Journal - International Journal of Systematic and Evolutionary


Journal - International Journal of Systematic and Evolutionary
International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2075–2081
DOI 10.1099/ijs.0.040394-0
Flavobacterium anatoliense sp. nov., isolated from
fresh water, and emended description of
Flavobacterium ceti
Murat Kacagan, Kadriye Inan, Ali Osman Belduz and Sabriye Canakci
Sabriye Canakci
[email protected]
Karadeniz Technical University, Faculty of Sciences, Department of Biology, 61080 Trabzon, Turkey
A Gram-staining-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped bacterial
strain isolated from fresh water in Trabzon, Turkey and designated MK3T was characterized by
phenotypic and molecular methods in order to determine its phylogenetic position. On the basis of
16S rRNA gene sequence similarity, strain MK3T was shown to belong to the genus
Flavobacterium, being most closely related to Flavobacterium ceti CECT 7184T (93.6 %).
Sequence similarity with other species of the genus Flavobacterium with validly published names
was less than 91.6 %. Phenotypic and chemotaxonomic data supported the affiliation of strain
MK3T to the genus Flavobacterium. The only menaquinone was MK-6; the major fatty acids were
iso-C15 : 0 (45.2 %), summed feature 9 (C16 : 0 10-methyl and/or iso-C17 : 1v9c; 20.4 %) and
summed feature 3 (C16 : 1v7c and/or C16 : 1v6c; 13.3 %) and the major polar lipids were
phosphatidylethanolamine, one unidentified aminophospholipid and two unidentified phospholipids. The G+C content of the genomic DNA was 38.6 mol%. The results of physiological and
biochemical tests allowed strain MK3T to be distinguished phenotypically from Flavobacterium
ceti CECT 7184T. Strain MK3T, therefore, represents a novel species of the genus
Flavobacterium, for which the name Flavobacterium anatoliense sp. nov. is proposed. The type
strain is MK3T (5LMG 26441T5NCCB 100384T). An emended description of Flavobacterium
ceti is also proposed.
The genus Flavobacterium, belonging to the phylum
Bacteroidetes (formerly the Cytophaga–Flavobacterium–
Bacteroides group), was proposed by Bergey et al. (1923)
and its description was considerably emended by Bernardet
et al. (1996). Flavobacterium species have been described
from diverse environmental habitats, such as microbial
mats from lakes, fresh water, seawater, sea ice, soil, the gut
of an earthworm, the lung and liver of a beaked whale,
sediments and wastewater treatment plants (McCammon
& Bowman, 2000; Van Trappen et al., 2002; Zhu et al.,
2003; Horn et al., 2005; Kim et al., 2006; Yi & Chun, 2006;
Yoon et al., 2006; Vela et al., 2007; Ryu et al., 2007).
Physiological characteristics of Flavobacterium strains are
also very diverse: they can be psychrophilic, psychrotolerant or mesophilic, and halotolerant, halophilic or
sensitive to salts. They produce a variety of enzymes
(Humphry et al., 2001; Tamaki et al., 2003; Aslam et al.,
2005; Zhang et al., 2006). These findings suggest that these
bacteria may have important roles in the uptake and
degradation of organic matter in aquatic environments
(Kirchman, 2002). Indeed, many species of the genus
Abbreviations: FAME, fatty acid methyl ester; PS, phosphatidylserine.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequences of Flavobacterium anatoliense MK3T is JF825522.
040394 G 2013 IUMS
Flavobacterium are able to hydrolyse organic polymers such
as complex polysaccharides (Bernardet et al., 1996).
Strain MK3T was isolated from a sample of fresh water
collected from the Yomra river 204 m above sea level and
7 km inland from the coast in Trabzon, Turkey. Temperature
and pH of the sample were 20 uC and pH 8.0, respectively.
