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Article

Research article

J. Microbiol. Biotechnol. 2019; 29(8): 1266-1272

Published online August 28, 2019 https://doi.org/10.4014/jmb.1812.12011

Copyright © The Korean Society for Microbiology and Biotechnology.

Flavobacterium jocheonensis sp. nov., Isolated from Marine Green Alga Ulva pertusa

Ha Ri Choi , So Hyun Park and Moon Soo Heo *

Department of Aquatic Life Medicine, Jeju National University, Jeju 63243, Republic of Korea

Correspondence to:Moon Soo  Heo
msheo@jejunu.ac.kr

Received: December 8, 2018; Accepted: July 29, 2019

Abstract

A bacterial strain, labeled UR11T, was isolated from green alga Ulva pertusa collected from Jeju Island, Korea. UR11T was identified as a gram-negative, rod-shaped, motile by gliding and aerobic bacterial strain with yellow colonies on R2A plates. The strain UR11T grew over at a temperature range of 10°C to 30°C (optimally at 25°C), a pH range of 6.0-11 (optimally at pH 7.0) and a Nacl range of 0.5-5% Nacl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain UR11T was a member of the genus Flavobacterium. Strain UR11T shared close similarity with F. jejuensis EC11T (98.0%) F. jumunjinense HME7102T (96.1%), F. haoranii LQY-7T (95.3%), F. dongtanense LW30T (95.1%), and F. ahnfeltiae 10Alg 130T (94.9%). The major fatty acids (>5%) were iso-C15:0 (33.9%), iso-C15:1 G (12.4%), iso-C17:0 3-OH (9.0%), iso- C16:0 (7.0%) and iso-C15:0 3-OH (6.3%). The major polar lipids were phosphatidylethanolamine, seven unknown aminolipids, two unknown aminopolarlipids and two unknown lipids. DNADNA hybridization value was 58% at strain UR11T w ith F. jejuensis EC11T . Based on phenotypic, chemotaxonomic and phylogenetic evidence, strain UR11T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium jocheonensis sp. nov. is proposed. The type strain is Flavobacterium jocheonensis is UR11T (=KCTC 52377 T =JCM 31512 T).

Keywords: Green alga, marine bacteria, Ulva pertusa, Flavobacterium, 16S rRNA gene

Introduction

Marine algae provide surfaces for consistent colonization by microbial communities and microorganisms that inhabit these surfaces and have a positive effect by interacting with the algae. [1-3] For example, the microbes that inhabit algae help the host grow normally while also helping to release, settle and grow the spores of algae [4, 5].

The genus Flavobacterium, type genus of the family Flavobacteriaceae and member of the phylum Bacteroidetes, was first proposed by Bergey et al. (1923) with emended description by Bernardet et al. (1996) Kang et al., and Dong et al. (2013). At the time of writing, this genus consists of 211 species with validly published names (http://www.bacterio.net/flavobacterium.html). The members of Flavobacterium have been isolated from a wide range of habitats including sea water [10, 11], freshwater [12, 13], and soil [14-17] as well as marine algae [18, 19]. They are gram-negative, aerobic, with gliding motility, yellow-pigmented, rod-shaped and contain menaquinone 6 (MK-6) as the major respiratory quinone. [20-22]. Members of Flavobacterium show a genomic DNA G+C content in the range of 30-52 mol%.

In this paper, we report the isolation and characterization of the genus Flavobacterium associated with marine green alga Ulva pertusa for which the name Flavobacterium jocheonensis sp. nov. is proposed.

Materials and Methods

Bacterial strains

Strain UR11T was isolated from the green alga Ulva pertusa collected on Jeju Island (Korea), by a standard dilution plating method. The sample of alga was serially diluted (10-fold dilutions) using sterile 0.85% (w/v) NaCl solution and 0.1ml homogenates of each dilution were spread onto R2A agar plates (Difco, USA) and incubated for 7 days at 25°C. The novel isolate UR11T was routinely cultivated on R2A agar at 25°C and preserved as R2A broth (Difco) supplemented with glycerol suspension (20%, v/v, glycerol in water) at -80°C. Flavobacterium jejuensis KCTC 42149T, Flavobacterium jumunjinense KCTC 23618T, Flavobacterium haoranii KCTC 23008T, Flavobacterium dongtanense KACC 15621T and Flavobacterium ahnfeltiae KCTC 32467T were obtained from Korean Collection for Type Cultures (KCTC) and Korean Agricultural Culture Collection (KACC) and used as reference strains.

