Maribacter flavus sp. nov., isolated from a cyanobacterial culture pond

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1 International Journal of Systematic and Evolutionary Microbiology (2015), 65, DOI /ijsem Maribacter flavus sp. nov., isolated from a cyanobacterial culture pond Mingxing Tang, 1,2 Guanghua Wang, 1 Wenzhou Xiang, 1 Chenghao Chen, 1,2 Jiayi Wu, 1 Shikun Dai, 1 Hualian Wu, 1 Tao Li 1 and Houbo Wu 1 Correspondence Houbo Wu wuhoubo@scsio.ac.cn 1 Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Key Laboratory of Marine Materia Medica (LMMM-GD), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou , PR China 2 University of Chinese Academy of Sciences, Beijing , PR China A Gram-stain-negative, yellow, non-spore-forming, strictly aerobic bacterium, designated C3 T, was isolated from a cyanobacterial culture pond. Cells were halophilic, rod-shaped and able to move by gliding. Growth of strain C3 T was observed at C (optimum 25 8C), ph (optimum ph 7.5), and in the presence of 1 7 % (w/v) NaCl (optimum 2 3 %). Phylogenetic analysis based on 16S rrna gene sequences revealed that strain C3 T formed a distinct lineage within the family Flavobacteriaceae and exhibited the highest similarity (95.21 %) to the type strains of Maribacter dokdonensis, Maribacter arcticus, Maribacter orientalis and Maribacter stanieri, and Maribacter caenipelagi HD-44. The only isoprenoid quinone present within strain C3 T was menaquinone 6 (MK-6). The G+C content of genomic DNA was 41.5 mol%. The major polar lipids were phosphatidylethanolamine and three unidentified lipids. The predominant cellular fatty acids (.5 % of the total fatty acids) were iso-c 15 : 1 G, iso-c 15 : 0, iso-c 15 : 0 3-OH, iso-c 17 : 0 3-OH, and summed feature 3 (comprising C 16 : 1 v7c and/or C 16 : 1 v6c). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, strain C3 T represents a novel species of the genus Maribacter, for which the name Maribacter flavus sp. nov. is proposed. The type strain is C3 T (5KCTC T 5CGMCC T ). The genus Maribacter, which was first established by Nedashkovskaya et al. in 2004, is a member of the family Flavobacteriaceae (Bernardet et al., 2002; Nedashkovskaya et al., 2004). Subsequently, the description of the genus Maribacter has been emended by Barbeyron et al. (2008), Nedashkovskaya et al. (2010), Weerawongwiwat et al. (2013) and Lo et al. (2013), respectively. Bacterial cells of this genus are yellow or orange, rod-shaped, Gram-negative and strictly aerobic. The isoprenoid quinone present is menaquinone 6 (MK-6). The DNA G+C content of species of this genus ranges from 34.4 to 41.1 mol%. At the time of writing, 13 species of the genus Maribacter have been described, namely Maribacter aestuarii (Lo et al., 2013), M. antarcticus (Zhang et al., 2009), M. aquivivus (Nedashkovskaya et al., 2004), M. arcticus (Cho et al., 2008), M. caenipelagi (Jung et al., 2014), M. chungangensis (Weerawongwiwat et al., 2013), M. dokdonensis (Yoon The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain C3 T is KR A supplementary figure is available with the online Supplementary Material. et al., 2005), M. forsetii (Barbeyron et al., 2008), M. orientalis (Nedashkovskaya et al., 2004), M. polysiphoniae (Nedashkovskaya et al., 2007), M. sedimenticola (Nedashkovskaya et al., 2004), M. stanieri (Nedashkovskaya et al., 2010) and M. ulvicola (Nedashkovskaya et al., 2004). All species of this genus originated from diverse marine environments. Strain C3 T was isolated from the culture broth of a seawater-dependent cyanobacterium Plectonema sp. This microalga was maintained using f/2 medium, which was composed of natural seawater, deionized fresh water, sodium bicarbonate, and other inorganic ions, such as ferric ions and calcium ions (Guillard & Ryther, 1962). The algae culture was diluted to 1000-fold using sterilized f/2 medium, and 100 ml diluted solution was spread on marine agar 2216 (MA; Difco) plates, which were subsequently incubated at 25 8C under aerobic conditions for 2 weeks. According to the shape and colour of bacterial colonies, a single colony was isolated and further subcultivated by streaking three times on new MA plates. The strain was maintained on MA plates at 25 8C and in marine broth 2216 (MB; Difco) supplemented with 30 % G 2015 IUMS Printed in Great Britain 3997