The water sample was filtered within 2 h of sampling through
Millipore membrane filters with a pore size of 0.45 mm. The
filters were deposited on Degryse 162 agar and incubated at
28 uC for 3 days (Kristjansson & Alfredsson, 1983). Degryse
162 agar contained (l21) the following: yeast extract, 2.5 g;
tryptone, 2.5 g; nitrilotriacetic acid, 1.0 g; CaSO4 . 2H2O,
0.4 g; MgSO4 . 7HO, 2.0 g; 15 ml 0.2M Na2HPO4 . 12H2O;
10 ml 0.2 M KH2PO4; 0.5 ml of 0.01 M Fe(III) citrate .
5H2O; 5.0 ml trace element solution, pH 7.5 and 28.0 g agar.
The trace element solution contained (l21) the following:
MnSO4 . H2O, 0.22 g; ZnSO4 . 7H2O, 0.05 g; H3BO3, 0.05 g;
CuSO4 . 5H2O, 0.0025 g; Na2MoO4 . 2H2O, 0.0025 g; and
CoCl2 . 6H2O, 0.0046 g (Degryse et al., 1978). Yellow
pigmented colonies were picked from the filters (which
otherwise contained mostly white bacterial colonies) and
purified by streaking on the same medium. The purity of
the isolate was assessed by using colony morphology
and microscopy. Strain MK3T was routinely cultured on
Degryse 162 agar and preserved at 280 uC as a suspension in
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M. Kacagan and others
Degryse 162 broth supplemented with 20 % glycerol (v/v).
Growth was tested on Degryse 162 agar, nutrient agar (NA;
Merck), Luria–Bertani agar (LB; Himedia), MacConkey agar
(Oxoid) and trypticase soy agar (TSA; Oxoid).
The 16S rRNA gene of strain MK3T was selectively amplified
from purified genomic DNA by using the oligonucleotide
primer pair UNI16S-L and UNI16S-R (Brosius et al., 1978).
PCRs were performed as follows: 2 min at 95 uC, 36 cycles of
94 uC for 1 min, 50 uC for 45 s, 72 uC for 2 min and finally
10 min at 72 uC. The PCR product was cloned into the
pGEM-T Easy vector system and the 16S rRNA gene
sequence was determined with a model 373A DNA sequencer
(Applied Biosystems) by using an ABI PRISM cycle
sequencing kit at Macrogen (South Korea). The results of
16S rRNA gene sequencing were analysed using the EzTaxon
server (; Chun et al., 2007). The 16S
rRNA gene sequences of related taxa were obtained from
GenBank and edited by using the BioEdit program (Hall,
1999) and multiple alignments were performed with the
CLUSTAL_X program (Thompson et al., 1997). Evolutionary
distances were calculated by using Kimura’s two-parameter
model (Kimura, 1980), and phylogenetic analyses were
performed by the neigbour-joining (Saitou & Nei, 1987) and
maximum-parsimony (Fitch, 1971) methods. Bootstrap
analysis based on 1000 replicates was also conducted in
order to obtain confidence levels for the branches
(Felsenstein, 1985). The phylogenetic trees were constructed
using the program MEGA4 (Tamura et al., 2007).
The almost-complete 16S rRNA gene sequence of strain
MK3T (1404 nt) was obtained and used for the phylogenetic analyses. Strain MK3T exhibited the highest level of 16S
rRNA gene sequence similarity with Flavobacterium ceti
CECT 7184T (93.6 %). Sequence similarity was distinctly
lower with the next closest relatives (i.e. 91.6 % with
Flavobacterium weaverense JCM 12384T, Flavobacterium
beibuense LMG 25233T, Flavobacterium rivuli DSM
21788Tand Flavobacterium cheniae CGMCC 1.6844T).
Sequence similarity with the type strain of the type species,
Flavobacterium aquatile ATCC 11947T, was 89.9 %. The
neigbour-joining phylogenetic tree (Fig. 1) revealed that
strain MK3T is a member of the genus Flavobacterium,
forming a cluster with Flavobacterium ceti CECT 7184T
(96 % bootstrap support). The topology of the maximumparsimony tree was essentially the same (data not shown).
