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Research article

J. Microbiol. Biotechnol. 2019; 29(12): 1969-1974

Published online December 28, 2019 https://doi.org/10.4014/jmb.1812.12004

Copyright © The Korean Society for Microbiology and Biotechnology.

Labrenzia callyspongiae sp. nov., Isolated from Marine Sponge Callyspongia elegans in Jeju Island

So Hyun Park 1, Ji Young Kim 2 and Moon Soo Heo 1*

1Department of Aquatic Life Medicine, Jeju National University, Jeju 63243, Republic of Korea, 2Research institute for Basic Science, Jeju National University, Jeju 63243, Republic of Korea

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

Received: December 5, 2018; Accepted: October 27, 2019

Abstract

A Gram-staining-negative, aerobic, light brown pigment bacterium, designated strain CE80T was isolated from marine sponge Callyspongia elegans in Jeju Island, Republic of Korea. Strain CE80T grew optimally at 25°C, in the range of pH 5.0-11.0 (optimum 7.0-8.0), and with 1.0-5.0% NaCl (optimum 1-3% (w/v)). Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain CE80T belonged to the genus Labrenzia and was closely related to L. suaedae YC6927T (98.3%), L. alexandrii DFL-11T (96.6%), L. aggregata IAM 12614T (96.6%) L. marina mano18T (96.5%) and L. alba CECT 5094T (96.2%). The major fatty acids of strain CE80T were C18:1 ω7c, and summed feature. The polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamin, one unidentified aminolipid, one phospholipid and four unidentified lipids. The DNA G+C content of strain CE80T was 55.9 mol%. The major respiratory quinone was Q-10. DNA-DNA relatedness between strain CE80T and L. suaedae YC6927T was 56.1±2.8%. On the basis of physiological and biochemical characterization and phylogenetic and chemotaxonomic analysis, strain CE80T represents a novel species of the Labrenzia, for which the name Labrenzia callyspongiae sp. nov., is proposed. The type strain is CE80T (=KCTC 42849T =JCM 31309T).

Keywords: 16S rRNA, Labrenzia, marine sponge, novel species

Introduction

The genus Labrenzia, established by Biebl et al. [1], belongs to the class Alphaproteobacteria and the family Rhodobacteraceae. At the time of writing, six species of the genus Labrenzia including the recently described species L. suaedae [2] and L. salina [3] have been identified, which were isolated from marine habitats and tidal flats, as dinoflagellates and halophytes, respectively. The members of genus Labrenzia are gram-negative, aerobic, and most species show the presence of glycolipid sulphoquinovosuldiacylglyceride (SQDG). Some species also synthesize bacteriochlorophyll a (BChl a). Previously, the genus Agrobacterium was reported by Stapp & Knosel [4] and Ruger & Hofle [5]; Agrobacterium aggregatum was described by Ahrens [6], and later the taxonomic position of the marine subdivision Agrobacterium was reassessed resulting in two species of the genus Agrobacterium getting transferred to the new genus Stappia as Stappia aggregata and Stappia stellulata [7] and finally reclassified as Labrenzia aggregata by Biebl et al. [1]. Stappia alba described by Pujalte et al. [8] and Stappia marina described by Kim et al. [9] were reclassified as Labrenzia alba and Labrenzia marina, respectively [1]. In the present study we determined the exact taxonomic position of strain ce80T by using a polyphasic characterization that included the determination of phenotypic, physiological and 16S rRNA gene sequence analysis, chemotaxonomic properties, and DNA-DNA hybridization.

Materials and Methods

Isolation

Strain CE80T was isolated from marine sponge Callyspongia elegans collected at Jeju Island. To isolate marine bacteria, the sponge sample of homogenized core tissue was 10-fold diluted with sterilized 0.85% (w/v) NaCl solution. Each dilution was inoculated onto marine agar 2216 (MA; Difco, USA) and incubated at 25°C for 7 days under aerobic conditions. The isolate was purified by repeated subculture and stored at -80°C as a suspension marine broth (MB; Difco, USA) containing 20% (v/v) glycerol. The type strains of the most closely related species, L. suaedae KACC 13772 and L. aggregate KACC 15203T were obtained from the Korean Agricultural Culture Collection (KACC); L. marina KCTC 12288T was obtained from the Korean Collection for Type Cultures (KCTC); L. alba DSM 18320T and L. alexandrii DSM 17067T were obtained from the Deutsche Sammlung von Mikroorganismenund Zellkulturen (DSM).