Morphology and Physiological Characteristics

Cell morphology of strain UR11T was examined by light microscopy (Nikon, Japan) and scanning electron microscopy (SUPRA 55VP, ZEISS) with cells grown aerobically for 3 days at 25°C on R2A agar. Gram staining was performed using the Gram Stain Kit (BBL, Difco, USA), according to the manufacturer’s instructions. Gliding motility was investigated on R2A broth with 0.5% agar, by the method of Bowman (2000). Anaerobic growth was determined in an anaerobic jar with the AnaeroPack (Oxoid, UK) on R2A agar at 25°C for 4 weeks. Growth was conducted at varied temperatures (5°C, 10°C 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, and 45°C) and pH 5.0-11.0 (intervals of 1.0 pH unit) in R2A broth at pH 5.0-11.0 (intervals of 1.0 pH unit) for 2 weeks at 25°C. Salt tolerance was tested on R2A agar containing various concentrations of NaCl (0-7%, w/v) at 1% intervals. Catalase activity was tested by observing bubble production using a catalase reagent (BioMérieux, UK). Oxidase activity was determined by oxidase reagent (BioMérieux). The hydrolysis of DNA (1%), starch (3%), cellulose (1%), Tween 20, 40, 60, and 80 (1%) and casein (1%) were tested after containing on R2A agar. Furthermore, these biochemical tests were determined using the API 20NE and API ZYM tests (BioMérieux), according to the manufacturer’s instructions.

Phylogenetic Analysis

Genomic DNA of strain UR11T was extracted and purified as described by Wilson (1987). Amplification of the 16S rRNA was carried out by PCR using universal 27F and 1522R primers [25]. The PCR product was then cloned using the TOPO Cloning Kit (Invitrogen, USA) and sequenced by Genotech (Korea). The complete 16S rRNA gene sequence (1,510 bp) was assembled with SeqMan software (DNASTAR). Similarity searches were achieved using the BLAST program (http://blast.ncbi.nlm,nih.gov/Blast.cgi) and EzTaxon-e server [http://eztaxon-e.ezbiocloud.net/; 26, 27]. Multiple sequence alignments were performed using the CLUSTAL X program, version 1.83 [28] and gaps were edited by the BioEdit program [29]. Phylogenetic analysis was performed using the software package MEGA version 6.0 [30]. Phylogenetic trees were constructed using the neighbor-joining [31], maximum-parsimony [32], and maximum-likelihood [33] methods. Bootstrap values were estimated based on 1000 replications [34]. DNA-DNA hybridization (DDH) was performed between UR11T and Flavobacterium jejuensis EC11 using the microplate method [35].

Chemotaxonomy

For analysis of cellular fatty acids, cells of strain UR11T and four reference strains were grown at 25°C on R2A agar for 3 days. Fatty acids were extracted, methylated as described by the standard protocol of the Sherlock Microbial Identification System (MIDI; version 4.5), analyzed by gas chromatography (GC 7890A; Agilent, USA), and then identified using the TSBA 5.0 library [36]. Polar lipids from strain UR11T were extracted as described by Minnikin et al. (1984) and analyzed by 2-dimensional thin-layer chromatography (TLC) [37]. The polar lipid pattern was identified by comparing results following staining with molybdophos-phoric acid, ninhydrin, Zinzadze and α-naphthol. For determining the G+C DNA content, genomic DNA was extracted according to standard procedures described by Wilson (1987) from cells that had been cultured on R2A agar for 3 days at 25°C and analyzed by the Korean Culture Center of Microorganisms (KCCM, Korea).

Results

Morphology and physiological characteristics

Strain UR11T cells were aerobic, gram-negative rods (0.16-0.23 μm × 1.43-1.62 μm), and gliding motility was observed. The colonies were yellow, circular, smooth and 1-2 mm in diameter after growth for 3 days at 25°C on R2A agar. Growth occurred at 5-35°C (optimum 25°C) at pH 6.0-10.0 (optimum pH, 7.0) but not at pH 5.0 and 11.0. and in the range of 0-5% NaCl (w/v), with optimal growth occurring in the absence of NaCl. The morphological, physiological and biochemical characteristics are explained below in the species description, and comparison with reference strains is presented in Table 1.

Table 1 . Biochemical characteristicd of strain UR11T and related type strains..

