Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0210-8
ORIGINAL PAPER
Flavimarina pacifica gen. nov., sp. nov., a new marine bacterium of the family Flavobacteriaceae, and emended descriptions of the genus Leeuwenhoekiella, Leeuwenhoekiella aequorea and Leeuwenhoekiella marinoflava Olga I. Nedashkovskaya • Andrey D. Kukhlevskiy Natalia V. Zhukova • Seung Bum Kim
•
Received: 27 March 2014 / Accepted: 3 June 2014 Ó Springer International Publishing Switzerland 2014
Abstract A facultatively anaerobic, Gram-stain negative, rod-shaped and yellow pigmented bacterium, designated strain IDSW-73T, was isolated from a seawater sample and subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strain formed a distinct phyletic line in the family Flavobacteriaceae and is most closely related to the members of the genus Leeuwenhoekiella, with 16S rRNA gene sequence similarity of 91.4–92.6 %. Strain IDSW-73T was found to be able to grow with 0–12 % NaCl and at 4–33 °C; and was able to hydrolyse gelatin, starch and Tweens 20, O. I. Nedashkovskaya (&) G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022 Vladivostok, Russia e-mail: [email protected]; [email protected] A. D. Kukhlevskiy N. V. Zhukova A.V. Zhirmunsky Institute of Marine Biology, Far-Eastern Branch of the Russian Academy of Sciences, Pal’chevskogo St. 17, 690032 Vladivostok, Russia A. D. Kukhlevskiy N. V. Zhukova Far Easten Federal University, Sukhanova St. 8, 690950 Vladivostok, Russia
40 and 80. The DNA G?C content was determined to be 42.2 mol%. The predominant cellular fatty acids were identified as branched-chain saturated and unsaturated and straight-chain unsaturated fatty acids such as isoC15:0, iso-C15:1, iso-C17:1 x9c, C15:1 x6c, iso-C15:0 3-OH, iso-C17:0 3-OH and summed feature 3 (as defined by MIDI), comprising iso-C15:0 2-OH and/or C16:1x7c. The polar lipids found were phosphatidylethanolamine, two unknown aminolipids and one unknown lipid. The major respiratory quinone was identified as MK-6. The significant molecular distinctiveness between the novel isolate and its nearest neighbours were strongly supported by notable differences in physiological and biochemical tests. Therefore, strain IDSW-73T is considered to represent a novel genus and species within the family Flavobacteriaceae, for which the name Flavimarina pacifica gen. nov., sp. nov. is proposed. The type strain is IDSW-73T (=KCTC 32466T = KMM 6759T). Emended descriptions of the recognized species of the genus Leeuwenhoekiella are also proposed. Keywords Flavimarina pacifica gen. nov., sp. nov. Flavobacteriaceae Marine bacteria Phylogeny Taxonomy
Introduction S. B. Kim Department of Microbiology and Molecular Biology, School of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, Republic of Korea
Members of the phylum Bacteroidetes are widely distributed in different marine ecosystems and often isolated from surface and deep waters (Alonso et al.
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Antonie van Leeuwenhoek
2007; Simonato et al. 2010; Vieira et al. 2008). They constitute one of the dominant taxonomic groups identified in studies of bacterioplankton communities and are characterized by high specific richness (Hahnke and Harder 2013; Diez-Vivas et al. 2014). The most cultivated Bacteroidetes belong to the family Flavobacteriaceae, which constitutes the largest phylogenetic lineage within the phylum and is comprised of more than 120 validly named genera at the time of writing (http://www.bacterio.net). The prevalence of phylotypes of the family Flavobacteriaceae affiliated to the genera Dokdonia, Formosa, Maribacter, Mesonia, Polaribacter, Psychroserpens, Tenacibaculum and Ulvibacter were reported in a study of bacterial communities inhabited the Yellow Sea (Dang et al. 2008). Representatives of the above listed genera, as well as Aquimarina, Arenibacter, Cellulophaga, Gramella, Kriegella, Leeuwenhoekiella, Maribacter, Muricauda and Zobellia, were identified among isolated bacteria collected from the German Bight in the North Sea (Hahnke and Harder 2013). However, a large number of the flavobacterial 16S rRNA gene sequences remain to be characterized as novel taxa with cultured representatives. In the course of a taxonomic survey of a coastal bacterioplankton community inhabiting the Troitsa Bay in the Sea of Japan, a Gram-negative, rod-shaped, non-motile and yellow-pigmented bacterial isolate, designated IDSW-73T, was obtained. Phylogenetic analysis based on 16S rRNA gene sequences revealed that its closest relatives were known species of the genus Leeuwenhoekiella with 91.4–92.6 % 16S rRNA sequence similarity. Noteworthy, a most nearest neighbour of the novel isolate was strain Flavobacteriaceae bacterium Hel_I_48 (GenBank accession number JX854131), isolated in the North Sea, with 16S rRNA gene sequence similarity of 99.3 % (Hahnke and Harder 2013). The aim of the present study was to describe the isolation and detailed characterization of the novel environmental isolate, IDSW-73T, using a polyphasic taxonomic approach.
Sea of Japan (also known as the East Sea) by a standard plating method. For the strain isolation, 0.1 ml sea water was spread onto marine agar 2216 (MA) plates. The novel isolate was obtained from a single colony after incubation of the plate at 28 °C for 7 days. After primary isolation and purification, the strain was cultivated at 28 °C on the same medium and stored at -80 °C in marine broth supplemented with 20 % (v/v) glycerol.
Materials and methods
Mol% G?C determination
Isolation and cultivation
Genomic DNA was isolated following the method of Marmur (1961) and the G?C content was determined by the thermal denaturation method (Marmur and Doty 1962).
Strain IDSW-73T was isolated from a seawater sample collected from Troitsa Bay, Gulf of Peter the Great,
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16S rRNA gene sequencing and phylogenetic analysis DNA was extracted from 0.1 to 0.2 g of the bacterial cells (wet weight), using the extraction protocol of Sambrook and Russell (2001). PCR was carried out using the universal oligonucleotide primers 11F (50 -GTTTGATCMTGGCTCAG-30 ) and 1492R (50 -TACGGYTACCTTGTTACGACTT30 ) as described by Lane (1991) and the GeneAmp PCR System 9700 (Applied Biosystems Inc.). PCR amplicons were used as templates for sequencing amplification using a BigDye Terminator version 3.1 Cycle sequencing kit (Applied Biosystems). The purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer and the sequence was assembled with SeqScape version 2.6 (Applied Biosystems). The sequences obtained were aligned with those of representative members of selected genera of the family Flavobacteriaceae by using PHYDIT version 3.2 (http://plaza.snu.ac.kr/*jchun/ phydit/). Phylogenetic trees were inferred by using suitable programs of the PHYLIP package (Felsenstein 1993). Phylogenetic distances were calculated from the Jukes-Cantor model (Jukes and Cantor 1969) and the trees were constructed on the basis of the neighbour-joining (Saitou and Nei 1987) and maximum-likelihood (Felsenstein 1993) algorithms. A bootstrap analysis was performed with 1,000 resampled datasets by using the SEQBOOT and CONSENSE programs of the PHYLIP package.