16S rRNA Gene Sequencing and Phylogenetic Analyses

For 16S rRNA gene [10] sequencing, genomic DNA [11] was extracted from strain CE80T and amplified by PCR with two universal primers, 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1522R (5’-AAGGAGGTGATCCAGCCGCA-3’). The amplified PCR product was cloned using the TOPO cloning kit (Invitrogen) and sequence analysis was requested from Genotech (Korea). Full sequence of the 16S rRNA gene was assembled using SeqMan software (DNASTAR) and we obtained the sequences for related taxa from GenBank and the EzTaxon-e server (www.ezbiocloud.net)[12]. The 16S rRNA gene sequences of strain CE80T and related type strains were aligned by using CLUSTAL_X [13], while the BioEdit program [14] was used to edit gaps. Phylogenetic analysis was performed using the MEGA 6.0 software package [15] and phylogenetic trees were reconstructed based on the maximum-likelihood [16], maximum-parsimony [17] and neighbor-joining [18] methods through bootstrap analysis based on 1000 replicates [19]. Evolutionary distances were calculated using Jukes-Cantor model. The whole genome sequence of CE80T was performed by Macrogen (Republic of Korea) sequencing service. The sequencing library was prepared using TruSeq DNA PCR Fee kit according to the manufacturer’s instructions. The genome sequence data were produced using the Illumina Hiseq 4000 platform and assembled de novo with SPAdes version 3.13.0 [20]. Also, the average nucleotide identity (ANI) was calculated using an ANI calculator (www.Ezbiocloud.net/tool/ani) [21].

Morphology and Physiological, and Biochemical Characterization

The cell morphology of cell was observed by light microscopy (Nikon, Japan), scanning electron microscopy and transmission electron microscopy (SUPRA66VP, ZEISS). Gliding motility was determined according to Schaal [22]. Gram staining was conducted using the Gram Stain Kit (BD Science, USA) according to the manufacturer’s instructions. Growth of strain CE80T was evaluated on MA at different temperatures (5, 10, 15, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C) for 5 days. The pH range for growth was determined in marine broth adjusted to pH 4.0-11.0 (0.5 pH unit intervals) using citric acid/sodium citrate buffer, KH2PO4/K2HPO4 and NaHCO2/Na2CO2 as buffering systems. Tolerance to NaCl was tested in synthetic marine ZoBell medium (5 g Bacto peptone, 1 g yeast extract, 0.1 g ferric citrate, 15 g agar in 1L distilled water)(ZoBell, 1941) supplemented with various NaCl concentrations (0-10%, w/v, intervals 1%). Anaerobic growth was determined on MA at 25°C for 2 weeks under anaerobic condition (in an anaerobic jar with the Anaerobic Pack (Oxid)). Catalase and oxidase activity were tested by bubble formation in 3% hydrogen peroxide (H2O2) solution and using 1% (w/v) tetramethyl-P-phenylenediamine. Hydrolysis tests were performed using MA containing starch, casein, Tween 20, 40, 60, 80, cellulose and DNase, respectively [23]. The enzyme activities and biochemical characteristics were tested by using API ZYM and API 20 NE kits (bioMerieux) according to the manufacturer’s instructions. These tests were performed at 25°C for 5 days.

Chemotaxonomy

For cellular fatty acid analysis, strain CE80T and one reference strain were harvested from MA after cultivation at 25°C for 5 days. Saponification, methylation and extraction of cellular fatty acids were performed by gas chromatography according to the protocol for the Sherlock Microbial Identification System (MIDI; version 6.1) and the TSBA6 database [24].

For analysis of the polar lipids, cell mass was harvested after incubation at 25°C for 5 days in MA and freeze-dried. Polar lipid analysis of strain CE80T was carried out using two-dimensional TLC as described by Minnikin et al. [25]. The polar lipids were extracted and separated using chloroform/methanol/water (65:25:4, by volume) for the first dimension and chloroform/methanol/acetic acid/water (80:12:15:4, by volume) for the second dimension [25], while molybdophosphoric acid (for total lipids), ninhydrin (for lipids containing free amino groups), molybdenum blue (for phosphorus-containing lipids) and α-naphthol (for glycolipids) were used for the detection reagents [26].

Analysis of the respiratory quinones was performed by reversed-phase high-performance liquid chromatography [25].

