboratory according to the BD
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formula. The following physiological and biochemical tests were conducted using cells
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grown on MA for 2 days at 25 °C. Gram staining was investigated using the bioMérieux
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Gram stain kit according to the manufacturer’s instructions. Oxidase activity was evaluated
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by the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck) and catalase activity
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was tested by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide
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solution (Smibert & Krieg, 1994). Cell morphology and the presence of flagella were
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investigated using phase-contract microscopy and transmission electron microscopy (JEM-
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1010, JEOL) with cells from an exponentially grown culture in marine broth at 25 °C.
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Anaerobic growth was assessed on MA under the anaerobic condition (with 4–10 % CO2)
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using the GasPak Plus system (BBL) at 25 °C for 10 days. The following properties of strain
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H29T and reference strains were evaluated in parallel under the same conditions at 25 °C.
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Hydrolysis of casein, starch, aesculin, CM-cellulose, tyrosine, Tween 20 and Tween 80 was 6
135
investigated as described by Lányí (1987) using MA as the basal medium. Nitrate reduction
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was assessed according to the method descried previously (Lányí, 1987). Additional
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enzymatic activities, biochemical features and oxidation of carbon sources were evaluated
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using the API ZYM, API 20NE kits (bioMérieux) and GN2 MicroPlate system (Biolog),
139
respectively, according to the instructions of the manufacturers, except that the inocula of
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strain H29T and reference strains were prepared by resuspending cells in artificial seawater
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(20 g NaCl, 2.9 g MgSO4, 4.53 g MgCl2·6H2O, 0.64 g KCl and 1.75 g CaCl2·2H2O per liter).
142
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Cells were Gram-staining-negative rods with 0.8 to 1.2 m in width and 1.5 to 1.8 m in
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length (Supplementary Fig. S1, available in IJSEM online). Cells are motile by the presence
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of a single polar flagellum. Anaerobic growth was not observed on MA after 10 days of
146
incubation at 25 °C. Physiological and biochemical characteristics of strain H29T are
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additionally described in the species description and compared with those of the closely
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related type strains in Table 1 and Supplementary Table S1 (available in IJSEM Online).
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Most of properties such as catalase and urease activities and hydrolysis of carbon compounds
150
in strain H29T were consistent with those of other related-Rheinheimera species, while some
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other properties such as colony color, no growth at 4 °C and many other phenotypic
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properties differentiated strain H29T from other related-Rheinheimera species (Table 1).
153 154
The DNA G+C content of strain H29T was determined by the fluorometric method (Gonzalez
155
& Saiz-Jimenez, 2002) using SYBR Green I and a real-time PCR thermocycler (Bio-Rad).
156
Isoprenoid quinones were extracted according to the method of Minnikin et al. (1984) and 7
157
were analyzed using a HPLC (model LC-20A, Shimadzu) equipped with a diode array
158
detector (SPD-M20A, Shimadzu) and a reversed-phase column (250×4.6 mm, Kromasil,
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Akzo Nobel) as described previously (Komagata & Suzuki, 1987). Strain H29T and four
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reference strains were cultivated on marine broth at 25 °C for 12 hours for the analysis of
161
their cellular fatty acids. The fatty acids of cells were saponified, methylated and extracted
162
according to the standard protocol of the Sherlock Microbial Identification System (MIDI)
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version 6.0. The fatty acid methyl esters were analyzed by GC (Hewlett Packard 7890) and
164
identified by the TSBA6 database of the Microbial Identification System (Sasser, 1990).
165
Polar lipids of strain H29T were extracted, resolved by two-dimensional TLC and identified
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as described by Minnikin et al. (1984). The following reagents were used to detect different
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polar lipids: 10 % (w/v) ethanolic molybdatophosphoric acid (for total polar lipids),
168
ninhydrin (for aminolipids) and the Dittmer-Lester reagent (for phospholipids).
169 170
The genomic DNA G+C content of strain H29T was approximately 46.5 mol%, which is a
171
little lower than the range of DNA G+C contents of previously reported type species of the
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genus Rheinheimera (Yoon et al., 2007; Zhang et al., 2008; Chen et al. 2010; Liu et al., 2012;
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Suarez et al., 2014). The only respiratory quinone detected in strain H29T was ubiquinone-8
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(Q-8), which is a good agreement with the major or only respiratory quinone detected in all
175
members of the genus Rheinheimera. The major cellular fatty acids (>5 % of the total fatty
176
acids) of strain H29T were iso-C15:0 3-OH (31.3 %), summed feature 3 (comprising C16:1
177
ω7c/C16:1 ω6c, 15.7 %), C16:0 (10.8 %), summed feature 8 (comprising C18:1 ω7c and C18:1
178
ω6c, 5.7 %) and C12:0 3-OH (5.6 %). The overall fatty acid profile of strain H29T was similar
179
to those of the reference strains of the genus Rheinheimera; there were some differences in 8
180
the respective proportions of some fatty acid components, especially in hydroxyl fatty acids
181
(Table 2). The major polar lipid of strain H29T was phosphatidylethanolamine and
182
phosphatidylglycerol (Supplementary Fig. S2, available at IJSEM Online). Two unidentified
183
aminolipids, one aminophospholipid, two unidentified lipids were also detected as the minor
184
polar lipids. These characteristics of strain H29T are consistent with the description of the
185
genus Rheinheimera (Brettar et al., 2002; Yoon et al., 2007; Zhang et al., 2008 ; Chen et al.,
186
2010 ; Liu et al., 2012). In conclusion, the physiological and chemotaxonomic features and
187
the phylogenetic inference of strain H29T support the proposition that strain H29T represents
188
a novel species of the genus Rheinheimera, for which the name Rheinheimera aestuarii sp.