2 M. Tang and others 93 Maribacter dokdonensis DSW-8 T (AY960749) Maribacter sedimenticola KMM 3903 T (AY271623) Maribacter ulvicola KMM 3951 T (AY271626) Maribacter forsetii KT02ds18-6 T (AM712900) 53 'Maribacter caenipelagi' HD-44 (KF748920) Maribacter aquivivus KMM 3949 T (AY271625) Maribacter stanieri KMM 6046 T (EU246691) Maribacter orientalis KMM 3947 T (AY271624) Maribacter arcticus KOPRI T (AY771762) Maribacter aestuarii GY20 T (JN642273) Maribacter antarcticus DSM T (JHZC ) Maribacter chungangensis CAU 1044 T (JN036550) Maribacter polysiphoniae LMG T (AM497875) 0.05 Pibocella ponti KMM 6031 T (AY576654) Maribacter flavus C3 T (KR058351) Kriegella aquimaris KMM 3665 T (AB084262) Pseudozobellia thermophila KMM 3531 T (AB084261) Zobellia russellii KMM 3677 T (AB121976) Zobellia galactanivorans DsiJ T (FP476056) 95 Zobellia uliginosa ATCC T (M62799) Zobellia amurskyensis KMM 3526 T (AB121974) 95 Zobellia laminariae KMM 3676 T (AB121975) Arenibacter latericius KMM 426 T (AF052742) Escherichia coli KCTC 2441 T (EU014689) Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rrna gene sequences showing the position of strain C3 T and related taxa. Escherichia coli KCTC 2441 T was used as an outgroup. The same topology was supported by the minimum-evolution tree. Lines in bold indicate branches found in the maximum-likelihood tree. Bootstrap values (expressed as percentages of 1000 replications).50 % are shown at branch points. Bar, 0.05 substitutions per nucleotide position. (v/v) glycerol at 280 8C. Maribacter dokdonensis DSW-8 T, Maribacter sedimenticola KMM 3903 T and Zobellia galactanivorans DsiJ T were obtained from the CGMCC (China General Microbiological Culture Collection Center) to be used as the reference strains in the comparative analysis. Genomic DNA of strain C3 T was extracted by following the method described by Barbeyron et al. (1984). An almostcomplete 16S rrna gene sequence was amplified by PCR with the universal primers 27F and 1492R (Lane, 1991). The 16S rrna gene sequences of strain C3 T and other closely related members of the family Flavobacteriaceae obtained from EzBioCloud (Kim et al., 2012) were aligned using the SINA software package (Pruesse et al., 2012) in SILVA rrna database. Phylogenetic trees were reconstructed using the neighbour-joining (Saitou & Nei, 1987), minimum-evolution (Rzhetsky & Nei, 1992) and maximum-likelihood (Felsenstein, 1981) methods in the software package MEGA version 6.0 (Tamura et al., 2013). The evolutionary distance matrices were estimated by Kimura s two-parameter model (Kimura, 1980). The topology of the phylogenetic tree was assessed by using the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. 16S rrna gene sequences blasted in the EzTaxon database revealed that strain C3 T shared % similarities to members of the genus Maribacter (Altschul et al., 1990; Chun et al., 2007). The most closely related strains were M. dokdonensis DSW-8 T, M. arcticus KOPRI T, 3998 International Journal of Systematic and Evolutionary Microbiology 65