Cells of strain MK3Tand F. ceti CECT 7184T were Gramstained using the method of Dussault (1955) and the KOH
method (Powers, 1995). Cell morphology and gliding
motility were examined by using phase-contrast microscopy (Nikon Eclipse E600) on an exponentially growing
liquid culture. Gliding mobility was investigated using the
hanging drop technique (Bernardet et al., 2002). The
morphology, size and pigmentation of colonies were
observed under optimal growth conditions on Degryse
162 agar after 1 day of incubation at 28 uC. Growth was
tested in Degryse 162 broth at 4–45 uC (at 5 uC intervals)
and at pH 4.0–11.0 (at 0.5 pH unit intervals). The pH of
the medium was adjusted using the following biological
buffers: 30 mM C2H3O2Na (pH 4.0–5.5); 30 mM MES
(pH 5.5–6.0); 30 mM K2HPO4/KH2PO4 (pH 6.5–7.5);
30 mM Tricine (pH 8.0–9.0) and 30 mM CAPS (pH 9.5–
11.0). The pH of each buffer was adjusted with HCl or
NaOH, the pH values were determined at room temperature, and the pH was readjusted after sterilization. Growth
was scored as the OD600 after 3 days of incubation.
Anaerobic growth was tested using a GasPak pouch
(Becton Dickinson) for 7 days. Salt tolerance was tested in
Degryse 162 broth supplemented with 0210 % NaCl (w/v,
at 1 % intervals) for 7 days at 28 uC. The sensitivity of strain
MK3T to antibiotics was examined on plates of Degryse 162
agar incubated at 28 uC. The following standard (6 mm)
antibiotic discs (Oxoid) were used: ampicillin (10 mg),
gentamicin (10 mg), cefuroxime (30 mg), cefazolin (30 mg),
trimethoprim/sulfamethoxazole (1.25/23.75 mg), imipenem
(10 mg), ciprofloxacin (5 mg), amikacin (30 mg), meropenem (10 mg), ceftriaxone (30 mg), piperacillin/tazobactam
(100/10 mg), cefepime (30 mg), levofloxacin (5 mg), cefoperazone (75 mg) and kanamycin (30 mg). The diameters of
inhibition zones were measured after 2 days. Oxidase
activity was tested by determining oxidation of 1 % (w/v)
tetramethyl-p-phenylenediamine (Merck) and catalase activity was evaluated by determining the production of oxygen
bubbles in a 3 % (v/v) aqueous hydrogen peroxide solution
(Baek et al., 2006). The production of flexirubin-type
pigments and of extracellular glycans was investigated using
the KOH and Congo red tests, respectively, according to the
minimal standards for the description of new taxa in the
family Flavobacteriaceae (Bernardet et al., 2002). Hydrolysis
of casein was determined by observing clear zones surrounding colonies on skim milk agar (Atlas, 1993).
Hydrolysis of starch, chitin, Tween 20, Tween 80, aesculin,
tyrosine, carboxymethylcellulose and DNA was investigated
according to previously described methods (Bernardet et al.,
2002; Smibert & Krieg, 1994; Bowman et al., 1996; Bowman,
2000). In addition the API 20E and Vitek2 Gram Negative
Identification Card (GNI) microtest systems (bioMérieux)
were used at 28 uC for 18–24 h according to the manufacturer’s instructions. Sporulation was tested on solid
maintenance medium (Smibert & Krieg, 1994).
Strain MK3T grew optimally on Degryse 162 agar at 28 uC,
yielding dark yellow, convex and circular colonies. Cells
were strictly aerobic, Gram-staining-negative, non-motile
rods that were able to grow at 6–40 uC and at pH 5.5–10.0.
Other biochemical and physiological properties of strain
MK3Tare presented in Table 1 and in the species
Fatty acid methyl esters (FAMEs) of strain MK3T and F.
ceti CECT 7184T were obtained from 40 mg wet cells
collected from Degryse 162 agar plates after incubation at
28 uC for 24 h in the late-exponential growth phase by
saponification, methylation and extraction using minor
modifications of described methods (Kuykendall et al.,
1988; Miller, 1982). The FAME mixtures were analysed
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Flavobacterium anatoliense sp. nov.