189
nov is proposed.
190
191
Description of Rheinheimera aestuarii sp. nov.
192
Rheinheimera aestuarii (a.es.tu.a´ri.i.L. gen. n. aestuarii of coastal sediment, from where the
193
organism was isolated).
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Colonies are white, circular, convex, smooth and entire margins with approximately 2 mm in
195
diameter after 2 days of incubation on MA agar. Cells are Gram-staining-negative, strictly
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aerobic rods and motile by the presence of a single polar flagellum. Growth occurs at 10–
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40 °C (optimum, 20–25 °C) and pH 6.0–9.0 (optimum, pH 7.0–8.0) and in the presence of 1–
198
4 % (w/v) NaCl (optimum, 2–3 %). Catalase and oxidase are positive. Nitrate is reduced to
199
nitrite. Casein, aesculin, starch, Tween 20 and Tween 80 are hydrolyzed, but CM-cellulose
200
and tyrosine are not. In the API 20NE strip, hydrolysis of aesculin and gelatin were detected,
201
but indole production, D-glucose fermentation, urease, arginine dihydrolase and β9
202
galactosidase activities, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-
203
acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid,
204
trisodium citrate and phenylacetic acid are absent. In the API ZYM strip, alkaline
205
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine
206
arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-
207
phosphophydrolase, N-acetyl-β-glucosaminidase activities are present. However, α-
208
chymotrypsin, α-galactosidase, β-glucuronidase, β-galactosidase, α-glucosidase, β-
209
glucosidase, α-mannosidase and β-fucosidase activities are absent. N-acetyl-D-galactosamine,
210
N-acetyl-D-glucosamine, L-arabitol, i-erythritol, L-fucose, gentiobiose, α-D-lactose,
211
lactulose, D-mellibiose, β-methyl-D-glucoside, turanose, xylitol, formic acid, D-galacturonic
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acid, D-glucosaminic acid, α-hydroxy butyric acid, β-hydroxy butyric acid, α-keto glutaric
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acid, L-alaninamide, L-alanine, L-alanyl-glycine, L-glutamic acid, glycyl-L-aspartic acid, L-
214
ornithine, L-proline, L-pyroglutamic acid, L-threonine, thymidine and phenyethylamine in
215
the GN2 MicroPlate are oxidized. The other organic substrates in the Biolog GN2
216
MicroPlates are not oxidized. The only respiratory quinone detected is Q-8 and the major
217
cellular fatty acids are iso-C15:0 3-OH, summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c),
218
C16:0, summed feature 8 (comprising C18:1 ω7c and C18:1 ω6c) and C12:0 3-OH. The major
219
polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content
220
of the type strain is 46.5 mol%.
221
The type strain is H29T (= KACC 18251T = JCM 30404T), which was isolated from coastal
222
sediment in Jeju Island, Republic Korea.
223
10
224
ACKNOWLEDGEMENTS
225
We thank Dr. S. Ratering in Justus-Liebig University Giessen for providing R.
226
muenzenbergensis E49T and R. hassiensis E48T. This work was supported by the Cooperative
227
Research Program for Agriculture Science & Technology Development (Project No.
228
PJ00999302), RDA, Republic of Korea.
229
230
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Uchimura, T. (2003). Rheinheimera pacifica sp. nov., a novel halotolerant bacterium isolated
283
from deep sea water of the Pacific. Int J Syst Evol Microbiol 53, 1973–1977.
284
Rosselló-Mora, R. & Amann, R. (2001). The species concept for prokaryotes. FEMS
285
Microbiol Rev 25, 39–67.
286
Ryu, S. H., Chung, B. S., Park, M., Lee, S. S., Lee, S. S. & Jeon, C. O. (2008). 13
287
Rheinheimera soli sp. nov., a gammaproteobacterium isolated from soil in Korea. Int J Syst
288
Evol Microbiol 58, 2271–2274.
289
Sasser, M. (1990). Identification of bacteria by gas chromatography of cellular fatty acids,
290
MIDI Technical Note 101. Newark, DE: MIDI Inc.
291
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General
292
and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt. Washington D.C.:
293
American Society for Microbiology.