Genomic Analyses

DNA G+C content of strain CE80T was detected by using HPLC analysis [27] at the Korean Culture Center of Microorganisms (KCCM). DNA–DNA hybridization of strain CE80T and type strain L. suaedae was performed with photobiotin-labelled DNA probes and microplate hybridization method by Ezaki et al. [28]. The experiment was with done five replications and the mean values were calculated for DNA–DNA relatedness.

To detect the presence of a gene related to production of bacteriochlorophyll a (BChl a), the pufLM gene was amplified using specific primers as described by Kim et al. [9].

Results and Discussion

Cells of strain CE80T were gram-negative, aerobic, bright brown rods (0.56-0.74 μm × 1.5-2.7 μm) and exhibited gliding motility. The growth occurred at 10-37°C (optimum 25°C) and pH 5.0–11.0 (optimum 7.5). The range of NaCl for growth was 1-5% (w/v) (optimum 1-3%). The detailed results of the morphological, physiological and biochemical characteristics are described and compared with type strains Labrenzia suaedae as listed in Table 1.

Table 1 . Comparisons of phenotype characteristics of strain CE80T and closely related type strains of the genus Labrenzia..

Characteristic123456
Growth at/with
0% NaCl-+--+-
10% NaCl----+-
Hydrolysis of :
DNA+-++--
Aesculin-+++++
API ZYM
Cystine arylamidase+++--+
α-chymotrypsin-+---+
β-glucuronidase-+-+--
α-glucosidase-+++-+
β-glucosidase-+----
N-acetyl-b-glucosamidase-+++--
α-mannosidase-+-+--
α-fucosidase-+----
API 20NE
D-Glucose-+---+
D-Arabinose-+---+
D-mannose-+---+
D-Mannitol-+---+
N-acetyl-D-glucosamine-+---+
Gluconate-+---+
malate-+---+
Citrate-+---+
Presence of pufLM genes-++ND-+
presence of SQDG--++*+*+*
DNA G+C content55.958.5*60*ND59*56*

Strains: 1, Strain CE80T (this study); 2, L. suaedae KACC 13772T; 3, L. marina KCTC12288T; 4, L. alba DSM 18320T; 5, L. aggregata KACC 15203T; 6, L. alexandrii DSM 17067T. All strains are positive for motility, oxidase, catalase, reduction of nitrates to nitrites, hydrolysis of gelatin, assimilation of β-galactosidase, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase and α-galactosidase. All strains are negative for: hydrolysis of starch, casein, Tween 20, 60, cellulose, indole production, glucose fermentation; arginine dihydrolase, assimilation of D-maltose, caprate, adipate, phenylacetate; lipase (C14) and Naphthol-AS-BI-β D-glucuronide activities..

*Data taken from Bibi et al. (2014)..



The almost full-length 16S rRNA gene sequence of strain CE80T (1,384 bp) was obtained. Based on phylogenetic analysis, strain CE80T was shown to be most closely related to members of genus Labrenzia exhibiting highest 16S rRNA gene sequence similarity to L. suaedae YC6927T (98.3%), L. alexandrii DFL-11T (96.6%), L. aggregata IAM 12614T (96.6%) L. marina mano18T (96.5%) and L. alba CECT 5094T (96.2%). A phylogenetic tree based on the neighbor-joining algorithm showed that strain CE80T formed a phylogenetic lineage distinct from but related closely to type strain Labrenzia suaedae YC6927T with 81% bootstrap support (Fig. 1).

Figure 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, for strain CE80T and genus Labrenzia. Agrobacterium agile IAM 12615T (D88521) was used as an outgroup. Numbers at branch nodes are bootstrap percentages based on 1000 resamplings; only values greater than 50% are shown. Filled circles indicate branches found in phylogenetic consensus trees generated with the Maximum-Likelihood and Maximum-Parsimony method. Bar, 0.02 substitutions per nucleotide position.

The draft assembled genome size of strain CE80T was 4,737,804 bp, contained seven contigs and an N50 length of 2,393,935 bp. The ANI value between strain CE80T and type strain L. suaedae YC6927T was 83.7%, which was lower than the standard cut-off of 95–96% [29] for species identity.

The presence of the pufLM genes can show ability to synthesize the photosynthetic reaction center. To confirm the presence pufLM genes of strain CE80T, we used a specific primer and the gene was not found. This result shows that the photosynthetic reaction center may not be functional in this strain, which is found in other reported members such as L. alexandrii, L. marina, L. suaedae, and L. salina [1, 2, 3, 9].