Antonie van Leeuwenhoek
Whole cell polar lipid, fatty acid and lipoquinone composition
Morphological, physiological and biochemical tests
For whole-cell fatty acid and polar lipid analysis, strain IDSW-73T and reference strains Leuwenhoekiella aequorea LMG 22550T, Leuwenhoekiella blandensis MED 217T, Leuwenhoekiella marinoflava LMG 1345T and Leuwenhoekiella palythoae KMM 6264T were grown under optimal physiological conditions for all strains (at 28 °C for 24 h on MA). The reference strains were obtained from our laboratory. Cellular fatty acid methyl esters (FAMEs) were prepared according to the methods described by Sasser (1990) using the standard protocol of the Sherlock Microbial Identification System (version 6.0, MIDI) and analysed using a GC-21A chromatograph (Shimadzu) equipped with a fused-silica capillary column (30 m 9 0.25 mm) coated with Supercowax-10 and SPB-5 phases (Supelco) at 210 °C. FAMEs were identified by using equivalent chain-length measurements and comparing the retention times to those of authentic standards. FAMEs were also analysed by GC–MS (QP5050A; Shimadzu) equipped with an MDN-5S capillary column (30 m 9 0.25 mm), the temperature program was from 140 to 250 °C, at a rate of 2 °C min-1. The polar lipids of strain IDSW-73T and the reference strains were extracted using the chloroform/methanol extraction method of Bligh and Dyer (1959). Twodimensional TLC of polar lipids was carried out on silica gel 60 F254 (10 9 10 cm; Merck) using chloroform/methanol/water (65:25:4, by vol) in the first dimension and chloroform/methanol/acetic acid/ water (80:12:15:4, by vol.) in the second dimension (Collins and Shah 1984). The spray reagents used to reveal the spots were 10 % sulfuric acid in methanol, molybdate reagent and ninhydrin. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v) and purified by TLC, using a mixture of n-hexane and diethyl ether (85:15, v/v) as the solvent. Isoprenoid quinone composition was characterized by HPLC (Shimadzu LC-10A) using a reversed-phase type Supelcosil LC-18 column (15 cm 9 4.6 mm) and acetonitrile/ 2-propanol (65:35, v/v) as a mobile phase at a flow rate of 0.5 ml min-1 as described previously (Komagata and Suzuki 1987). The column was kept at 40 °C. Quinones were detected by monitoring at 270 nm.
Cell morphology was examined by light microscopy (Olympus CX41) and transmission electron microscopy (Libra 120; Carl Zeiss). Gram-staining was done as described by Gerhardt et al. (1994). Oxidative and fermentative utilization of glucose was determined using the medium of Hugh and Leifson modified for marine bacteria (Lemos et al. 1985). Catalase activity was tested by addition of 3 % (v/v) H2O2 solution to a bacterial colony and observation for the appearance of gas. The presence of oxidase was tested using N,N,N,N-tetramethyl-p-phenylenediamine. Degradation of agar, starch, casein, gelatin, chitin, DNA, Tweens 20, 40 and 80 and urea, production of acid from carbohydrates, nitrate reduction and production of hydrogen sulphide were tested according to standard methods (Gerhardt et al. 1994). Growth at 4, 10, 25, 28, 30, 32, 35 and 37 °C was measured on MA. Tolerance to NaCl was assessed in medium A containing 5 g Bacto Peptone (Difco), 2 g Bacto Yeast Extract (Difco), 1 g glucose, 0.02 g KH2PO4 and 0.05 g MgSO47H2O per liter of distilled water with 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12 and 15 % (w/v) of NaCl. The pH range for growth was investigated in MB adjusted to pH from 5.0 to 11.0 with HCl and Na2CO3. Physiological and biochemical properties of the novel isolate was also tested using the API 20E, API 20NE, API 50CH and API ZYM systems (bioMe´rieux), according to the manufacturer’s instructions except that the galleries were incubated at 28 °C. Carbon source utilization was tested (1) using commercial API 20E, API 20 NE and API 50CH (bioMe´rieux) identification strips, and (2) using a medium that contained 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g Yeast Extract (Difco) and 0.4 % (w/v) carbon source per liter of artificial seawater. Susceptibility to antibiotics was examined by the routine diffusion plate method. Discs were impregnated with the following antibiotics (lg per disc): ampicillin (10), benzylpenicillin (10U), carbenicillin (100), cefalexin (30), cefazolin (30), chloramphenicol (30), erythromycin (15), doxycycline (10), gentamicin (10), kanamycin (30), lincomycin (15), oleandomycin (15), nalidixic acid (30), neomycin (30), ofloxacin (5), oxacillin (10), polymyxin B (300 U), rifampicin (5), streptomycin (30), tetracycline (5) and vancomycin (30).
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Antonie van Leeuwenhoek T
Galbibacter mesophilus Mok-17 (AB255367) T
Arenibacter latericius KMM 426 (AF052742)
100 *
T
100
Maribacter sedimenticola KMM 3903 (AY271623)
*
T
Maribacter arcticus KOPRI 20941 (AY771762) T
Aquimarina brevivitae SMK-19 (AY987367)
100
T
*
Aquimarina gracilis PSC32 (HM998909) *
T
Aquimarina muelleri KMM 6020 (AY608406)
100
T
Aquimarina intermedia KMM 6258 (AM113977)
*
T
99 *
100
*
50
Dokdonia genika Cos-13 (AB198086) T
Dokdonia donghaensis DSW-1 (DQ003276)
100 Dokdonia eikasta PMA-26T (AB198088) * Dokdonia diaphoros MSKK-32T (AB198089) T
Marixanthomonas ophiurae KMM 3046 (AB261012) T
Gramella echinicola KMM 6050 (AY608409) 85
60
*
T
Salegentibacter salarius ISL-6 (EF486353)
100 * *
T
Salegentibacter mishustinae KMM 6049 (AY576653) T
Salegentibacter holothurioum KMM 3524 (AB116148) T
Salegentibacter salegens DSM 5424 (M92279) 54 * T * Salegentibacter agarivorans KMM 7019 (DQ191176) 76 * T 81 Salegentibacter salinarum ISL-4 (EF612764)
73 *
T
Zunongwangia profunda SM-A87 (DQ855467) 73 *
T
100
Salinimicrobium gaetbulicola BB-My20 (JF340052)
*
T
Salinimicrobium catena HY1 (DQ640642) T
Nonlabens tegetincola UST030701-324 (AY987349)
100
*
T
*
Nonlabens spongiae UST030701-156 (DQ064789) T
100
68
*
Nonlabens agnitus JC2678 (HM475136) T
Nonlabens marina S1-08 (AB602426) T
100
* * 95 *
Leeuwenhoekiella marinoflava ATCC 19326 (M58770) T
Leeuwenhoekiella aequorea LMG 22550 (AJ278780) T
Leeuwenhoekiella blandensis MED217 (DQ294291) T
Leeuwenhoekiella palythoae KMM 6264 (FJ405187) T
0.01
IDSW-73 (KJ152756)
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of Flavimarina pacifica gen. nov., sp. nov. IDSW-73T and related members of the family Flavobacteriaceae. The asterisks
indicate clades at genus level that were also recovered from the maximum-likelihood tree. Bootstrap values is based on 1,000 replications. Bar 0.01 substitutions per nucleotide position
Results and discussion
recognized species of the genus Leeuwenhoekiella (Fig. 1). The 16S rRNA gene sequence similarities of the novel isolate and L. palythoae KMM 6264T, L. marinoflava LMG 1345T, L. blandensis MED 217T, and L. aequorea LMG 22550T were 92.6, 92.5, 92.4 and 91.4 %. Other recognized species of the family Flavobacteriaceae exhibited less than 91 % 16S rRNA gene sequence similarity. Significant molecular differences found between strain IDSW-73T and the type strains of the Leeuwenhoekiella species (8.6–7.4 % sequence divergence), from which it can
Molecular phylogenetic analysis An almost-complete 16S rRNA gene sequence (1448 nt) of strain IDSW-73T was determined (GenBank/ EMBL/DDBJ accession number KJ152756). Phylogenetic analysis revealed that strain IDSW-73T is a member of the family Flavobacteriaceae, class Flavobacteriia, phylum Bacteroidetes and formed a distinct evolutionary lineage adjoining to the