294
Suarez, C., Ratering, S., Geissler-Plaum, R. & Schnell, S. (2014). Rheinheimera hassiensis
295
sp. nov. and Rheinheimera muenzenbergensis sp. nov., two species from the rhizosphere of
296
Hordeum secalinum. Int J Syst Evol Microbiol 64, 1202–1209.
297
Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6:
298
molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30, 2725–2729.
299
Yoon, J. H., Bae, S. E., Kang, S. J. & Oh, T. K. (2007). Rheinheimera aquimaris sp. nov.,
300
isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 1386–1390.
301
Zhang, X., Sun, L., Qiu, F., McLean, R. J. C., Jiang, R. & Song, W. (2008). Rheinheimera
302
tangshanensis sp. nov., a rice root-associated bacterium. Int J Syst Evol Microbiol 58, 2420–
303
2424.
304
Zhong, Z. P., Liu, Y., Liu, L. Z., Wang, F., Zhou, Y. G. & Liu, Z. P. (2014). Rheinheimera
305
tuosuensis sp. nov., isolated from a saline lake. Int J Syst Evol Microbiol 64, 1142–1148.
14
306
Figure Legend
307
Fig. 1. A neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic
308
relationships of strain H29T and related taxa. Bootstrap values are shown on nodes in
309
percentages of 1,000 replicates; only values over 70% are given. Closed circles are indicated,
310
when the corresponding nodes were recovered in the maximum-likelihood and maximum-
311
parsimony algorithms. Serratia entomophila DSM 12358T (AJ233427) was used as an
312
outgroup (not shown). The scale bar equals 0.01 changes per nucleotide position.
313
15
314 315
Table 1. Phenotypic characteristics of strain H29T and related type strains of the genus Rheinheimera
316 317 318 319 320 321 322 323
Strains: 1, H29T; 2, R. pacifica CCUG 46544T (Romanenko et al., 2003); 3, R. muenzenbergensis E49T (Suarez et al., 2014); 4, R. hassiensis E48T (Suarez et al., 2014); 5, R. baltica DSM 14885T (Brettar et al., 2002). All strains are positive for the following characteristics: hydrolysis of Tween 20, Tween 80, aesculin and starch and oxidase, urease, alkaline phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase activities. All strains are negative for the following characteristics: Gram staining, hydrolysis of CM-cellulose and tyrosine, indole production, D-glucose fermentation, assimilation of D-mannose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid and phenylacetic acid and α-galactosidase, β-galactosidase, β-glucuronidase, αglucosidase, β-glucosidase and α-fucosidase activities. Symbols: +, positive; , negative. Characteristic
1*
2
3
4
5
white
Dark purple
yellow
brown
yellow
0.8–1.2 ×
0.4–0.8 ×
0.5–0.8 ×
0.4–0.8 ×
0.5–1.5 ×
1.5–1.8 + 10–40 1–4 6.0–9.0 + + +
1.8–2.0 + 4–37 0–8 5.5–10.5 + + +
1.3–2.4 + 4–37 0–5 7.0–9.5 –
0.7–2.0 + 4–37 0–6 6.5–10.0 – +
0.9–2.5 4–30 0–6 5.7–10.0 + + +
+
+
+
+
–
+
+
+
–
–
+
+
–
–
–
+
+
+
–
+
α-Chymotrypsin
–
+
+
+
α-Mannosidase
–
+
–
+
–
–
–
– + + + 50.5
– – – – 48.9
Colony color Cell size (μm) Motility Temperature range (°C) NaCl range (%) pH range Catalase Nitrate reduction* Hydrolysis of casein* Enzyme activity (API ZYM)* of: Esterase (C4) Esterase lipase (C8), cystine arylamidase Lipase (C14), valine acrylamidase Trypsine
324
Acid phosphatase, N-acetyl-β+ + – glucosaminidase Assimilation (API 20NE)* of: D-Glucose – + – L-Arabinose – + – N-acetyl glucosamine, D-Maltose + + Trisodium citrate – – – DNA G+C content (mol%) 46.5 49.6 50.0 * These analyses were performed using the same conditions in this study.
16
325 326
Table 2. Cellular fatty acid compositions (%) of strain H29T and related type strains of the genus Rheinheimera
327 328 329 330
Strains: 1, H29T; 2, R. pacifica CCUG 46544T; 3, R. muenzenbergensis E49T; 4, R. hassiensis E48T;5, R. baltica DSM 14885T. All data were from this study. All strains were grown on marine broth at 25 °C for 12 hrs. Data are expressed as percentages of total fatty acids. Major components (> 5.0 %) are highlighted in bold. Fatty acids amounting to less than 0.5 % in all strains are not shown. tr, trace amount (
International Journal of Systematic and Evolutionary Microbiology Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal sediment --Manuscript Draft-Manuscript Number:
IJS-D-15-00226R1
Full Title:
Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal sediment
Short Title:
Rheinheimera aestuarii sp. nov.