Characteristic123456
Temp. range for growth (°C)5-355-305-3015-355-355-30
Nitrate reduction-+---+
Catalase+++-++
Oxidase+++---
Hydrolysis of :
DNA++----
CAS (casein)++++-+
Tween 80--++++
Assimilation of:
Esculin degradation++++-+
D-Glucose++++--
D-Arabinose----+-
D-mannose++++-+
D-Mannitol-----+
N-acetyl-D-glucosamine+----+
D-maltose++++-+
Gluconate-----+
Adipate+-++-+
Malate+-----
Citrate+--+--
Enzyme activities:
α-chymotrypsin++-++-
β-glucuronidase+--+--
α-glucosidase++-+--
β-glucosidase++-+--
N-acetyl-β-glucosamidase+-----
G+C mol%32.628.1a36.5b34c30d34.3e

Strain: 1, UR11T 2, F. Jejuensis KCTC42149T(this study) 3, F. jumunjinense KCTC23618T(this study)4, F. haoranii KCTC23008T(this study) 5, F. dongtanense KACC15621T(this study)6, F. ahnfeltiae KCTC32467T(this study). All data were obtained in this study as +, Positive; -, negative. All strains are gram-negative, motile by gliding. All strains were positive for Gelatin, Alkaline phosphatase, Esterase (C4), Esterase Lipase (C8), Leucinearylamidase, Valinearylamidase, Crystinearylamidase, Trypsin, Acidphospatase and Naphtol-AS-BI-phosphohydrolase. All strains were negative for Indole production, Glucose fermentation, Arginine dihydrolase, Urease, Caprate, Phenylacetate, Lipase (C14), α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase..

Data from aPark et al., (2015); bJoung et al., (2013); cZhang et al.,(2010); dXiao et al., (2011);eNedashkovskaya et al., (2014)..



Phylogenetic Analysis

The 16S rRNA gene sequence of strain UR11T showed the highest similarity to F. jejuensis EC11T (98.0%), F. jumunjinense HME7102T (96.1%), F. haoranii LQY-7T (95.3%), F. dongtanense LW30T (95.1%), and F. ahnfeltiae 10Alg 130T (94.9%). Phylogenetic analysis based on 16s rRNA gene sequences using the neighbor-joining algorithm showed that strain UR11T formed a phyletic lineage distinct from other members of the genus Flavobacterium (Fig. 1). Strain UR11T of DNA-DNA hybridization (DDH) value was 58% that showed less than 70% DDH comparing Flavobacterium jejuensis EC11T.

Figure 1. Phylogenetic tree of the 16S rRNA gene sequences of strain UR11T and other related taxa. GenBank accession numbers are placed in parentheses. Boostrap values (>50%) are based on 1,000 replications. Bar 0.02 nucleotide substitutions per nucleotide position.

Chemotaxonomy

The major fatty acids (>5% of the total fatty acids) of strain UR11T were iso-C15:0 (33.9%), iso-C15:1 G (12.4%), iso-C17:0 3-OH (9.0%), iso-C16:0 (7.0%) and iso-C15:0 3-OH (6.3%) (Table 2). The polar lipid profile consisted of phosphatidyl-ethanolamine (PE), seven unknown aminolipids (AL1-7), two unknown aminopolarlipids (APL1 and APL2) and two unknown lipids (L1 and L2; Fig. 2). The polar lipid profile of strain UR11T was very similar to those of the reference strains F. jejuensis EC11T, F. jumunjinense HME7102T, F. haoranii LQY-7T, F. dongtanense LW30T and F. ahnfeltiae 10Alg 130T but could be differentiated by the presence or absence of several other polar lipids. The major respiratory quinone of the UR11T strain was menaquinone-6 (MK6). The G+C content of the genomic DNA was found to be 32.6 mol, as shown in Table 1.

Table 2 . Cellular fatty acid composition of strain UR11T and type strain..

Fatty acid123456
Saturated   
C14:00.6TR0.6TR1.20.6
C16:00.6TR0.81.03.71.2
Hydroxylated
C15:0 3-OH1.1--1.00.80.8
C16:0 3-OH0.9TR0.60.81.11.2
C17:0 3-OH0.6----TR
iso-C14:0 3-OH0.6TRTR0.6-TR
iso-C15:0 3-OH6.37.68.84.515.311.6
iso-C16:0 3-OH3.92.61.04.0TR1.2
iso-C17:0 3-OH9.011.210.88.6TR10.9
Branched
iso-C13:01.00.91.00.9TR1.5
iso-C14:03.2----0.8
iso-C15:033.941.439.932.2TR44.9
iso-C15:1 G12.49.813.920.38.910.0
iso-C16:07.03.82.25.31.41.3
iso-C16:1 H2.42.41.3---
iso-C17:1ω9c1.22.44.41.33.03.7
anteiso-C15:01.51.21.88.01.63.3
Unsaturated
C15:1ω6c4.13.33.31.0TR0.9
C17:1ω6c1.10.90.80.2--
Summed features
3TRTR2.91.0TR1.1