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Antonie van Leeuwenhoek Table 1 Cellular fatty acid contents of Flavimarina pacifica gen. nov., sp. nov. and the validly named species of the genus Leeuwenhoekiella Fatty acid
1
2
iso-C15:0
3
4
5
15.0
12.4
18.7
12.4
anteiso-C15:0
1.2
3.9
2.7
3.4
24.1 5.9
iso-C15:1
5.5
9.7
21.5
11.6
4.7
iso-C16:0
1.3
2.7
2.1
tr
8.4
iso-C17:1 x9c
8.6
9.1
8.2
7.8
4.7
anteiso-C17:1 x9c
1.8
tr
tr
tr
–
C15:0
2.0
5.4
5.5
4.3
7.4
C15:1 x6c
5.5
1.4
tr
1.8
1.6
C16:0
tr
tr
1.2
1.1
tr
C17:0
tr
1.3
tr
tr
1.1
C17:1 x6c iso-C15:0-3OH
tr 8.1
1.0 5.0
tr 4.6
tr 6.5
2.2 4.7
iso-C16:0-3OH
tr
5.3
tr
1.5
5.6
iso-C17:0-3OH
23.9
16.0
15.5
17.4
13.6
anteiso-C17:0-3OH
2.1
5.8
tr
3.9
1.4
C17:0-2OH
2.1
5.8
1.0
3.9
1.2
24.5
14.4
13.0
19.5
10.1
Summed feature 3*
* Summed feature 3 consists of the following fatty acids which could not be separated by the microbial identification system: one or more of C16:1x7c and iso-C15:0 2-OH Strains: 1, Flavimarina pacifica gen. nov., sp. nov. IDSW-73T; 2, Leeuwenoekiella aequorea LMG 22550T; 3, L. blandensis MED 217T; 4. L. marinoflava LMG 1345T; 5, L. palythoae KMM 6264T. All data from this study. tr, Trace amount (\1 %). Values are percentages of total fatty acids; those fatty acids for which the mean amount in all taxa was less than 1 % are not given
be concluded that the environmental isolate represents a novel genus and species of the family Flavobactericeae. Mol% G?C determination The DNA G?C content of strain KMM 6759T was determined to be 42.2 mol%. Chemotaxonomic characterization The predominant fatty acids of strain IDSW-73T ([5 % of the total fatty acids) were identified as branched-chain saturated and unsaturated and straightchain unsaturated fatty acids: iso-C15:0 (15.0 %), isoC15:1 (5.5 %), C15:1 x6c (5.5 %), iso-C17:1 x9c (8.6 %), iso-C15:0 3-OH (8.1 %), iso-C17:0 3-OH
(23.9 %) and summed feature 3 (comprising C16:1 x7c and/or iso-C15:0 2-OH fatty acids as defined by the MIDI system; 24.5 %). The fatty acid profile of strain IDSW-73T strain was similar to those of the type strains of the genus Leeuwenhoekiella, while the differences in the proportions of some fatty acids, including anteiso-C15:0, anteiso-C17:1 x9c, C15:0, C15:1 x6c and iso-C16:0-3OH, were marked (Table 1). The polar lipid profile of the novel isolate was found to be composed of phosphatidylethanolamine, two unknown lipids and one unknown aminolipid, which is in line with those of the reference strains (Fig. 2). However, the presence of two unknown lipids (L1 and L2) and the absence of another unknown lipid (L) could distinguish strain IDSW-73T from its phylogenetic relatives except L. aequorea LMG 22550T. The sole respiratory quinone found to be present in the isolate was menaquinone 6, which is a characteristic lipoquinone for members of the family Flavobacteriaceae (Bernardet et al. 2002). Morphological, physiological and biochemical characteristics The physiological and biochemical characteristics of strain IDSW-73T are given in the genus and species descriptions and Table 2. The isolate was observed to be a rod-shaped, motile by gliding, facultatively anaerobic and oxidase- and catalase-positive organism. Strain IDSW-73T was found to be susceptible to ampicillin, carbenicillin, cefalexin, chloramphenicol, erythromycin, lincomycin nalidixic acid, ofloxacin, oleandomycin and rifampicin; and resistant to benzylpenicillin, cefazolin, doxycycline, gentamicin, kanamycin, neomycin, streptomycin, tetracycline, oxacillin, polymyxin B and vancomycin. The novel strain can be clearly distinguished from its closest relatives by the fermentative type of metabolism, maximum growth temperature (33 vs. 37–41 °C) and the ability to produce acid from mannose and raffinose and to utilize inositol. Such phenotypic properties as the absence of a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase and a-mannosidase activities and acid formation from glycerol are also useful tests for separation of strain IDSW-73T from the members of the genus Leeuwenhoekiella. Other phenotypic characteristics that differentiate the isolate from its nearest neighbours are shown in Table 2.
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Antonie van Leeuwenhoek
Fig. 2 Thin-layer chromatogram showing the polar lipid profiles of strain IDSW-73T (a), L. aequorea LMG 22550T (b); L. blandensis MED 217T (c); L. marinoflava LMG 1345T (d); 5, L. palythoae KMM 6264T (e) following separation by two-dimensional TLC. Solvent systems: (1) chloroform–
methanol–water (65:25:4, v/v/v.); (2) chloroform–acetic acid– methanol–water (80:15:12:4, v/v/v/v.). PE, phosphatidylethanolamine; AL, an unknown aminolipid; L, L1 and L2, the unknown lipids
Polyphasic taxonomic conclusion
nov., sp, nov. is proposed. The emended descriptions of the genus Leeuwenhoekiella, L. aequorea and L. marinoflava are also proposed due to newly obtained data.
The results obtained in the present study indicated that strain IDSW-73T demonstrates low 16S rRNA gene sequence similarities with its closest phylogenetic relatives, members of the genus Leeuwenhoekiella, and other validly named representatives of the family Flavobacteriaceae (91.4–92.6 % and less than 91 %, respectively). Furthermore, the novel strain can be clearly differentiated from validly named Leeuwenhoekiella by the type of metabolism (fermentative vs. aerobic), maximum growth temperature (33 vs. 37–41 °C), acid production from mannose and raffinose, inositol utilization and the absence of agalactosidase, b-galactosidase, a-glucosidase, b-glucosidase and a-mannosidase activities. The abovementioned findings, including phylogenetic and phenotypic characteristics, strongly support the discrimination of the novel strain from its close relatives at the generic level. Therefore, on the basis of the phylogenetic, genotypic, chemotaxonomic and phenotypic data presented here, we suggest that strain IDSW-73T should be classified as representing a novel genus and species within the family Flavobacteriaceae, for which the name Flavimarina pacifica gen.
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Description of Flavimarina gen. nov. Flavimarina (Fla.vi.ma.ri’na. L. adj. flavus, yellow; L. fem. adj. marina, marine; N.L. fem. n. Flavimarina, a yellow-coloured organism of marine origin). Cells are Gram-negative, facultatively anaerobic, rod-shaped and non-motile. Do not form endospores. Non-diffusible pigments are produced. Chemo-organotroph. Cytochrome oxidase-, catalase and alkaline phosphatase-positive. The predominant cellular fatty acids are branched-chain saturated and unsaturated and straight-chain unsaturated fatty acids such as iso-C15:0, iso-C15:1, iso-C17:1 x9c, C15:1 x6c, iso-C15:0 3-OH, iso-C17:0 3-OH and summed feature 3 (comprising iso-C15:0 2-OH and/or C16:1x7c). Polar lipids are phosphatidylethanolamine, two unknown aminolipids and one unknown lipid. The major respiratory quinone is MK-6. The DNA G?C content of the type strain of the type species is 42.2 mol%. 16S rRNA
Antonie van Leeuwenhoek Table 2 Differential phenotypic characteristics of Flavimarina pacifica gen. nov., sp. nov. and the validly named species of the genus Leeuwenhoekiella Characteristic
1
2
3
4
5
Source of isolation
Seawater
Seawater
Seawater
Seawater
Soft coral -
Glucose fermentation
?
-
-
-
Temperature range for growth (°C)
4–33
4–37
10–41
4–37
4–38
Salinity range for growth (% NaCl)
0–12
0–15
0–17
0–15
0–12
Casein
-
?
?
?
-
Tween 20
?
?
?
?
-
Arabinose, maltose
?
-
-
-
?
Cellobiose
-
-
-
-
?
Galactose
?
?
-
?
?
Glucose
?
-
?
-
?
Lactose
-
-
-
-
?
Mannose, raffinose
?
-
-
-
-
Rhamnose Sucrose
?
?
-
-
? ?
Xylose
?
-
?
-
-
Glycerol
-
?
?
?
?
Mannitol
-
?
-
-
-
Inositol
?
-
-
-
-
Mannitol
?
?