Article Type:
Note
Section/Category:
New taxa - Proteobacteria
Corresponding Author:
Che Ok Jeon Chung-Ang University Seoul, KOREA, REPUBLIC OF
First Author:
Kyunghwa Baek
Order of Authors:
Kyunghwa Baek Che Ok Jeon
Manuscript Region of Origin:
KOREA, REPUBLIC OF
Abstract:
A Gram-staining-negative, strictly aerobic, non-pigmented and motile bacterium with a single polar flagellum, designated H29T, was isolated from coastal sediment of Jeju Island, South Korea. Cells were non-spore forming rods showing catalase- and oxidase-positive reactions. Growth of strain H29T was observed at 10-40 C (optimum, 20-25 C) and pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 1-4 % (w/v) NaCl (optimum, 2-3 %). Strain H29T contained C16:0, iso-C15:0 3-OH and summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c) as the major fatty acids and ubiquinone-8 (Q-8) as the sole isoprenoid quinone. Phosphatidylethanolamine and phosphatidylglycerol were identified as the major polar lipids. The G+C content of the genomic DNA was 46.5 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain H29T formed a phyletic lineage with Rheinheimera hassiensis E48T within the genus Rheinheimera of the family Chromatiaceae. Strain H29T was most closely related to Rheinheimera pacifica KMM 1406T, Rheinheimera muenzenbergensis E49T, Rheinheimera hassiensis E48T and Rheinheimera baltica OSBAC1T with 97.8 %, 97.6 %, 97.4 % and 97.2 % of 16S rRNA gene sequence similarities, respectively, but DNA-DNA hybridization values of strain H29T with these type species were lower than 70 %. On the basis of the phenotypic, chemotaxonomic and molecular properties, strain H29T represents a novel species of the genus Rheinheimera, for which the name Rheinheimera aestuarii sp. nov is proposed. The type strain is H29T (= KACC 18251T = JCM 30404T).
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1
Rheinheimera aestuarii sp. nov., a marine bacterium isolated from coastal
2
sediment
3
Kyunghwa Baek1,2 and Che Ok Jeon1,*
4 5
1
Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea 2
Marine Microorganisms Team, National Marine Biodiversity Institute of Korea,
6
Chungcheongnamdo 325-902, Republic of Korea
7 8
*Author for correspondence: Che Ok Jeon.
9
Tel: +82-2-820-5864, Fax: +82-2-825-5206.
10
E-mail: [email protected]
11 12
Running title: Rheinheimera aestuarii sp. nov.
13
Subject category: New taxa (Gammaproteobacteria)
14 15
The GenBank accession number for the 16S rRNA gene sequence of strain H29T is
16
KM588222.
17
Transmission electron micrographs, a picture of polar lipid analysis and the results of Biolog
18
GN2 MicroPlate assay are available as supplementary data in the online version of this paper.
19 1
20
A Gram-staining-negative, strictly aerobic, non-pigmented and motile bacterium with a
21
single polar flagellum, designated H29T, was isolated from coastal sediment of Jeju
22
Island, South Korea. Cells were non-spore forming rods showing catalase- and oxidase-
23
positive reactions. Growth of strain H29T was observed at 10–40 C (optimum, 20–25
24
C) and pH 6.0–9.0 (optimum, pH 7.0–8.0) and in the presence of 1–4 % (w/v) NaCl
25
(optimum, 2–3 %). Strain H29T contained C16:0, iso-C15:0 3-OH and summed feature 3
26
(comprising C16:1 ω7c/C16:1 ω6c) as the major fatty acids and ubiquinone-8 (Q-8) as the
27
sole isoprenoid quinone. Phosphatidylethanolamine and phosphatidylglycerol were
28
identified as the major polar lipids. The G+C content of the genomic DNA was 46.5
29
mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain
30
H29T formed a phyletic lineage with Rheinheimera hassiensis E48T within the genus
31
Rheinheimera of the family Chromatiaceae. Strain H29T was most closely related to
32
Rheinheimera pacifica KMM 1406T, Rheinheimera muenzenbergensis E49T,
33
Rheinheimera hassiensis E48T and Rheinheimera baltica OSBAC1T with 97.8 %, 97.6 %,
34
97.4 % and 97.2 % of 16S rRNA gene sequence similarities, respectively, but DNA-DNA
35
hybridization values of strain H29T with these type species were lower than 70 %. On
36
the basis of the phenotypic, chemotaxonomic and molecular properties, strain H29T
37
represents a novel species of the genus Rheinheimera, for which the name Rheinheimera
38
aestuarii sp. nov is proposed. The type strain is H29T (= KACC 18251T = JCM 30404T).