3 Maribacter flavus sp. nov. Table 1. Differential characteristics of strain C3 T and the type strains of related species Strains: 1, C3 T (data from this study); 2, M. dokdonensis DSW-8 T (Yoon et al., 2005; Jung et al., 2014); 3, M. sedimenticola KMM 3903 T (Nedashkovskaya et al., 2004; Jung et al., 2014); 4, M. arcticus KCTC T (Cho et al., 2008; Jung et al., 2014); 5, Z. galactanivorans DsiJ T (Barbeyron et al., 2001); 6, P. ponti KMM 6031 T (Nedashkovskaya et al., 2005). All strains were positive for hydrolysis of Tween 20 and negative for urease activity. +, Positive; 2, negative; NA, no available data. Characteristic NaCl for growth (%, w/v) Range Optimum NA Temperature for growth (8C) Range Optimum ph for growth Range NA NA NA Optimum NA Pigmentation Yellow Yellow Yellow Yellow Yellow orange Yellow Flexirubin Hydrolysis of: Gelatin Chitin Starch Tween Tween Casein Reduction of nitrate Assimilation of: D-Glucose L-Arabinose D-Mannose D-Mannitol N-Acetylglucosamine NA Maltose NA Sucrose D-Xylose NA NA Enzyme activity L-Arginine dihydrolase NA 2 NA b-glucosidase NA b-galactosidase NA DNA G+C content (mol%) M. orientalis KMM 3947 T, M. stanieri KMM 6046 T and M. caenipelagi HD-44, all of which shared the highest 16S rrna gene sequence similarity (95.21 %) to strain C3 T, followed by M. aestuarii GY20 T (94.85 %), M. ulvicola KMM 3951 T (94.78 %), and M. forsetii KT02ds18-6 T (94.61 %). Phylogenetic analysis based on 16S rrna gene sequences using the neighbour-joining algorithm illustrated that strain C3 T formed a distinct evolutionary lineage within the family Flavobacteriaceae and converged with members of the genera Maribacter and Pibocella into one clade (Fig. 1). The phylogenetic trees built by the minimum-evolution method also showed the same topology, while small differences occurred in the maximum-likelihood tree. The morphology of exponential growth phase cells was examined using transmission electron microscopy (Hitachi TEM System-H7650) after negative staining with 1 % (w/v) phosphotungstic acid. Cell motility was determined by the hanging-drop method (Bernardet et al., 2002). The Gram reaction was performed according to the method described by Gerhardt et al. (1994) with cells grown on MA plates at 30 8C for 72 h. Endospore formation and other morphological characteristics were examined by the procedures described by Dong and Cai (2001). Production of flexirubin-type pigments was detected according to the method of Reichenbach et al. (1974). Production of H 2 S was assayed using the procedure described by Bruns et al. (2001). Anaerobic growth

4 M. Tang and others was tested in accordance with the protocols of Barbeyron et al. (2001). Oxidase activity was determined by using oxidase test strips (Huankai). Catalase activity was assayed by bubble production in a 3 % (v/v) hydrogen peroxide solution (Smibert & Krieg, 1994). The temperature range for growth was determined by observing colony formation after streaking the strain on MA plates and incubating at 4, 10, 15, 20, 25, 30, 37 and 45 8C under aerobic conditions for 72 h. Growth at different ph was examined by measuring the turbidity of the broth after incubating the strain in MB adjusted to ph (in increments of 0.5 ph units), using the following buffering solutions to regulate the final ph: 0.1 M KH 2 PO 4 /K 2 HPO 4 (for ph ), 0.1 M Glycine/NaOH (for ph ), and 0.1 M Na 2 CO 3 /NaOH (for ph ) (Wang et al., 2013). Growth in the presence of 0, 0.5, 1, 3, 5, 7, 9, 11 and 15 % (w/v) NaCl was tested in MB. Hydrolysis of gelatin, chitin, Tweens 20, 40 and 80, casein and starch were assayed by incubating the strain on MA plates supplemented with the corresponding substrates. Growth on sole carbon sources and enzymic activities were determined using Biolog GEN III MicroPlate and API 20NE systems, respectively, after 24 h pre-incubation of strain C3 T in sterile seawater at 27 8C. Cells of strain C3 T were rod-shaped and motile by gliding, mm in diameter and mm in length. No flagella were observed on the bacterial cells under transmission electron microscopy (Fig. S1, available in the online Supplementary Material). Cells were oxidase- and catalase-positive, Gram-stain-negative and chemo-organotrophic with a respiratory-type metabolism. Colonies on MA plates were yellow and shiny with entire edges. Endospores and flexirubin-type pigments were not observed. Growth of strain C3 T occurred at C, ph , and with 1 7 % (w/v) NaCl, while the optimal growth