-
-
-
Esterase (C4)
?
?
?
?
-
Lipase (C14)
?
-
?
?
-
Cystine arylamidase
?
-
?
?
-
a-Chymotrypsin
-
-
?
-
-
Trypsin
?
-
?
?
?
Naphtol-AS-BI-phosphohydrolase
?
?
-
?
?
a-Galactosidase, b-galactosidase a-Glucosidase, b-glucosidase
-
? ?
? ?
? ?
? ?
N-Acetyl-b-glucosaminidase, a-mannosidase
-
?
?
?
?
DNA G?C content (mol%)
42.2
35–36
42.5
38
Hydrolysis of
Acid formation from
Utilization of
Enzyme activity (API ZYM)
T
41.2 T
Strains: 1, Flavimarina pacifica gen. nov., sp. nov. IDSW-73 ; 2, Leeuwenhoekiella aequorea LMG 22550 ; 3 L. blandensis MED 217T; 4. L. marinoflava LMG 1345T; 5, L. palythoae KMM 6264T. Data from Nedashkovskaya et al. (2005, 2009), Pinhassi et al. (2006), and this study. All strains are positive for gliding motility; oxidase, catalase, alkaline phosphatase, acid phosphatase, esterase lipase (C8), leucine arylamidase and valine arylamidase activities; gelatin, starch and Tweens 40 and 80 hydrolysis; utilization of arabinose, glucose, lactose, mannose and sucrose. All strains are negative for nitrate reductase, a-fucosidase and b-glucuronidase activities, flexirubin-type pigments production, H2S and indole production, agar, DNA, chitin, urea and cellulose hydrolysis
gene sequence analysis indicates that the genus Flavimarina is a member of the family Flavobacteriaceae, the class Flavobacteriia within the phylum Bacteroidetes. The type species is Flavimarina pacifica.
Description of Flavimarina pacifica sp. nov. Flavimarina pacifica [pa.ci’fi.ca. L. fem. adj. pacifica, peaceful; pertaining to the Pacific Ocean from where the type strain was isolated].
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Antonie van Leeuwenhoek
Flavimarina pacifica demonstrates the following characteristics in addition to those given for the genus. Cells are 0.4–0.6 lm in diameter and 1.2–3.5 lm in length. On marine agar, colonies are 2–3 mm in diameter, circular, with entire edges, shiny and yellow in colour. Growth occurs at 4–33 °C (optimum, 25–28 °C), at pH 5.5–10.0 (optimum, pH 7.5–8.0) and with 0–12 % NaCl (optimum, 2.0–5.0 %). Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities are absent. Gelatin, starch and Tweens 20, 40 and 80 are hydrolysed but agar, casein, DNA, urea, chitin and cellulose are not hydrolysed. Acid is produced from Larabinose, D-galactose, D-glucose, maltose, mannose, raffinose, sucrose and D-xylose, but not from cellobiose, D-fructose, D-lactose, melibiose, L-rhamnose, ribose, trehalose, N-acetylglucosamine, glycerol, inositol, mannitol, sorbitol and citrate. Cellobiose, melibiose, L-rhamnose, trehalose and N-acetylglucosamine are utilized, but inositol, mannitol, sorbitol, citrate, methionine and tryptophan are not utilized. Growth is observed on L-alanine, L-asparagine, Lglutamine, L-leucine, L-proline, L-threonine, L-tyrosine and L-valine, but no growth occurs on DLmethionine and L-tryptophan. In the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, valine arylamidase, trypsin, acid phosphatase and naphtol-AS-BI-phosphohydrolase activities are present; but a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, bglucosidase N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. Nitrate is not reduced to nitrite. Hydrogen sulphide, indole and acetoin are not produced. In the API 20E gallery strain IDSW-73T is positive for ONPG, gelatin hydrolysis and acid production from glucose, rhamnose, sucrose, melibiose, amygdalin and arabinose. In API 20NE, positive results are obtained for glucose fermentation, aesculin and gelatin hydrolysis, the PNPG test (bglucosidase) and assimilation of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate and adipate. In API 50CH, positive results are obtained for L-arabinose, D-xylose, galactose, glucose, fructose, mannose, methyl-a-D-glucopyranoside, methyl-a-D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, Dcellobiose, maltose, D-lactose, D-melibiose, sucrose, trehalose, raffinose, amidon, D-turanose and D-lixose.
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The DNA G?C content of the type strain is 42.2 mol%. The type strain, IDSW-73T (= KCTC 32466T = KMM 6759T), was isolated from seawater collected from Troitsa Bay, Gulf of Peter the Great, Sea of Japan (also is known as East Sea), Pacific Ocean, Russia. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F. pacifica IDSW-73T is KJ152756.
Emended description of the genus Leeuwenhoekiella Nedashkovskaya et al. (2005) The description of the genus Leeuwehhoekiella is as given by Nedashkovskaya et al. (2005) with the following additions. The major polar lipids are phosphatidylethanolamine and one unknown aminolipid; unidentified lipids are also present.
Emended description of Leeuwenhoekiella aequorea Nedashkovskaya et al. (2005) The description of L. aequorea is as given by Nedashkovskaya et al. (2005) with the following additions. Using the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase and a-mannosidase activities are present but lipase (C14), cystine arylamidase, a-chymotrypsin, trypsin, a-fucosidase and bglucuronidase activities are absent. Polar lipids are phosphatidylethanolamine, one unknown aminolipid and two unknown lipids.
Emended description of Leeuwenhoekiella marinoflava Nedashkovskaya et al. (2005) The description of L. marinoflava is as given by Nedashkovskaya et al. (2005) with the following additions. Using the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, a-galactosidase, b-
Antonie van Leeuwenhoek
galactosidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase and a-mannosidase activities are present but a-chymotrypsin, a-fucosidase and bglucuronidase activities are absent. Polar lipids are phosphatidylethanolamine, one unknown aminolipid and one unknown lipid. Acknowledgments This research was supported by grants of the Presidium of the Russian Academy of Sciences ‘‘Molecular and Cell Biology’’, Russian Scientific Fund no. 14-14-00030, the Presidium of the Far-Eastern Branch of the Russian Academy of Sciences no. 12-III-A-06-105 and the government of Russian Federation for the state support of scientific investigations conducting under the guidance of the leading researchers at the Russian education institutions of the high professional education, agreement no. 11.G34.31.0010.