39 40
The genus Rheinheimera, a member of the family Chromatiaceae of the phylum
41
Gammaproteobacteria, was first described by Brettar et al. (2002) with Rheinheimera baltica
42
isolated from the central Baltic Sea. The genus Rheinheimera features Gram-staining2
43
negative, oxidase-positive, catalase-variable, aerobic and flagellated rod-shaped to coccoid
44
(Brettar et al., 2002). Cells of the genus Rheinheimera contain straight- and branched-
45
saturated fatty acids as the major cellular fatty acids, ubiquinone-8 (Q-8) as the major
46
isoprenoid quinone and phosphatidylethanolamine and phosphoglycerol as the major polar
47
lipids and have a range of 47–52 mol% DNA G+C content (Yoon et al., 2007; Zhang et al.,
48
2008; Chen et al. 2010; Liu et al., 2012; Suarez et al., 2014). At the time of writing, the genus
49
Rheinheimera includes 16 type species with validly published names (Parte, 2014), which
50
have been isolated from various environmental habitats such as seawater (Brettar et al., 2002,
51
2006; Yoon et al., 2007; Romanenko et al., 2003), marine sediment (Li et al., 2011; Park et
52
al., 2014), soil (Ryu et al., 2008), rhizosphere (Zhang et al., 2008; Suarez et al., 2014),
53
chiromomid egg mass (Halpern et al., 2007) and freshwater (Merchant et al., 2007; Chen et
54
al., 2010, 2013; Liu et al., 2012; Zhong et al., 2014), which suggests that members of the
55
genus Rheinheimera may have diverse metabolic functions in environments. In this study, we
56
isolated a presumably new Rheinheimera strain, designated H29T, from coastal sediment of
57
Jeju Island in Korea and determined its taxonomic position using a polyphasic approach.
58 59
Strain H29T was isolated from coastal sea sediment (33° 30' 46.24" N 126° 53' 54.49" E),
60
located in Jeju Island, Republic of Korea. A coastal sediment sample was obtained and
61
serially diluted in 2 % (w/v) NaCl solution. The diluted samples were spread on marine agar
62
2216 (MA, BD) and incubated at 25 °C for 3 days under an aerobic condition. The 16S rRNA
63
genes of colonies grown on MA were PCR-amplified using the universal primers, F1 and
64
R13, as described previously (Jeong et al., 2013). The amplicons were double-digested using
65
a mixture of HaeIII and HhaI and their fragment patterns were analyzed on 2.5 % (w/v) 3
66
agarose gels. The representative PCR products showing unique fragment patterns were
67
partially sequenced using the F1 primer and the resulting 16S rRNA gene sequences were
68
compared with those of validly reported all type strains using the Nucleotide Similarity
69
Search program in the EzTaxon-e server (Kim et al., 2012). A putative novel species
70
belonging to the genus Rheinheimera, designated strain H29T, was selected for further
71
analysis of the phenotypic and phylogenetic properties. Rheinheimera pacifica CCUG 46544T
72
and Rheinheimera baltica DSM 14885T were purchased from their culture collection centers
73
and Rheinheimera muenzenbergensis E49T and Rheinheimera hassiensis E48T were provided
74
by Dr. Ratering in Justus-Liebig University Giessen, Germany as gifts. All four strains were
75
used as reference strains for the comparisons of phenotypic properties and fatty acid
76
composition and DNA-DNA hybridization.
77 78
The PCR amplicon from the 16S rRNA gene of strain H29T was ligated into the pCR2.1
79
vector using a TOPO cloning kit (Invitrogen) and sequenced with the M13 reverse and T7
80
primers of the pCR2.1 vector to obtain almost complete 16S rRNA gene sequence. The
81
resulting 16S rRNA gene sequence (1419 nucleotides) of strain H29T was manually quality-
82
checked and compared with those of all type strains using the Nucleotide Similarity Search
83
program in the EzTaxon-e server. The 16S rRNA gene sequences of strain H29T and closely
84
related taxa were aligned using the fast secondary-structure aware infernal aligner available
85
in Ribosomal Database Project (RDP) (Nawrocki & Eddy, 2007). Phylogenetic trees based on
86
the neighbor-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP)
87
algorithms were built using the MEGA6 software package (Tamura et al., 2013).
88
Evolutionary distances for the NJ algorithm were calculated with the Juke-Cantor model and 4
89
the nearest-neighbor-interchange (NNI) metric was applied to calculate the distances in the
90
ML analysis. MP analysis was conducted using a branch swapping heuristic search option
91
based on Tree-Bisection Reconnection (TBR). The phylogenetic tree topologies were
92
evaluated using bootstrap analyses based on 1,000 resampled datasets. DNA-DNA
93
hybridization analyses between strain H29T and four reference strains, R. pacifica CCUG
94
46544T, R. muenzenbergensis E49T, R. hassiensis E48T and R. baltica DSM 14885T, showing
95
greater than 97 % 16S rRNA gene sequence similarities were performed in triplicate to
96
evaluate their DNA-DNA relatedness according to the procedure described previously (Lee et
97
al., 2011) using the non-radioactive DIG-High Prime system (Roche). Hybridization signals
98
were scanned and analyzed using an Adobe Photoshop CS6 (ver. 13.0). Hybridization signals
99
produced by the hybridization of the probes to the homologous target DNA were taken to be
100
100 %, and the signal intensities from self-hybridizations of serial dilutions were used for the
101
calculation of the DNA-DNA relatedness. The DNA-DNA relatedness was confirmed by
102
reciprocal interchange of the probe and the target DNA.