Flavobacterium gillisiae ACAM 601 (U85989)
Flavobacterium degerlachei LMG 21915 (AJ557886)
Flavobacterium frigoris LMG 21922 (AJ557887)
Flavobacterium x injiangense JCM 11314 (AF433173)
Flavobacterium limicola DSM 15094 (AB075230)
Flavobacterium x anthum ACAM 81 (AF030380)
Flavobacterium omnivorum JCM 11313 (AF433174)
Flavobacterium frigidarium ATCC 700810 (AF162266)
Flavobacterium psychrophilum ATCC 49418 (AM398681)
Flavobacterium micromati LMG 21919 (AJ557888)
Flavobacterium pectinovorum NCIMB 9059 (AM230590)
Flavobacterium hibernum ATCC 51468 (L39067)
Flavobacterium hydatis ATCC 29551 (M230487)
Flavobacterium aquidurense WB 1.1-56 (AM177392)
Flavobacterium frigidimaris KUC-1 (AB183888)
Flavobacterium hercynium WB 4.2-33 (AM265623)
Flavobacterium saccharophilum NCIMB 2072 (AM230491)
Flavobacterium succinicans IFO 14905 (AM230492)
Flavobacterium soli DS-6 (DQ178976)
Flavobacterium daejeonense DSM 17708 (DQ222427)
Flavobacterium johnsoniae ATCC 17061 (CP000685)
Flavobacterium flevense ATCC 27944 (AM230486)
Flavobacterium antarcticum AT1026 (AY581113)
Flavobacterium tegetincola ACAM 602 (U85887)
Flavobacterium weaverense JCM 12384 (AY581114)
Flavobacterium segetis AT1048 (AY581115)
Flavobacterium gelidilacus LMG 21477 (AJ440996)
Flavobacterium aquatile ATCC 11947 (M62797)
Flavobacterium cheniae NBRC 103934 (EF407880)
Flavobacterium croceum DSM 17960 (DQ372982)
Flavobacterium indicum DSM 17447 (AY904351)
Flavobacterium columnare IAM 14301 (AB078047)
Flavobacterium saliperosum JCM 13331 (DQ021903)
Flavobacterium anatoliense MK3 (J F825522)
Flavobacterium ceti CECT 7184 (AM292800)
Flavobacterium beibuense LMG 25233 (GQ245972)
Flavobacterium rivuli DSM 21788 (AM934661)
Leeuwenhoekiella marinoflava ATCC 19326 (AB680577)
Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between strain
MK3T and the type strains of representative species of the genus Flavobacterium. Bootstrap values (expressed as percentages
of 1000 replications) greater than 70 % are shown at the branch points. Leeuwenhoekiella marinoflava ATCC 19326T was
used as an outgroup. Bar, 0.01 substitutions per nucleotide position.
using the instant FAME method of the Sherlock Microbial
Identification System (MIDI) version 6.0B using an
AgilentTec model 6890N gas chromatogram and identified
with the MIDI RTSBA60 database (Sasser, 1990). Peaks
were automatically integrated and percentages were
calculated using the MIS Standard Software (Microbial
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M. Kacagan and others
Table 1. Differential characteristics of strain MK3T and
Flavobacterium ceti CECT 7184T
Strains: 1, Flavobacterium anatoliense sp. nov. MK3T; 2,
Flavobacterium ceti CECT 7184T . +, Positive reaction; 2, negative;
W, weakly positive (all data from this study).
Cell length (mm)
Colony pigmentation
Growth on nutrient agar
Growth at 15 uC
Growth with 5 % NaCl
Optimum temperature (uC)
Enzyme activity (VITEK 2 GNI)
Hydrolysis of:
Arginine (API 20E)
DNA G+C content (mol%)
ID). The major fatty acids of strain MK3T were iso-C15 : 0
(45.2 %), summed feature 9 (C16 : 0 10-methyl and/or isoC17 : 1v9c; 20.4 %) and summed feature 3 (C16 : 1v6c and/or
C16 : 1v7c; 13.3 %). The fatty acid composition of F. ceti
CECT 7184T was similar, with only minor differences in
the respective proportions of the components. The
complete fatty acid profiles are given in Table 2. The
major fatty acids are in accordance with those of members
of the genus Flavobacterium (Bernardet et al., 1996, 2002;
Bernardet & Bowman, 2011; Vela et al., 2007).