References Alonso C, Warnecke F, Amann R, Pernthaler J (2007) High local and global diversity of Flavobacteria in marine plankton. Environ Microbiol 9:1253–1266 Bernardet JF, Nakagawa Y, Holmes B (2002) Proposal minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. IntJ Syst Evol Microbiol 52:1049–1070 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 Collins MD, Shah HM (1984) Fatty acid, menaquinone and polar lipid composition of Rothia dentosacariosa. Arch Microbiol 137:247–249 Dang H, Li T, Chen M, Huang G (2008) Cross-ocean distribution of Rhodobacteriales bacteria as primary surface colonizers in temperate coastal marine waters. Appl Environ Microbiol 74:52–60 Diez-Vivas C, Gasol JM, Acinas SJ (2014) Spatial and temporal variability among marine Bacteroidetes populations in the NW Mediterranean Sea. Syst Appl Microbiol 37:68–78 Felsenstein J (1993) PHYLIP (phylogenetic inference package), version 3.5c. Department of Genetic, University of Washington, Seattle, USA Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) (1994) Methods for general and molecular bacteriology. American Society for Microbiology, Washington Hahnke RL, Harder J (2013) Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media. Syst Appl Microbiol 36:497–504
Jukes TH, Cantor CR (1969) Evolution of protein molecules. In: Munro HN (ed) Mammalian protein metabolism, vol 3. Academic Press, New York, pp 21–213 Komagata K, Suzuki K-I (1987) Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19:161–207 Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175 Lemos ML, Toranzo AE, Barja JL (1985) Modified medium for oxidation-fermentation test in the identification of marine bacteria. Appl Environ Microbiol 40:1541–1543 Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218 Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5:109–118 Nedashkovskaya OI, Vancanneyt M, Dawyndt P, Engelbeen K, Vandemeulebroecke K, Cleenwerck I, Hoste B, Mergaert J, Tan TL, Frolova GM, Mikhailov VV, Swings J (2005) Reclassification of [Cytophaga] marinoflava Reichenbach 1989 as Leeuwenhoekiella marinoflava gen. nov., comb. nov. and description of Leeuwenhoekiella aequorea sp. nov. Int J Syst Evol Microbiol 55:1033–1038 Nedashkovskaya OI, Vancanneyt M, Kim SB, Zhukova NV, Han JH, Mikhailov VV (2009) Leeuwenhoekiella palythoae sp. nov., a new member of the family Flavobacteriaceae. Int J Syst Evol Microbiol 59:3074–3077 Pinhassi J, Bowman JP, Nedashkovskaya OI, Lekunberri I, Gomez-Comsarnau L, Pedro´s-Alio´ C (2006) Leeuwenhoekiella blandensis sp. nov., a genome-sequenced marine member of the family Flavobacteriaceae. Int J Syst Evol Microbiol 56:1489–1493 Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. USFCC Newsl 20:16 Simonato F, Gome´z-Pereira PR, Fuchs BM, Amann R (2010) Bacterioplankton diversity and community composition in the Southern Lagoon in Venice. Syst Appl Microbiol 33:128–138 Vieira RP, Gonzalez AM, Cardoso AM, Oliveira DN, Albano RM, Clementino MM, Martins OB, Paranhos R (2008) Relationships between bacterial diversity and environmental variables in a tropical marine environment, Rio de Janeiro. Environ Microbiol 10:189–199
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ORIGINAL PAPER
Flavimarina pacifica gen. nov., sp. nov., a new marine bacterium of the family Flavobacteriaceae, and emended descriptions of the genus Leeuwenhoekiella, Leeuwenhoekiella aequorea and Leeuwenhoekiella marinoflava Olga I. Nedashkovskaya • Andrey D. Kukhlevskiy Natalia V. Zhukova • Seung Bum Kim
•
Received: 27 March 2014 / Accepted: 3 June 2014 Ó Springer International Publishing Switzerland 2014
Abstract A facultatively anaerobic, Gram-stain negative, rod-shaped and yellow pigmented bacterium, designated strain IDSW-73T, was isolated from a seawater sample and subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strain formed a distinct phyletic line in the family Flavobacteriaceae and is most closely related to the members of the genus Leeuwenhoekiella, with 16S rRNA gene sequence similarity of 91.4–92.6 %. Strain IDSW-73T was found to be able to grow with 0–12 % NaCl and at 4–33 °C; and was able to hydrolyse gelatin, starch and Tweens 20, O. I. Nedashkovskaya (&) G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, 690022 Vladivostok, Russia e-mail: [email protected]; [email protected] A. D. Kukhlevskiy N. V. Zhukova A.V. Zhirmunsky Institute of Marine Biology, Far-Eastern Branch of the Russian Academy of Sciences, Pal’chevskogo St. 17, 690032 Vladivostok, Russia A. D. Kukhlevskiy N. V. Zhukova Far Easten Federal University, Sukhanova St. 8, 690950 Vladivostok, Russia
40 and 80. The DNA G?C content was determined to be 42.2 mol%. The predominant cellular fatty acids were identified as branched-chain saturated and unsaturated and straight-chain unsaturated fatty acids such as isoC15:0, iso-C15:1, iso-C17:1 x9c, C15:1 x6c, iso-C15:0 3-OH, iso-C17:0 3-OH and summed feature 3 (as defined by MIDI), comprising iso-C15:0 2-OH and/or C16:1x7c. The polar lipids found were phosphatidylethanolamine, two unknown aminolipids and one unknown lipid. The major respiratory quinone was identified as MK-6. The significant molecular distinctiveness between the novel isolate and its nearest neighbours were strongly supported by notable differences in physiological and biochemical tests. Therefore, strain IDSW-73T is considered to represent a novel genus and species within the family Flavobacteriaceae, for which the name Flavimarina pacifica gen. nov., sp. nov. is proposed. The type strain is IDSW-73T (=KCTC 32466T = KMM 6759T). Emended descriptions of the recognized species of the genus Leeuwenhoekiella are also proposed. Keywords Flavimarina pacifica gen. nov., sp. nov. Flavobacteriaceae Marine bacteria Phylogeny Taxonomy
Introduction S. B. Kim Department of Microbiology and Molecular Biology, School of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, Republic of Korea
Members of the phylum Bacteroidetes are widely distributed in different marine ecosystems and often isolated from surface and deep waters (Alonso et al.
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2007; Simonato et al. 2010; Vieira et al. 2008). They constitute one of the dominant taxonomic groups identified in studies of bacterioplankton communities and are characterized by high specific richness (Hahnke and Harder 2013; Diez-Vivas et al. 2014). The most cultivated Bacteroidetes belong to the family Flavobacteriaceae, which constitutes the largest phylogenetic lineage within the phylum and is comprised of more than 120 validly named genera at the time of writing (http://www.bacterio.net). The prevalence of phylotypes of the family Flavobacteriaceae affiliated to the genera Dokdonia, Formosa, Maribacter, Mesonia, Polaribacter, Psychroserpens, Tenacibaculum and Ulvibacter were reported in a study of bacterial communities inhabited the Yellow Sea (Dang et al. 2008). Representatives of the above listed genera, as well as Aquimarina, Arenibacter, Cellulophaga, Gramella, Kriegella, Leeuwenhoekiella, Maribacter, Muricauda and Zobellia, were identified among isolated bacteria collected from the German Bight in the North Sea (Hahnke and Harder 2013). However, a large number of the flavobacterial 16S rRNA gene sequences remain to be characterized as novel taxa with cultured representatives. In the course of a taxonomic survey of a coastal bacterioplankton community inhabiting the Troitsa Bay in the Sea of Japan, a Gram-negative, rod-shaped, non-motile and yellow-pigmented bacterial isolate, designated IDSW-73T, was obtained. Phylogenetic analysis based on 16S rRNA gene sequences revealed that its closest relatives were known species of the genus Leeuwenhoekiella with 91.4–92.6 % 16S rRNA sequence similarity. Noteworthy, a most nearest neighbour of the novel isolate was strain Flavobacteriaceae bacterium Hel_I_48 (GenBank accession number JX854131), isolated in the North Sea, with 16S rRNA gene sequence similarity of 99.3 % (Hahnke and Harder 2013). The aim of the present study was to describe the isolation and detailed characterization of the novel environmental isolate, IDSW-73T, using a polyphasic taxonomic approach.
Sea of Japan (also known as the East Sea) by a standard plating method. For the strain isolation, 0.1 ml sea water was spread onto marine agar 2216 (MA) plates. The novel isolate was obtained from a single colony after incubation of the plate at 28 °C for 7 days. After primary isolation and purification, the strain was cultivated at 28 °C on the same medium and stored at -80 °C in marine broth supplemented with 20 % (v/v) glycerol.
Materials and methods
Mol% G?C determination
Isolation and cultivation
Genomic DNA was isolated following the method of Marmur (1961) and the G?C content was determined by the thermal denaturation method (Marmur and Doty 1962).
Strain IDSW-73T was isolated from a seawater sample collected from Troitsa Bay, Gulf of Peter the Great,
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16S rRNA gene sequencing and phylogenetic analysis DNA was extracted from 0.1 to 0.2 g of the bacterial cells (wet weight), using the extraction protocol of Sambrook and Russell (2001). PCR was carried out using the universal oligonucleotide primers 11F (50 -GTTTGATCMTGGCTCAG-30 ) and 1492R (50 -TACGGYTACCTTGTTACGACTT30 ) as described by Lane (1991) and the GeneAmp PCR System 9700 (Applied Biosystems Inc.). PCR amplicons were used as templates for sequencing amplification using a BigDye Terminator version 3.1 Cycle sequencing kit (Applied Biosystems). The purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer and the sequence was assembled with SeqScape version 2.6 (Applied Biosystems). The sequences obtained were aligned with those of representative members of selected genera of the family Flavobacteriaceae by using PHYDIT version 3.2 (http://plaza.snu.ac.kr/*jchun/ phydit/). Phylogenetic trees were inferred by using suitable programs of the PHYLIP package (Felsenstein 1993). Phylogenetic distances were calculated from the Jukes-Cantor model (Jukes and Cantor 1969) and the trees were constructed on the basis of the neighbour-joining (Saitou and Nei 1987) and maximum-likelihood (Felsenstein 1993) algorithms. A bootstrap analysis was performed with 1,000 resampled datasets by using the SEQBOOT and CONSENSE programs of the PHYLIP package.