103 104
Comparative analysis of the 16S rRNA gene sequences showed that strain H29T was most
105
closely related to R. pacifica KMM 1406T, R. muenzenbergensis E49T, R. hassiensis E48T
106
and R. baltica OSBAC1T with similarities of 97.8 %, 97.6 % 97.4 % and 97.2 %,
107
respectively. The phylogenetic tree using the NJ algorithm showed that strain H29T formed a
108
phyletic lineage with R. hassiensis E48T within the genus Rheinheimera of the family
109
Chromatiaceae (Fig. 1), which was also recovered by the phylogenetic inferences based on
110
the MP and ML algorithms. The DNA-DNA relatedness of strain H29T with R. pacifica
111
CCUG 46544T, R. muenzenbergensis E49T, R. hassiensis E48T and R. baltica DSM 14885T 5
112
were 17.7 ± 4.5 %, 21.1 ± 6.0 %, 17.7 ± 4.5 % and 15.0 ± 4.1 %, respectively, which were
113
clearly lower than the 70 % threshold generally accepted for the new species delineation
114
(Rosselló-Mora & Amann, 2001). These results indicated that strain H29T could be
115
considered as a novel species of the genus Rheinheimera.
116 117
Growth of strain H29T at different temperatures (5–45 °C at 5 °C intervals) and pH (5.0–10.0
118
at 0.5 pH unit intervals) was assessed in marine broth. Marine broth with pH below 8.0 and
119
pH 8.0–10.0 was prepared using the Na2HPO4-NaH2PO4 and Tris-HCl buffers, respectively.
120
The pH values of the marine broth were adjusted again if necessary after sterilization
121
(121 °C, 15 min). Growth of strain H29T at different NaCl concentrations (0, 0.5, 1, 2, 3, 4, 5
122
and 6 % (w/v)) was tested in marine broth prepared in the laboratory according to the BD
123
formula. The following physiological and biochemical tests were conducted using cells
124
grown on MA for 2 days at 25 °C. Gram staining was investigated using the bioMérieux
125
Gram stain kit according to the manufacturer’s instructions. Oxidase activity was evaluated
126
by the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck) and catalase activity
127
was tested by the production of oxygen bubbles in 3 % (v/v) aqueous hydrogen peroxide
128
solution (Smibert & Krieg, 1994). Cell morphology and the presence of flagella were
129
investigated using phase-contract microscopy and transmission electron microscopy (JEM-
130
1010, JEOL) with cells from an exponentially grown culture in marine broth at 25 °C.
131
Anaerobic growth was assessed on MA under the anaerobic condition (with 4–10 % CO2)
132
using the GasPak Plus system (BBL) at 25 °C for 10 days. The following properties of strain
133
H29T and reference strains were evaluated in parallel under the same conditions at 25 °C.
134
Hydrolysis of casein, starch, aesculin, CM-cellulose, tyrosine, Tween 20 and Tween 80 was 6
135
investigated as described by Lányí (1987) using MA as the basal medium. Nitrate reduction
136
was assessed according to the method descried previously (Lányí, 1987). Additional
137
enzymatic activities, biochemical features and oxidation of carbon sources were evaluated
138
using the API ZYM, API 20NE kits (bioMérieux) and GN2 MicroPlate system (Biolog),
139
respectively, according to the instructions of the manufacturers, except that the inocula of
140
strain H29T and reference strains were prepared by resuspending cells in artificial seawater
141
(20 g NaCl, 2.9 g MgSO4, 4.53 g MgCl2·6H2O, 0.64 g KCl and 1.75 g CaCl2·2H2O per liter).
142
143
Cells were Gram-staining-negative rods with 0.8 to 1.2 m in width and 1.5 to 1.8 m in
144
length (Supplementary Fig. S1, available in IJSEM online). Cells are motile by the presence
145
of a single polar flagellum. Anaerobic growth was not observed on MA after 10 days of
146
incubation at 25 °C. Physiological and biochemical characteristics of strain H29T are
147
additionally described in the species description and compared with those of the closely
148
related type strains in Table 1 and Supplementary Table S1 (available in IJSEM Online).
149
Most of properties such as catalase and urease activities and hydrolysis of carbon compounds
150
in strain H29T were consistent with those of other related-Rheinheimera species, while some
151
other properties such as colony color, no growth at 4 °C and many other phenotypic
152
properties differentiated strain H29T from other related-Rheinheimera species (Table 1).
153 154
The DNA G+C content of strain H29T was determined by the fluorometric method (Gonzalez
155
& Saiz-Jimenez, 2002) using SYBR Green I and a real-time PCR thermocycler (Bio-Rad).