of this strain was observed at 25 8C, ph 7.5, and in the presence of 2 3 % (w/v) NaCl. Tweens 20 and 40 could be hydrolysed, but Tween 80, casein, gelatin, starch and chitin could not be hydrolysed. Other phenotypic characteristics of strain C3 T and the reference strains are listed in Table 1 and the species description. Biomass for analysis of polar lipids and isoprenoid quinones, and DNA base composition determination was obtained from MA broth after incubation for 72 h at 27 8C. Polar lipids were extracted using the method of Kamekura (1993), and identified by two-dimensional thin-layer chromatography followed by spraying with corresponding detection reagents (Tindall, 1990). Isoprenoid quinones were extracted according to Collins (1985) and determined by reversed phase HPLC as described by Komagata & Suzuki (1987). Total DNA extraction was performed according to Barbeyron et al. (1984), and the G+C content was assayed by using the HPLC method (Mesbah et al., 1989). Cell biomass for analysis of fatty acids were acquired from MA plates after incubation for 72 h at 30 8C. Cellular fatty acid composition was assayed by using the Sherlock Microbial Identification System (MIDI) according to the manufacturer s instructions. The fatty acid profile was analysed by GC (Agilent G6890N) and identified by using the Microbial Identification software package (Sherlock version 6.0). The major polar lipids of strain C3 T were phosphatidylethanolamine and three unidentified lipids. Minor amounts of four unidentified aminophospholipids and one phospholipid were also present (Fig. 2). Although, the polar lipid profile of strain C3 T was consistent with those of most species of the genus Maribacter in that phosphatidylethanolamine and two unidentified lipids (L2 and L3) were the major polar lipids, it was still apparently distinguishable due to differences in the number of polar lipids detected (Jung et al., 2014). Strain C3 T harboured four major polar lipids including phosphatidylethanolamine, while M. arcticus KCTC T and M. chungangensis CAU 1044 T contained phosphatidylethanolamine as the only major polar lipid (Weerawongwiwat et al., 2013); M. caenipelagi HD-44, M. aquivivus KCTC T and M. ulvicola KCTC T contained three major polar lipids (Jung et al., 2014). Comparison of polar lipid patterns of these species not only supported affiliation of strain C3 T to the genus Maribacter, but also implied that this strain is different from other species of the genus Maribacter. The only isoprenoid quinone detected in strain C3 T was MK- 6, which was in accordance with that of other species of the genus Maribacter. The DNA G+C content of strain C3 T was 41.5 mol%, which was slightly higher than that of other APL2 APL4 PL L1 L2 APL3 PE L3 APL1 Fig. 2. Thin-layer chromatogram of the total polar lipids of strain C3 T after staining with 10 % ethanolic molybdatophosphoric acid. PE, Phosphatidylethanolamine; PL, unidentified phospholipid; APL1 4, unidentified aminophospholipids; L1 3, unidentified lipids International Journal of Systematic and Evolutionary Microbiology 65

5 Maribacter flavus sp. nov. species of the genus Maribacter. The cellular fatty acid analysis of strain C3 T showed a profile of 13 fatty acids with a dominance of (.5 %)iso-c 15 : 1 G, iso-c 15 : 0,iso-C 15 : 0 3-OH, iso-c 17 : 0 3-OH and summed feature 3 (C 16 : 1 v7c and/or C 16 : 1 v6c) (Table 2). The relative content of iso-c 15 : 1 G, iso-c 15 : 0 and iso-c 17 : 0 3-OH showed that the bacterium was similar to M. sedimenticola KMM 3903 T and M. dokdonensis DSW-8 T. Moreover, data in bold in Table 2 demonstrated that the major fatty acid pattern of strain C3 T was dramatically different from that of Pibocella ponti KMM 6031 T and Z. galactanivorans DsiJ T, both of which were related Table 2. Comparison of the fatty acid profiles of strain C3 T and type strains of related species Strains: 1, C3 T (data from this study); 2, M. sedimenticola