Antonie van Leeuwenhoek
Whole cell polar lipid, fatty acid and lipoquinone composition
Morphological, physiological and biochemical tests
For whole-cell fatty acid and polar lipid analysis, strain IDSW-73T and reference strains Leuwenhoekiella aequorea LMG 22550T, Leuwenhoekiella blandensis MED 217T, Leuwenhoekiella marinoflava LMG 1345T and Leuwenhoekiella palythoae KMM 6264T were grown under optimal physiological conditions for all strains (at 28 °C for 24 h on MA). The reference strains were obtained from our laboratory. Cellular fatty acid methyl esters (FAMEs) were prepared according to the methods described by Sasser (1990) using the standard protocol of the Sherlock Microbial Identification System (version 6.0, MIDI) and analysed using a GC-21A chromatograph (Shimadzu) equipped with a fused-silica capillary column (30 m 9 0.25 mm) coated with Supercowax-10 and SPB-5 phases (Supelco) at 210 °C. FAMEs were identified by using equivalent chain-length measurements and comparing the retention times to those of authentic standards. FAMEs were also analysed by GC–MS (QP5050A; Shimadzu) equipped with an MDN-5S capillary column (30 m 9 0.25 mm), the temperature program was from 140 to 250 °C, at a rate of 2 °C min-1. The polar lipids of strain IDSW-73T and the reference strains were extracted using the chloroform/methanol extraction method of Bligh and Dyer (1959). Twodimensional TLC of polar lipids was carried out on silica gel 60 F254 (10 9 10 cm; Merck) using chloroform/methanol/water (65:25:4, by vol) in the first dimension and chloroform/methanol/acetic acid/ water (80:12:15:4, by vol.) in the second dimension (Collins and Shah 1984). The spray reagents used to reveal the spots were 10 % sulfuric acid in methanol, molybdate reagent and ninhydrin. Isoprenoid quinones were extracted with chloroform/methanol (2:1, v/v) and purified by TLC, using a mixture of n-hexane and diethyl ether (85:15, v/v) as the solvent. Isoprenoid quinone composition was characterized by HPLC (Shimadzu LC-10A) using a reversed-phase type Supelcosil LC-18 column (15 cm 9 4.6 mm) and acetonitrile/ 2-propanol (65:35, v/v) as a mobile phase at a flow rate of 0.5 ml min-1 as described previously (Komagata and Suzuki 1987). The column was kept at 40 °C. Quinones were detected by monitoring at 270 nm.
Cell morphology was examined by light microscopy (Olympus CX41) and transmission electron microscopy (Libra 120; Carl Zeiss). Gram-staining was done as described by Gerhardt et al. (1994). Oxidative and fermentative utilization of glucose was determined using the medium of Hugh and Leifson modified for marine bacteria (Lemos et al. 1985). Catalase activity was tested by addition of 3 % (v/v) H2O2 solution to a bacterial colony and observation for the appearance of gas. The presence of oxidase was tested using N,N,N,N-tetramethyl-p-phenylenediamine. Degradation of agar, starch, casein, gelatin, chitin, DNA, Tweens 20, 40 and 80 and urea, production of acid from carbohydrates, nitrate reduction and production of hydrogen sulphide were tested according to standard methods (Gerhardt et al. 1994). Growth at 4, 10, 25, 28, 30, 32, 35 and 37 °C was measured on MA. Tolerance to NaCl was assessed in medium A containing 5 g Bacto Peptone (Difco), 2 g Bacto Yeast Extract (Difco), 1 g glucose, 0.02 g KH2PO4 and 0.05 g MgSO47H2O per liter of distilled water with 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12 and 15 % (w/v) of NaCl. The pH range for growth was investigated in MB adjusted to pH from 5.0 to 11.0 with HCl and Na2CO3. Physiological and biochemical properties of the novel isolate was also tested using the API 20E, API 20NE, API 50CH and API ZYM systems (bioMe´rieux), according to the manufacturer’s instructions except that the galleries were incubated at 28 °C. Carbon source utilization was tested (1) using commercial API 20E, API 20 NE and API 50CH (bioMe´rieux) identification strips, and (2) using a medium that contained 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g Yeast Extract (Difco) and 0.4 % (w/v) carbon source per liter of artificial seawater. Susceptibility to antibiotics was examined by the routine diffusion plate method. Discs were impregnated with the following antibiotics (lg per disc): ampicillin (10), benzylpenicillin (10U), carbenicillin (100), cefalexin (30), cefazolin (30), chloramphenicol (30), erythromycin (15), doxycycline (10), gentamicin (10), kanamycin (30), lincomycin (15), oleandomycin (15), nalidixic acid (30), neomycin (30), ofloxacin (5), oxacillin (10), polymyxin B (300 U), rifampicin (5), streptomycin (30), tetracycline (5) and vancomycin (30).
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Antonie van Leeuwenhoek T
Galbibacter mesophilus Mok-17 (AB255367) T
Arenibacter latericius KMM 426 (AF052742)
100 *
T
100
Maribacter sedimenticola KMM 3903 (AY271623)
*
T
Maribacter arcticus KOPRI 20941 (AY771762) T
Aquimarina brevivitae SMK-19 (AY987367)
100
T
*
Aquimarina gracilis PSC32 (HM998909) *
T
Aquimarina muelleri KMM 6020 (AY608406)
100
T
Aquimarina intermedia KMM 6258 (AM113977)
*
T
99 *
100
*
50
Dokdonia genika Cos-13 (AB198086) T
Dokdonia donghaensis DSW-1 (DQ003276)
100 Dokdonia eikasta PMA-26T (AB198088) * Dokdonia diaphoros MSKK-32T (AB198089) T
Marixanthomonas ophiurae KMM 3046 (AB261012) T
Gramella echinicola KMM 6050 (AY608409) 85
60
*
T
Salegentibacter salarius ISL-6 (EF486353)
100 * *
T
Salegentibacter mishustinae KMM 6049 (AY576653) T
Salegentibacter holothurioum KMM 3524 (AB116148) T
Salegentibacter salegens DSM 5424 (M92279) 54 * T * Salegentibacter agarivorans KMM 7019 (DQ191176) 76 * T 81 Salegentibacter salinarum ISL-4 (EF612764)
73 *
T
Zunongwangia profunda SM-A87 (DQ855467) 73 *
T
100
Salinimicrobium gaetbulicola BB-My20 (JF340052)
*
T
Salinimicrobium catena HY1 (DQ640642) T
Nonlabens tegetincola UST030701-324 (AY987349)
100
*
T
*
Nonlabens spongiae UST030701-156 (DQ064789) T
100
68
*
Nonlabens agnitus JC2678 (HM475136) T
Nonlabens marina S1-08 (AB602426) T
100
* * 95 *
Leeuwenhoekiella marinoflava ATCC 19326 (M58770) T
Leeuwenhoekiella aequorea LMG 22550 (AJ278780) T
Leeuwenhoekiella blandensis MED217 (DQ294291) T
Leeuwenhoekiella palythoae KMM 6264 (FJ405187) T
0.01
IDSW-73 (KJ152756)
Fig. 1 Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of Flavimarina pacifica gen. nov., sp. nov. IDSW-73T and related members of the family Flavobacteriaceae. The asterisks
indicate clades at genus level that were also recovered from the maximum-likelihood tree. Bootstrap values is based on 1,000 replications. Bar 0.01 substitutions per nucleotide position
Results and discussion