Strain: 1, UR11T 2, F. Jejuensis KCTC42149T 3, F. jumunjinense KCTC23618T 4, F. haoranii KCTC23008T 5, F. dongtanense KACC15621T 6, F. ahnfeltiae KCTC32467T. All data incurred in this study are recorded as -, not detected; TR, trace amount (<0.5%). All strains were incubated in R2A agar plate at 25°C for 3days. Fatty acids that account <0.5% of the total fatty acids in all strains were deleted..

*Summed features were represent when two fatty acids that cannot be separated by MIDI system. Summed features 3,iso-C15:0 2-OH/C16:1ω7c..


Figure 2. Two dimensional thin-layer chromatogram of total polar lipids of strain UR11T. Total polar lipids were identified by spraying with molybdophosphoric acid reagent. PE, phosphatidylethanolamine; AL1-7, unknown aminolipids; APL1-2, unkown aminopolarlipids; L 1–2, unknown lipid.

Discussion

Flavobacterium jocheonensis (jo.cheon.en’sis.N.L. fem. adj. jocheonensis refers to Jocheon in Jeju Island, where the type strain was isolated).

Strain UR11T are gram-negative, motile by gliding, aerobic and rod-shaped, approximately 0.16-0.23 μm wide and 1.43-1.62 μm long. Colonies were yellow, circular and smooth after 3 days of incubation at 25°C on R2A agar. Growth occurs at 5-35°C, but not at 37°C, 40°C, and 45°C (optimum temperature, 25°C) and pH 6.0-10.0 (optimum pH, 7.0), but not at pH 5.0 and 11.0. Growth was observed at 0-5% NaCl (w/v). Oxidase and catalase activities are positive. Strain UR11T hydrolyzes aesculin, gelatin, casein, DNA, Tween 40 and 60, but does not hydrolyze starch, cellulose, Tween 20 and 80. The strain tested negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, β-galactosidase, L-arabinose, D-mannitol, gluconate, caprate and phenylacetate but positive for D-glucose, D-mannose, N-acetyl-D-glucosamine, D-maltose, adipate, malate and citrate. In ZYM testing, alkaline phosphatase, esterase(C4), esterase lipase(C8), leucine arylamidase, valine arylamidase, crystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, α-glucosidase, β-glusidase and N-acetyl-β-glucosamidase are present, but lipase(C14), α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase are not present. The main fatty acids (>5%) of strain UR11T were iso-C15:0 (33.9%), iso-C15:1 G (12.4%), iso-C17:0 3-OH (9.0%), iso-C16:0 (7.0%) and iso-C15:0 3-OH (6.3%). The major fatty acid composition of strain UR11T was similar to that of F. jejuensis EC11T, with minor differences in their respective proportions. The polar lipids profile consisted of phosphatidylethanolamine, seven unknown aminolipids, two unknown aminopolarlipids and two unknown lipids. They all contained phosphatidyl-ethanolamine (PE). PE is present in many species of the genus Flavobacterium [38]. Menaquinone-6 is the predominant quinone. The major respiratory quinone of the UR11T strain was menaquinone-6 (MK6), which is also characteristic for members of the genus Flavobacterium. The G+C content is 32.6 mol. A DDH experiment was performed with F. jejuensis EC11T as closest phylogenetic neighbor. DNA-DNA hybridization (DDH) between the strain UR11T and Flavobacterium jejuensis EC11T showed 58% relatedness. The DNA-DNA hybridization value of less than 70% shows that the new isolates belong to a novel species. The results demonstrate that strain UR11T is a novel species of the genus Flavobacterium [39, 40].

The type strain, UR11T (=KCTC 52377T = JCM 31512T), was isolated from green alga Ulva pertusa in Jeju Island, Republic of Korea. The NCBI GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain UR11T is KX24431.