KMM 3903 T (this study); 3, M. dokdonensis DSW-8 T (this study);4, Z. galactanivorans DsiJ T (this study); 5, P. ponti KMM 6031 T (Nedashkovskaya et al., 2005). Data are reported as percentages of the total fatty acids. Major differences are highlighted in bold. ND, Not detected; 2, trace amount. Fatty acid Straight-chain C 12 : 0 ND ND ND 0.67 ND C 13 : 0 ND ND ND ND ND C 14 : C 15 : C 16 : C 18 : ND Unsaturated C 15 : 1 v6c ND C 18 : 3 v6c ND ND 0.63 ND ND Branched iso-c 13 : 0 ND ND ND iso-c 15 : 1 G ND iso-c 15 : anteiso-c 15 : 0 ND ND ND Hydroxy iso-c 15 : 0 3-OH C 16 : 0 3-OH iso-c 17 : 0 3-OH Summed features* D ND 5 ND ND 2.55 ND ND 7 ND ND 0.83 ND ND ND *Summed features represent groups of two or three fatty acids that could not be separated by GLC with the MIDI system. Summed feature 3 comprises C 16 : 1 v7c and/or C 16 : 1 v6c; summed feature 4 comprises iso-c 17 : 1 I and/or anteiso-c 17 : 1 B; summed feature 5 comprises C 18 : 2 v6, 9c and/or ante-c 18 : 0 ; summed feature 7 comprises C 19 : 1 v6c and/or cyclo-c 19 : 0 v10c and/or unknown ECL (equivalent chainlength) ; summed feature 9 comprises iso-c 17 : 1 v9c and/or 10-methyl C 16 : 0. DComprises C 16 : 1 v7c and/or iso-c 15 : 0 2-OH. to strain C3 T and other species of the genus Maribacter.Morphological, biochemical and physiological data of this bacterial strain, combined with phylogenetic analysis, are sufficient to support the creation of a novel species of the genus Maribacter, for which the name Maribacter flavus sp. nov. is proposed. Description of Maribacter flavus sp. nov. Maribacter flavus (fla9vus. L. adj. flavus yellow). Cells are Gram-stain-negative, non-spore-forming, strictly aerobic rods, mm in width and mm in length, and motile by gliding. Colonies on MA are circular, yellow and convex with entire margins. Flexirubin-type pigments are not produced. Growth occurs at uc, at ph , and with 1 7 % (w/v) NaCl. Grows optimally at 25 uc, at ph 7.5, and with 2 3 % (w/v) NaCl. Oxidase and catalase activities are positive. Tweens 20 and 40 are hydrolysed, but Tween 80, casein, gelatin, starch and chitin are not. Nitrate is reduced to nitrite. Indole and H 2 S are not produced. Utilizes D-glucose, L-arabinose, D- mannose, D-mannitol, N-acetylglucosamine, maltose, trehalose, pyruvic acid, sucrose, potassium gluconate, adipic acid, malic acid and phenylacetic acid, but not capric acid, glycine, D-xylose, lactose, myo-inositol or trisodium citrate. b-glucosidase and b-galactosidase activities are positive, while L-arginine dihydrolase, urease and protease activities are negative. The major polar lipids are phosphatidylethanolamine and three unidentified lipids. The only isoprenoid quinone is MK-6. The predominant cellular fatty acids (w5 % of the total fatty acids) are iso-c 15:1 G, iso-c 15:0, iso-c 15:0 3-OH, iso-c 17:0 3-OH and summed feature 3 (C 16:1 v7c/c 16:1 v6c). The type strain C3 T (5KCTC T 5CGMCC T ), was isolated from the culture broth of a cyanobacterium Plectonema sp., which was cultivated in the South China Sea Institute of Oceanology, Guangzhou, China. The DNA G+C content of the type strain is 41.5 mol%. Acknowledgements We thank the reviewers of this manuscript for their contribution to this work, and would also like to acknowledge editorial support. This research was supported by the Public Science and Technology Research Funds Projects of Ocean ( ), National Natural Science Foundation of China (nos , ), Guangdong Province and Chinese Academy of Science Cooperation Foundation (2012B ), Funds for Marine Renewable Energy (GHME2011SW04) and Guangdong Ocean Innovative Demonstration Area of Economic Development Project (SZHY2012-B01-003). References Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, Barbeyron, T., Kean, K. & Forterre, P. (1984). DNA adenine methylation of GATC sequences appeared recently in the Escherichia coli lineage. J Bacteriol 160,

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