recognized species of the genus Leeuwenhoekiella (Fig. 1). The 16S rRNA gene sequence similarities of the novel isolate and L. palythoae KMM 6264T, L. marinoflava LMG 1345T, L. blandensis MED 217T, and L. aequorea LMG 22550T were 92.6, 92.5, 92.4 and 91.4 %. Other recognized species of the family Flavobacteriaceae exhibited less than 91 % 16S rRNA gene sequence similarity. Significant molecular differences found between strain IDSW-73T and the type strains of the Leeuwenhoekiella species (8.6–7.4 % sequence divergence), from which it can
Molecular phylogenetic analysis An almost-complete 16S rRNA gene sequence (1448 nt) of strain IDSW-73T was determined (GenBank/ EMBL/DDBJ accession number KJ152756). Phylogenetic analysis revealed that strain IDSW-73T is a member of the family Flavobacteriaceae, class Flavobacteriia, phylum Bacteroidetes and formed a distinct evolutionary lineage adjoining to the
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Antonie van Leeuwenhoek Table 1 Cellular fatty acid contents of Flavimarina pacifica gen. nov., sp. nov. and the validly named species of the genus Leeuwenhoekiella Fatty acid
1
2
iso-C15:0
3
4
5
15.0
12.4
18.7
12.4
anteiso-C15:0
1.2
3.9
2.7
3.4
24.1 5.9
iso-C15:1
5.5
9.7
21.5
11.6
4.7
iso-C16:0
1.3
2.7
2.1
tr
8.4
iso-C17:1 x9c
8.6
9.1
8.2
7.8
4.7
anteiso-C17:1 x9c
1.8
tr
tr
tr
–
C15:0
2.0
5.4
5.5
4.3
7.4
C15:1 x6c
5.5
1.4
tr
1.8
1.6
C16:0
tr
tr
1.2
1.1
tr
C17:0
tr
1.3
tr
tr
1.1
C17:1 x6c iso-C15:0-3OH
tr 8.1
1.0 5.0
tr 4.6
tr 6.5
2.2 4.7
iso-C16:0-3OH
tr
5.3
tr
1.5
5.6
iso-C17:0-3OH
23.9
16.0
15.5
17.4
13.6
anteiso-C17:0-3OH
2.1
5.8
tr
3.9
1.4
C17:0-2OH
2.1
5.8
1.0
3.9
1.2
24.5
14.4
13.0
19.5
10.1
Summed feature 3*
* Summed feature 3 consists of the following fatty acids which could not be separated by the microbial identification system: one or more of C16:1x7c and iso-C15:0 2-OH Strains: 1, Flavimarina pacifica gen. nov., sp. nov. IDSW-73T; 2, Leeuwenoekiella aequorea LMG 22550T; 3, L. blandensis MED 217T; 4. L. marinoflava LMG 1345T; 5, L. palythoae KMM 6264T. All data from this study. tr, Trace amount (\1 %). Values are percentages of total fatty acids; those fatty acids for which the mean amount in all taxa was less than 1 % are not given
be concluded that the environmental isolate represents a novel genus and species of the family Flavobactericeae. Mol% G?C determination The DNA G?C content of strain KMM 6759T was determined to be 42.2 mol%. Chemotaxonomic characterization The predominant fatty acids of strain IDSW-73T ([5 % of the total fatty acids) were identified as branched-chain saturated and unsaturated and straightchain unsaturated fatty acids: iso-C15:0 (15.0 %), isoC15:1 (5.5 %), C15:1 x6c (5.5 %), iso-C17:1 x9c (8.6 %), iso-C15:0 3-OH (8.1 %), iso-C17:0 3-OH
(23.9 %) and summed feature 3 (comprising C16:1 x7c and/or iso-C15:0 2-OH fatty acids as defined by the MIDI system; 24.5 %). The fatty acid profile of strain IDSW-73T strain was similar to those of the type strains of the genus Leeuwenhoekiella, while the differences in the proportions of some fatty acids, including anteiso-C15:0, anteiso-C17:1 x9c, C15:0, C15:1 x6c and iso-C16:0-3OH, were marked (Table 1). The polar lipid profile of the novel isolate was found to be composed of phosphatidylethanolamine, two unknown lipids and one unknown aminolipid, which is in line with those of the reference strains (Fig. 2). However, the presence of two unknown lipids (L1 and L2) and the absence of another unknown lipid (L) could distinguish strain IDSW-73T from its phylogenetic relatives except L. aequorea LMG 22550T. The sole respiratory quinone found to be present in the isolate was menaquinone 6, which is a characteristic lipoquinone for members of the family Flavobacteriaceae (Bernardet et al. 2002). Morphological, physiological and biochemical characteristics The physiological and biochemical characteristics of strain IDSW-73T are given in the genus and species descriptions and Table 2. The isolate was observed to be a rod-shaped, motile by gliding, facultatively anaerobic and oxidase- and catalase-positive organism. Strain IDSW-73T was found to be susceptible to ampicillin, carbenicillin, cefalexin, chloramphenicol, erythromycin, lincomycin nalidixic acid, ofloxacin, oleandomycin and rifampicin; and resistant to benzylpenicillin, cefazolin, doxycycline, gentamicin, kanamycin, neomycin, streptomycin, tetracycline, oxacillin, polymyxin B and vancomycin. The novel strain can be clearly distinguished from its closest relatives by the fermentative type of metabolism, maximum growth temperature (33 vs. 37–41 °C) and the ability to produce acid from mannose and raffinose and to utilize inositol. Such phenotypic properties as the absence of a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase and a-mannosidase activities and acid formation from glycerol are also useful tests for separation of strain IDSW-73T from the members of the genus Leeuwenhoekiella. Other phenotypic characteristics that differentiate the isolate from its nearest neighbours are shown in Table 2.
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Fig. 2 Thin-layer chromatogram showing the polar lipid profiles of strain IDSW-73T (a), L. aequorea LMG 22550T (b); L. blandensis MED 217T (c); L. marinoflava LMG 1345T (d); 5, L. palythoae KMM 6264T (e) following separation by two-dimensional TLC. Solvent systems: (1) chloroform–
methanol–water (65:25:4, v/v/v.); (2) chloroform–acetic acid– methanol–water (80:15:12:4, v/v/v/v.). PE, phosphatidylethanolamine; AL, an unknown aminolipid; L, L1 and L2, the unknown lipids
Polyphasic taxonomic conclusion
nov., sp, nov. is proposed. The emended descriptions of the genus Leeuwenhoekiella, L. aequorea and L. marinoflava are also proposed due to newly obtained data.
The results obtained in the present study indicated that strain IDSW-73T demonstrates low 16S rRNA gene sequence similarities with its closest phylogenetic relatives, members of the genus Leeuwenhoekiella, and other validly named representatives of the family Flavobacteriaceae (91.4–92.6 % and less than 91 %, respectively). Furthermore, the novel strain can be clearly differentiated from validly named Leeuwenhoekiella by the type of metabolism (fermentative vs. aerobic), maximum growth temperature (33 vs. 37–41 °C), acid production from mannose and raffinose, inositol utilization and the absence of agalactosidase, b-galactosidase, a-glucosidase, b-glucosidase and a-mannosidase activities. The abovementioned findings, including phylogenetic and phenotypic characteristics, strongly support the discrimination of the novel strain from its close relatives at the generic level. Therefore, on the basis of the phylogenetic, genotypic, chemotaxonomic and phenotypic data presented here, we suggest that strain IDSW-73T should be classified as representing a novel genus and species within the family Flavobacteriaceae, for which the name Flavimarina pacifica gen.