Supplemental Materials

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2017R1A2B4005688). 2019 scientific promotion program funded by Jeju National University.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Phylogenetic tree of the 16S rRNA gene sequences of strain UR11T and other related taxa. GenBank accession numbers are placed in parentheses. Boostrap values (>50%) are based on 1,000 replications. Bar 0.02 nucleotide substitutions per nucleotide position.
Journal of Microbiology and Biotechnology 2019; 29: 1266-1272https://doi.org/10.4014/jmb.1812.12011

Fig 2.

Figure 2.Two dimensional thin-layer chromatogram of total polar lipids of strain UR11T. Total polar lipids were identified by spraying with molybdophosphoric acid reagent. PE, phosphatidylethanolamine; AL1-7, unknown aminolipids; APL1-2, unkown aminopolarlipids; L 1–2, unknown lipid.
Journal of Microbiology and Biotechnology 2019; 29: 1266-1272https://doi.org/10.4014/jmb.1812.12011

Table 1 . Biochemical characteristicd of strain UR11T and related type strains..

Characteristic123456
Temp. range for growth (°C)5-355-305-3015-355-355-30
Nitrate reduction-+---+
Catalase+++-++
Oxidase+++---
Hydrolysis of :
DNA++----
CAS (casein)++++-+
Tween 80--++++
Assimilation of:
Esculin degradation++++-+
D-Glucose++++--
D-Arabinose----+-
D-mannose++++-+
D-Mannitol-----+
N-acetyl-D-glucosamine+----+
D-maltose++++-+
Gluconate-----+
Adipate+-++-+
Malate+-----
Citrate+--+--
Enzyme activities:
α-chymotrypsin++-++-
β-glucuronidase+--+--
α-glucosidase++-+--
β-glucosidase++-+--
N-acetyl-β-glucosamidase+-----
G+C mol%32.628.1a36.5b34c30d34.3e

Strain: 1, UR11T 2, F. Jejuensis KCTC42149T(this study) 3, F. jumunjinense KCTC23618T(this study)4, F. haoranii KCTC23008T(this study) 5, F. dongtanense KACC15621T(this study)6, F. ahnfeltiae KCTC32467T(this study). All data were obtained in this study as +, Positive; -, negative. All strains are gram-negative, motile by gliding. All strains were positive for Gelatin, Alkaline phosphatase, Esterase (C4), Esterase Lipase (C8), Leucinearylamidase, Valinearylamidase, Crystinearylamidase, Trypsin, Acidphospatase and Naphtol-AS-BI-phosphohydrolase. All strains were negative for Indole production, Glucose fermentation, Arginine dihydrolase, Urease, Caprate, Phenylacetate, Lipase (C14), α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase..

Data from aPark et al., (2015); bJoung et al., (2013); cZhang et al.,(2010); dXiao et al., (2011);eNedashkovskaya et al., (2014)..


Table 2 . Cellular fatty acid composition of strain UR11T and type strain..

Fatty acid123456
Saturated   
C14:00.6TR0.6TR1.20.6
C16:00.6TR0.81.03.71.2
Hydroxylated
C15:0 3-OH1.1--1.00.80.8
C16:0 3-OH0.9TR0.60.81.11.2
C17:0 3-OH0.6----TR
iso-C14:0 3-OH0.6TRTR0.6-TR
iso-C15:0 3-OH6.37.68.84.515.311.6
iso-C16:0 3-OH3.92.61.04.0TR1.2
iso-C17:0 3-OH9.011.210.88.6TR10.9
Branched
iso-C13:01.00.91.00.9TR1.5
iso-C14:03.2----0.8
iso-C15:033.941.439.932.2TR44.9
iso-C15:1 G12.49.813.920.38.910.0
iso-C16:07.03.82.25.31.41.3
iso-C16:1 H2.42.41.3---
iso-C17:1ω9c1.22.44.41.33.03.7
anteiso-C15:01.51.21.88.01.63.3
Unsaturated
C15:1ω6c4.13.33.31.0TR0.9
C17:1ω6c1.10.90.80.2--
Summed features
3TRTR2.91.0TR1.1

Strain: 1, UR11T 2, F. Jejuensis KCTC42149T 3, F. jumunjinense KCTC23618T 4, F. haoranii KCTC23008T 5, F. dongtanense KACC15621T 6, F. ahnfeltiae KCTC32467T. All data incurred in this study are recorded as -, not detected; TR, trace amount (<0.5%). All strains were incubated in R2A agar plate at 25°C for 3days. Fatty acids that account <0.5% of the total fatty acids in all strains were deleted..

*Summed features were represent when two fatty acids that cannot be separated by MIDI system. Summed features 3,iso-C15:0 2-OH/C16:1ω7c..


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