Table 2. Fatty acid compositions (%) of Flavobacterium
anatoliense sp. nov. MK3T and Flavobacterium ceti CECT
Strains: 1, F. anatoliense MK3T; 2, F. ceti CECT 7184T. The fatty acids
amounting to ,1 % of the total fatty acids in the two strains are not
shown. TR, Trace (,1 % of total). Summed feature 3 comprises
(C16 : 1v7c and/or C16 : 1v6c), summed feature 9 comprises (C16 : 0 10methyl and/or iso-C17 : 1v9c).
Fatty acid
iso-C14 : 0
iso-C15 : 0
iso-C15 : 0 3-OH
C15 : 0 2-OH
anteiso-C15 : 0
C16 : 0
iso-C16 : 0
iso-C17 : 0 3-OH
Summed feature 9
Summed feature 3
Isoprenoid quinones of strain MK3T and polar lipids of
strain MK3T and F. ceti CECT 7184T were extracted from
100 mg freeze-dried cells using the two-stage method
described by Tindall (1990a, b). Polar lipids were separated
by two-dimensional TLC on silica gel (no. 818 135;
Macherey–Nagel). The first direction was developed with
chloroform : methanol : water (65 : 25 : 4, v/v) and the
second direction with chloroform : methanol : acetic acid : water (80 : 12 : 15 : 4, v/v). Detection was performed using
5 % ethanolic molybdophosphoric acid for the total lipids,
molybdenum blue for phospholipids, ninhydrin for
aminolipids, the periodate-Schiff reagent for a-glycols
and a-naphthol sulphuric acid for glycolipids (Tindall
et al., 2007). One spot that reacted with ninhydrin and
molybdenum blue showed 2D migration rate (RF) values
very similar to those of phosphatidylserine (PS) used as a
reference on the control TLC plate. Though the RF value of
the first dimension was exactly the same as the reference,
there was a small deviation in the RF value of the second
dimension. Therefore, the spot was considered an unidentified aminophospholipid (APL7) instead of PS.
The only isoprenoid quinone of strain MK3T was MK-6, in
line with all other members of the family Flavobacterıaceae
(Bernardet & Bowman, 2011). The major polar lipids of
strain MK3T were phosphatidylethanolamine, one unidentified aminophospholipid and two unidentified phospholipids (Fig. 2). Minor amounts of one unidentified aminolipid
and other unidentified aminophospholipids and phospholipids were also detected. The polar lipid profile F. ceti CECT
7184T was similar, except that the major phospholipid in
strain MK3T (PL4) was only present in minor amounts and
that three additional unidentified aminolipids were present.
For determination of the G+C content of strain MK3T,
genomic DNA was extracted and purified using the method of
Marmur (1961), and the G+C content was determined from
the midpoint value (Tm) of the thermal denaturation profile
(Mandel & Marmur, 1968). The DNA of Escherichia coli K-12
was used as standard. The DNA G+C content of strain MK3T
was 38.6 mol%, a value consistent with that of members of the
genus Flavobacterium (Bernardet & Bowman, 2011).
Hence, on the basis of the phylogenetic distance from all
recognized species of the genus Flavobacterium and the
combination of unique phenotypic characteristics (Table
1), strain MK3T represents a novel species of the genus
Flavobacterium, for which the name Flavobacterium
anatoliense sp. nov. is proposed. In addition, an emended
description of Flavobacterium ceti is proposed on the basis
of new data obtained in this study.
Description of Flavobacterium anatoliense
sp. nov.
Flavobacterium anatoliense ( N.L. neut. adj.
anatoliense of or belonging to Anatolia).