156
Isoprenoid quinones were extracted according to the method of Minnikin et al. (1984) and 7
157
were analyzed using a HPLC (model LC-20A, Shimadzu) equipped with a diode array
158
detector (SPD-M20A, Shimadzu) and a reversed-phase column (250×4.6 mm, Kromasil,
159
Akzo Nobel) as described previously (Komagata & Suzuki, 1987). Strain H29T and four
160
reference strains were cultivated on marine broth at 25 °C for 12 hours for the analysis of
161
their cellular fatty acids. The fatty acids of cells were saponified, methylated and extracted
162
according to the standard protocol of the Sherlock Microbial Identification System (MIDI)
163
version 6.0. The fatty acid methyl esters were analyzed by GC (Hewlett Packard 7890) and
164
identified by the TSBA6 database of the Microbial Identification System (Sasser, 1990).
165
Polar lipids of strain H29T were extracted, resolved by two-dimensional TLC and identified
166
as described by Minnikin et al. (1984). The following reagents were used to detect different
167
polar lipids: 10 % (w/v) ethanolic molybdatophosphoric acid (for total polar lipids),
168
ninhydrin (for aminolipids) and the Dittmer-Lester reagent (for phospholipids).
169 170
The genomic DNA G+C content of strain H29T was approximately 46.5 mol%, which is a
171
little lower than the range of DNA G+C contents of previously reported type species of the
172
genus Rheinheimera (Yoon et al., 2007; Zhang et al., 2008; Chen et al. 2010; Liu et al., 2012;
173
Suarez et al., 2014). The only respiratory quinone detected in strain H29T was ubiquinone-8
174
(Q-8), which is a good agreement with the major or only respiratory quinone detected in all
175
members of the genus Rheinheimera. The major cellular fatty acids (>5 % of the total fatty
176
acids) of strain H29T were iso-C15:0 3-OH (31.3 %), summed feature 3 (comprising C16:1
177
ω7c/C16:1 ω6c, 15.7 %), C16:0 (10.8 %), summed feature 8 (comprising C18:1 ω7c and C18:1
178
ω6c, 5.7 %) and C12:0 3-OH (5.6 %). The overall fatty acid profile of strain H29T was similar
179
to those of the reference strains of the genus Rheinheimera; there were some differences in 8
180
the respective proportions of some fatty acid components, especially in hydroxyl fatty acids
181
(Table 2). The major polar lipid of strain H29T was phosphatidylethanolamine and
182
phosphatidylglycerol (Supplementary Fig. S2, available at IJSEM Online). Two unidentified
183
aminolipids, one aminophospholipid, two unidentified lipids were also detected as the minor
184
polar lipids. These characteristics of strain H29T are consistent with the description of the
185
genus Rheinheimera (Brettar et al., 2002; Yoon et al., 2007; Zhang et al., 2008 ; Chen et al.,
186
2010 ; Liu et al., 2012). In conclusion, the physiological and chemotaxonomic features and
187
the phylogenetic inference of strain H29T support the proposition that strain H29T represents
188
a novel species of the genus Rheinheimera, for which the name Rheinheimera aestuarii sp.
189
nov is proposed.
190
191
Description of Rheinheimera aestuarii sp. nov.
192
Rheinheimera aestuarii (a.es.tu.a´ri.i.L. gen. n. aestuarii of coastal sediment, from where the
193
organism was isolated).
194
Colonies are white, circular, convex, smooth and entire margins with approximately 2 mm in
195
diameter after 2 days of incubation on MA agar. Cells are Gram-staining-negative, strictly
196
aerobic rods and motile by the presence of a single polar flagellum. Growth occurs at 10–
197
40 °C (optimum, 20–25 °C) and pH 6.0–9.0 (optimum, pH 7.0–8.0) and in the presence of 1–
198
4 % (w/v) NaCl (optimum, 2–3 %). Catalase and oxidase are positive. Nitrate is reduced to
199
nitrite. Casein, aesculin, starch, Tween 20 and Tween 80 are hydrolyzed, but CM-cellulose
200
and tyrosine are not. In the API 20NE strip, hydrolysis of aesculin and gelatin were detected,
201
but indole production, D-glucose fermentation, urease, arginine dihydrolase and β9
202
galactosidase activities, assimilation of D-glucose, L-arabinose, D-mannose, D-mannitol, N-
203
acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid,
204
trisodium citrate and phenylacetic acid are absent. In the API ZYM strip, alkaline
205
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine
206
arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-
207
phosphophydrolase, N-acetyl-β-glucosaminidase activities are present. However, α-
208
chymotrypsin, α-galactosidase, β-glucuronidase, β-galactosidase, α-glucosidase, β-
209
glucosidase, α-mannosidase and β-fucosidase activities are absent. N-acetyl-D-galactosamine,
210
N-acetyl-D-glucosamine, L-arabitol, i-erythritol, L-fucose, gentiobiose, α-D-lactose,
211
lactulose, D-mellibiose, β-methyl-D-glucoside, turanose, xylitol, formic acid, D-galacturonic
212
acid, D-glucosaminic acid, α-hydroxy butyric acid, β-hydroxy butyric acid, α-keto glutaric
213
acid, L-alaninamide, L-alanine, L-alanyl-glycine, L-glutamic acid, glycyl-L-aspartic acid, L-
214
ornithine, L-proline, L-pyroglutamic acid, L-threonine, thymidine and phenyethylamine in
215
the GN2 MicroPlate are oxidized. The other organic substrates in the Biolog GN2
216
MicroPlates are not oxidized. The only respiratory quinone detected is Q-8 and the major
217
cellular fatty acids are iso-C15:0 3-OH, summed feature 3 (comprising C16:1 ω7c/C16:1 ω6c),
218
C16:0, summed feature 8 (comprising C18:1 ω7c and C18:1 ω6c) and C12:0 3-OH. The major
219
polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content
220
of the type strain is 46.5 mol%.