The cellular fatty acids (>2 % of the total fatty acids) of strain CE80T were C18:1 ω7c (66.7%), summed feature 2 (C14:03-OH/ iso- C16:1 I) (10.2%), C18:0 (4.7%) 11-methyl C18:1 ω7c (4.4%), C20:1 ω7c (2.6%), and C18:0 3-OH (2.2%) (Table 2). The predominant fatty acids of strain CE80T and L. suaedae were C18:1 ω7c and summed features 2. The fatty acids C18:1 ω9c and C20:1 ω7c were present in strain CE80T, but absent in L. suaedae. The predominant respiratory quinone of strain CE80T was ubiquinone 10 (Q-10), which was in line with all members of the genus Labrenzia.

Table 2 . Cellular fatty acid compositions (%) of strain CE80T and closely related type strains of the genus Labrenzia..

Fatty acid123456
Saturated
C10:0tr2.1trtrtrtr
C16:0tr2.1trtrtrtr
C18:04.7tr6.26.43.53.2
Unsaturated
C18:1 ω7c66.866.455.970.569.967.7
C20:1 ω7c2.6-4.77.57.414.3
11-methyl C18:1 ω7c4.53.29.96.88.05.7
Cyclo C19:0 ω8c--6.2---
Hydroxy
C18:0 3-OH2.36.72.0trtrtr
Summed features
210.38.83.13.74.13.7
3trtr8.5-trtr

Strains: 1, strain CE80T (this study); 2, L.suaedae KACC 13772T; 3, L. marina KCTC12288T; 4, L. alba DSM 18320T; 5, L. aggregata KACC 15203T; 6, L. alexandrii DSM 17067T. All data were obtained from this study. Values are percentages of total fatty acids (less than 2% are not show); tr, Traces (<2.0%); -, not detected. *Summed features represent groups of two or three fatty acids that cannot be separated by gas chromatography with the MIDI system. Summed features 2 comprised C14:0 3-OH and/or iso- C16:1 I; 3, C16:1 ω7c and/or iso- C15:0 2-OH..



The predominant polar lipids of the strain CE80T were diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylmonomethylethanolamin (PMME), one unknown aminolipid, one unknown phospholipid and four unknown lipids. These polar lipids profiles were the most similar pattern observed in all members of the genus Labrenzia. The presence of sulphoquinovosyldiacylglyceride (SQDG) was known to be characteristic in species of Labrenzia, but L. suaedae lacked SQDG. Similarly, SQDG was detected in strain CE80T in this study.

The DNA G+C content of strain CE80T was calculated as 55.9 mol%, which is lower than the defined range values (58-63 mol%) reported for members of the genus Labrenzia [1, 2, 3, 8, 9]. The DNA-DNA hybridization value between strain CE80T and type strain L. suaedae was 56.1 ± 2.8. This value was less than the 70% threshold value recommended for the delineation genomic species of novel strains by Wayne et al. [30]. Based on the results of phylogenetic, phenotypic, biochemical and chemotaxonomic analyses, strain CE80T represents a novel species of the genus Labrenzia, for which the name Labrenzia callyspongiae sp. nov., is proposed.

Description of Labrenzia callysponigae sp. nov.

Labrenzia callyspongiae (cal.ly.spon'gi.ae. N.L. gen. n. callyspongiae of the sponge Callyspongia).

Strain CE80T is gram-negative, aerobic, and exhibited gliding motility. The organisms were light-brown, rod-shaped, approximately 0.56-0.74 μm wide and 1.51-2.69 μm long and translucent after 3 days of incubation at 25°C on marine agar. Growth occurs at 10-37°C (the optimum temperature is 25°C), pH 5.0-11.0 (the optimum pH is 7.5) and in the 1-5% of NaCl (w/v) (optimum at 1-3%, w/v), respectively. The catalase and oxidase tests were positive. Nitrate reduced to nitrite. The strains also tested positive for hydrolysis of gelatin, Tween 80, urea and DNA but starch, casein, cellulose, aesculin, Tween 20, 40 and 60 tested negative. In the 20 NE tests, a positive result showed for assimilation of β-galactosidase, but indole production, glucose fermentation, arginine dihydrolase, assimilated of D-glucose, D-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, D-maltose, gluconate, caprate, adipate malate, citrate and phenylacetate tested negative. In the ZYM tests, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucinearylamidase, valinearylamidse, cysteine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-galactosidase were present, but lipase (C14), α-chymotrypsin, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosamidase, α-mannosidase and α-fucosidase were absent. The main fatty acids of strain CE80T are C18:1 ω7c, and summed feature 2. The polar lipids profile showed presence of diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phos-phatidylglycerol, phosphatidylmono-methylethanolamin, one unidentified aminolipid, one phospholipid and four unidentified lipids. The major respiratory lipoquinone was ubiquinone 10 (Q-10) and the DNA G+C content was 55.9 mol %.