Cells are Gram-staining-negative, strictly aerobic, nonmotile, non-spore-forming rods approximately 0.2–0.4 mm
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Flavobacterium anatoliense sp. nov.
Fig. 2. Two-dimensional TLC of the total polar
lipids of strain MK3T(a) and Flavobacterium ceti
CECT 7184T (b). PE, Phosphatidylethanolamine;
APL1–7, unidentified aminophospholipids; AL1–
5, unidentified aminolipids; PL1–4, unidentified
in diameter and 0.8–1.2 mm in length. Optimal growth
occurs on Degryse 162 agar. Colonies are convex, circular
and dark yellow with regular edges. Good growth also
occurs on TSA and NA, and weak growth occurs on LB
agar. No growth occurs on MacConkey agar. Growth
occurs at 6–40 uC (optimum, 28 uC) but no growth occurs
4 uC and 45 uC. Optimal pH for growth is pH 7.0; growth
occurs at pH 5.5–10.0. Growth occurs in the absence of
NaCl and in the presence of up to 6 % (w/v) NaCl, but not
with 7 % and 10 % (w/v) NaCl. Flexirubin pigments are
produced (KOH test-positive) and Congo red is not
absorbed by colonies. Catalase and oxidase tests are
positive. Casein is hydrolysed but starch, aesculin, chitin,
DNA, carboxymethylcellulose and Tweens 20 and 80 are
not. Tyrosine is degraded and a brown pigment is also
produced on tyrosine agar. In the API 20E strip, OPNG
hydrolysis, production of urease, tryptophan deaminase
and lysine and ornithine decarboxylases, nitrate reduction,
citrate utilization, hydrogen sulfide and indole production
are negative; gelatin and arginine hydrolysis, production of
arginine dihydrolase and the Voges–Proskauer reaction are
positive. Acid is not produced from D-glucose, D-mannitol,
inositol, D-sorbitol, L-rhamnose, sucrose, melibiose, amygdalin or L-arabinose (API 20E). The following carbon
sources are not assimilated in the VITEK 2 GNI System:
adonitol, L-arabitol, D-glucose, maltose, D-mannitol, Dmannose, palatinose, D-sorbitol, sucrose, D-tagatose, trehalose, malonate, 5-keto-D-gluconate, coumarate, L-histidine,
L-malate, L-lactate and succinate. Also in the VITEK 2
system, lipase, phosphatase, Ala–Phe–Pro-arylamidase, glutamyl arylamidase, tyrosine arylamidase, L-proline arylamidase, glycine arylamidase and Glu–Gly–Arg-arylamidase
activities are present; b-galactosidase, b-glucosidase, bxylosidase, a-glucosidase, b-N-acetyl-galactosaminidase, agalactosidase and b-glucuronidase activities are absent.
Resistant to ampicillin, gentamicin, ceftriaxone, cefoperazone, cefazolin and cefuroxime; Sensitive to kanamycin,
imipenem, ciprofloxacin, amikacin, meropenem, piperacillin/tazobactam, cefepime, levofloxacin and trimethoprim/
sulfamethoxazole. The polar lipid profile includes phosphatidylethanolamine, one unidentified aminophospholipid,
two unidentified phospholipids and minor amounts of
one unidentified aminolipid and other unidentified aminophospholipids and phospholipids. The only isoprenoid
quinone is MK-6. The major cellular fatty acids (.10.0 %)
are iso-C15 : 0, summed feature 9 (C16 : 0 10-methyl and/or
iso-C17 : 1v9c) and summed feature 3 (C16 : 1v6c and/or
C16 : 1v7c).
The type strain is MK3T (5LMG 26441T5NCCB
100384T), isolated from fresh water in Trabzon, Turkey.
The DNA G+C content of the type strain is 38.6 mol%.
Emended description of Flavobacterium ceti Vela
et al. 2007
The description is as given by Vela et al. (2007) with the
following amendment. The major polar lipids are phosphatidylethanolamine, two unidentified aminophospholipids and one unidentified phospholipid. Significant
amounts of unidentified aminolipids, unidentified phospholipids and unidentified aminophospholipids are also
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