221
The type strain is H29T (= KACC 18251T = JCM 30404T), which was isolated from coastal
222
sediment in Jeju Island, Republic Korea.
223
10
224
ACKNOWLEDGEMENTS
225
We thank Dr. S. Ratering in Justus-Liebig University Giessen for providing R.
226
muenzenbergensis E49T and R. hassiensis E48T. This work was supported by the Cooperative
227
Research Program for Agriculture Science & Technology Development (Project No.
228
PJ00999302), RDA, Republic of Korea.
229
230
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14
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Figure Legend
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Fig. 1. A neighbor-joining tree based on 16S rRNA gene sequences showing the phylogenetic
308
relationships of strain H29T and related taxa. Bootstrap values are shown on nodes in
309
percentages of 1,000 replicates; only values over 70% are given. Closed circles are indicated,
310
when the corresponding nodes were recovered in the maximum-likelihood and maximum-
311
parsimony algorithms. Serratia entomophila DSM 12358T (AJ233427) was used as an
312
outgroup (not shown). The scale bar equals 0.01 changes per nucleotide position.
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15
314 315
Table 1. Phenotypic characteristics of strain H29T and related type strains of the genus Rheinheimera
316 317 318 319 320 321 322 323
Strains: 1, H29T; 2, R. pacifica CCUG 46544T (Romanenko et al., 2003); 3, R. muenzenbergensis E49T (Suarez et al., 2014); 4, R. hassiensis E48T (Suarez et al., 2014); 5, R. baltica DSM 14885T (Brettar et al., 2002). All strains are positive for the following characteristics: hydrolysis of Tween 20, Tween 80, aesculin and starch and oxidase, urease, alkaline phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase activities. All strains are negative for the following characteristics: Gram staining, hydrolysis of CM-cellulose and tyrosine, indole production, D-glucose fermentation, assimilation of D-mannose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid and phenylacetic acid and α-galactosidase, β-galactosidase, β-glucuronidase, αglucosidase, β-glucosidase and α-fucosidase activities. Symbols: +, positive; , negative. Characteristic
1*
2
3
4
5
white
Dark purple
yellow
brown
yellow
0.8–1.2 ×
0.4–0.8 ×
0.5–0.8 ×
0.4–0.8 ×
0.5–1.5 ×
1.5–1.8 + 10–40 1–4 6.0–9.0 + + +
1.8–2.0 + 4–37 0–8 5.5–10.5 + + +
1.3–2.4 + 4–37 0–5 7.0–9.5 –
0.7–2.0 + 4–37 0–6 6.5–10.0 – +
0.9–2.5 4–30 0–6 5.7–10.0 + + +
+
+
+
+
–
+
+
+
–
–
+
+
–
–
–
+
+
+
–
+
α-Chymotrypsin
–
+
+
+
α-Mannosidase
–
+
–
+
–
–
–
– + + + 50.5
– – – – 48.9
Colony color Cell size (μm) Motility Temperature range (°C) NaCl range (%) pH range Catalase Nitrate reduction* Hydrolysis of casein* Enzyme activity (API ZYM)* of: Esterase (C4) Esterase lipase (C8), cystine arylamidase Lipase (C14), valine acrylamidase Trypsine
324
Acid phosphatase, N-acetyl-β+ + – glucosaminidase Assimilation (API 20NE)* of: D-Glucose – + – L-Arabinose – + – N-acetyl glucosamine, D-Maltose + + Trisodium citrate – – – DNA G+C content (mol%) 46.5 49.6 50.0 * These analyses were performed using the same conditions in this study.
16
325 326
Table 2. Cellular fatty acid compositions (%) of strain H29T and related type strains of the genus Rheinheimera
327 328 329 330
Strains: 1, H29T; 2, R. pacifica CCUG 46544T; 3, R. muenzenbergensis E49T; 4, R. hassiensis E48T;5, R. baltica DSM 14885T. All data were from this study. All strains were grown on marine broth at 25 °C for 12 hrs. Data are expressed as percentages of total fatty acids. Major components (> 5.0 %) are highlighted in bold. Fatty acids amounting to less than 0.5 % in all strains are not shown. tr, trace amount (