The type strain CE80T (=KCTC 42849T =JCM 31309T) was isolated from marine sponge Callyspongia elegans in Jeju Island, Republic of Korea.

Supplemental Materials

Acknowledgements

This research was supported by the Leading Human Resource Training Program of Regional Neo Industry through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016H1D5A1911152 & 2017R1A2B4005688).

GenBank Accession Number

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and whole genome sequence of Labrenzia callyspongiae CE80T are KT596062 and WAJT00000000.

Conflicts of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, for strain CE80T and genus Labrenzia. Agrobacterium agile IAM 12615T (D88521) was used as an outgroup. Numbers at branch nodes are bootstrap percentages based on 1000 resamplings; only values greater than 50% are shown. Filled circles indicate branches found in phylogenetic consensus trees generated with the Maximum-Likelihood and Maximum-Parsimony method. Bar, 0.02 substitutions per nucleotide position.
Journal of Microbiology and Biotechnology 2019; 29: 1969-1974https://doi.org/10.4014/jmb.1812.12004

Table 1 . Comparisons of phenotype characteristics of strain CE80T and closely related type strains of the genus Labrenzia..

Characteristic123456
Growth at/with
0% NaCl-+--+-
10% NaCl----+-
Hydrolysis of :
DNA+-++--
Aesculin-+++++
API ZYM
Cystine arylamidase+++--+
α-chymotrypsin-+---+
β-glucuronidase-+-+--
α-glucosidase-+++-+
β-glucosidase-+----
N-acetyl-b-glucosamidase-+++--
α-mannosidase-+-+--
α-fucosidase-+----
API 20NE
D-Glucose-+---+
D-Arabinose-+---+
D-mannose-+---+
D-Mannitol-+---+
N-acetyl-D-glucosamine-+---+
Gluconate-+---+
malate-+---+
Citrate-+---+
Presence of pufLM genes-++ND-+
presence of SQDG--++*+*+*
DNA G+C content55.958.5*60*ND59*56*

Strains: 1, Strain CE80T (this study); 2, L. suaedae KACC 13772T; 3, L. marina KCTC12288T; 4, L. alba DSM 18320T; 5, L. aggregata KACC 15203T; 6, L. alexandrii DSM 17067T. All strains are positive for motility, oxidase, catalase, reduction of nitrates to nitrites, hydrolysis of gelatin, assimilation of β-galactosidase, activities of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cysteine arylamidase, trypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase and α-galactosidase. All strains are negative for: hydrolysis of starch, casein, Tween 20, 60, cellulose, indole production, glucose fermentation; arginine dihydrolase, assimilation of D-maltose, caprate, adipate, phenylacetate; lipase (C14) and Naphthol-AS-BI-β D-glucuronide activities..

*Data taken from Bibi et al. (2014)..


Table 2 . Cellular fatty acid compositions (%) of strain CE80T and closely related type strains of the genus Labrenzia..

Fatty acid123456
Saturated
C10:0tr2.1trtrtrtr
C16:0tr2.1trtrtrtr
C18:04.7tr6.26.43.53.2
Unsaturated
C18:1 ω7c66.866.455.970.569.967.7
C20:1 ω7c2.6-4.77.57.414.3
11-methyl C18:1 ω7c4.53.29.96.88.05.7
Cyclo C19:0 ω8c--6.2---
Hydroxy
C18:0 3-OH2.36.72.0trtrtr
Summed features
210.38.83.13.74.13.7
3trtr8.5-trtr

Strains: 1, strain CE80T (this study); 2, L.suaedae KACC 13772T; 3, L. marina KCTC12288T; 4, L. alba DSM 18320T; 5, L. aggregata KACC 15203T; 6, L. alexandrii DSM 17067T. All data were obtained from this study. Values are percentages of total fatty acids (less than 2% are not show); tr, Traces (<2.0%); -, not detected. *Summed features represent groups of two or three fatty acids that cannot be separated by gas chromatography with the MIDI system. Summed features 2 comprised C14:0 3-OH and/or iso- C16:1 I; 3, C16:1 ω7c and/or iso- C15:0 2-OH..


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