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Description of Flavimarina gen. nov. Flavimarina (Fla.vi.ma.ri’na. L. adj. flavus, yellow; L. fem. adj. marina, marine; N.L. fem. n. Flavimarina, a yellow-coloured organism of marine origin). Cells are Gram-negative, facultatively anaerobic, rod-shaped and non-motile. Do not form endospores. Non-diffusible pigments are produced. Chemo-organotroph. Cytochrome oxidase-, catalase and alkaline phosphatase-positive. The predominant cellular fatty acids are branched-chain saturated and unsaturated and straight-chain unsaturated fatty acids such as iso-C15:0, iso-C15:1, iso-C17:1 x9c, C15:1 x6c, iso-C15:0 3-OH, iso-C17:0 3-OH and summed feature 3 (comprising iso-C15:0 2-OH and/or C16:1x7c). Polar lipids are phosphatidylethanolamine, two unknown aminolipids and one unknown lipid. The major respiratory quinone is MK-6. The DNA G?C content of the type strain of the type species is 42.2 mol%. 16S rRNA
Antonie van Leeuwenhoek Table 2 Differential phenotypic characteristics of Flavimarina pacifica gen. nov., sp. nov. and the validly named species of the genus Leeuwenhoekiella Characteristic
1
2
3
4
5
Source of isolation
Seawater
Seawater
Seawater
Seawater
Soft coral -
Glucose fermentation
?
-
-
-
Temperature range for growth (°C)
4–33
4–37
10–41
4–37
4–38
Salinity range for growth (% NaCl)
0–12
0–15
0–17
0–15
0–12
Casein
-
?
?
?
-
Tween 20
?
?
?
?
-
Arabinose, maltose
?
-
-
-
?
Cellobiose
-
-
-
-
?
Galactose
?
?
-
?
?
Glucose
?
-
?
-
?
Lactose
-
-
-
-
?
Mannose, raffinose
?
-
-
-
-
Rhamnose Sucrose
?
?
-
-
? ?
Xylose
?
-
?
-
-
Glycerol
-
?
?
?
?
Mannitol
-
?
-
-
-
Inositol
?
-
-
-
-
Mannitol
?
?
-
-
-
Esterase (C4)
?
?
?
?
-
Lipase (C14)
?
-
?
?
-
Cystine arylamidase
?
-
?
?
-
a-Chymotrypsin
-
-
?
-
-
Trypsin
?
-
?
?
?
Naphtol-AS-BI-phosphohydrolase
?
?
-
?
?
a-Galactosidase, b-galactosidase a-Glucosidase, b-glucosidase
-
? ?
? ?
? ?
? ?
N-Acetyl-b-glucosaminidase, a-mannosidase
-
?
?
?
?
DNA G?C content (mol%)
42.2
35–36
42.5
38
Hydrolysis of
Acid formation from
Utilization of
Enzyme activity (API ZYM)
T
41.2 T
Strains: 1, Flavimarina pacifica gen. nov., sp. nov. IDSW-73 ; 2, Leeuwenhoekiella aequorea LMG 22550 ; 3 L. blandensis MED 217T; 4. L. marinoflava LMG 1345T; 5, L. palythoae KMM 6264T. Data from Nedashkovskaya et al. (2005, 2009), Pinhassi et al. (2006), and this study. All strains are positive for gliding motility; oxidase, catalase, alkaline phosphatase, acid phosphatase, esterase lipase (C8), leucine arylamidase and valine arylamidase activities; gelatin, starch and Tweens 40 and 80 hydrolysis; utilization of arabinose, glucose, lactose, mannose and sucrose. All strains are negative for nitrate reductase, a-fucosidase and b-glucuronidase activities, flexirubin-type pigments production, H2S and indole production, agar, DNA, chitin, urea and cellulose hydrolysis
gene sequence analysis indicates that the genus Flavimarina is a member of the family Flavobacteriaceae, the class Flavobacteriia within the phylum Bacteroidetes. The type species is Flavimarina pacifica.
Description of Flavimarina pacifica sp. nov. Flavimarina pacifica [pa.ci’fi.ca. L. fem. adj. pacifica, peaceful; pertaining to the Pacific Ocean from where the type strain was isolated].
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Flavimarina pacifica demonstrates the following characteristics in addition to those given for the genus. Cells are 0.4–0.6 lm in diameter and 1.2–3.5 lm in length. On marine agar, colonies are 2–3 mm in diameter, circular, with entire edges, shiny and yellow in colour. Growth occurs at 4–33 °C (optimum, 25–28 °C), at pH 5.5–10.0 (optimum, pH 7.5–8.0) and with 0–12 % NaCl (optimum, 2.0–5.0 %). Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase activities are absent. Gelatin, starch and Tweens 20, 40 and 80 are hydrolysed but agar, casein, DNA, urea, chitin and cellulose are not hydrolysed. Acid is produced from Larabinose, D-galactose, D-glucose, maltose, mannose, raffinose, sucrose and D-xylose, but not from cellobiose, D-fructose, D-lactose, melibiose, L-rhamnose, ribose, trehalose, N-acetylglucosamine, glycerol, inositol, mannitol, sorbitol and citrate. Cellobiose, melibiose, L-rhamnose, trehalose and N-acetylglucosamine are utilized, but inositol, mannitol, sorbitol, citrate, methionine and tryptophan are not utilized. Growth is observed on L-alanine, L-asparagine, Lglutamine, L-leucine, L-proline, L-threonine, L-tyrosine and L-valine, but no growth occurs on DLmethionine and L-tryptophan. In the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, valine arylamidase, trypsin, acid phosphatase and naphtol-AS-BI-phosphohydrolase activities are present; but a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, bglucosidase N-acetyl-b-glucosaminidase, a-mannosidase and a-fucosidase activities are absent. Nitrate is not reduced to nitrite. Hydrogen sulphide, indole and acetoin are not produced. In the API 20E gallery strain IDSW-73T is positive for ONPG, gelatin hydrolysis and acid production from glucose, rhamnose, sucrose, melibiose, amygdalin and arabinose. In API 20NE, positive results are obtained for glucose fermentation, aesculin and gelatin hydrolysis, the PNPG test (bglucosidase) and assimilation of glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, maltose, gluconate and adipate. In API 50CH, positive results are obtained for L-arabinose, D-xylose, galactose, glucose, fructose, mannose, methyl-a-D-glucopyranoside, methyl-a-D-mannopyranoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salicin, Dcellobiose, maltose, D-lactose, D-melibiose, sucrose, trehalose, raffinose, amidon, D-turanose and D-lixose.
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The DNA G?C content of the type strain is 42.2 mol%. The type strain, IDSW-73T (= KCTC 32466T = KMM 6759T), was isolated from seawater collected from Troitsa Bay, Gulf of Peter the Great, Sea of Japan (also is known as East Sea), Pacific Ocean, Russia. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of F. pacifica IDSW-73T is KJ152756.
Emended description of the genus Leeuwenhoekiella Nedashkovskaya et al. (2005) The description of the genus Leeuwehhoekiella is as given by Nedashkovskaya et al. (2005) with the following additions. The major polar lipids are phosphatidylethanolamine and one unknown aminolipid; unidentified lipids are also present.
Emended description of Leeuwenhoekiella aequorea Nedashkovskaya et al. (2005) The description of L. aequorea is as given by Nedashkovskaya et al. (2005) with the following additions. Using the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase and a-mannosidase activities are present but lipase (C14), cystine arylamidase, a-chymotrypsin, trypsin, a-fucosidase and bglucuronidase activities are absent. Polar lipids are phosphatidylethanolamine, one unknown aminolipid and two unknown lipids.
Emended description of Leeuwenhoekiella marinoflava Nedashkovskaya et al. (2005) The description of L. marinoflava is as given by Nedashkovskaya et al. (2005) with the following additions. Using the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, cystine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, a-galactosidase, b-
Antonie van Leeuwenhoek
galactosidase, a-glucosidase, b-glucosidase, N-acetylb-glucosaminidase and a-mannosidase activities are present but a-chymotrypsin, a-fucosidase and bglucuronidase activities are absent. Polar lipids are phosphatidylethanolamine, one unknown aminolipid and one unknown lipid. Acknowledgments This research was supported by grants of the Presidium of the Russian Academy of Sciences ‘‘Molecular and Cell Biology’’, Russian Scientific Fund no. 14-14-00030, the Presidium of the Far-Eastern Branch of the Russian Academy of Sciences no. 12-III-A-06-105 and the government of Russian Federation for the state support of scientific investigations conducting under the guidance of the leading researchers at the Russian education institutions of the high professional education, agreement no. 11.G34.31.0010.
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