Marivirga atlantica sp. nov., isolated from seawater and emended description of the genus Marivirga

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1 International Journal of Systematic and Evolutionary Microbiology (2015), 65, DOI /ijs Marivirga atlantica sp. nov., isolated from seawater and emended description of the genus Marivirga Chao-Yi Lin, 1,2 3 Xi-Ying Zhang, 1,2 3 Ang Liu, 1,2 Chang Liu, 1,2 Xiao-Yan Song, 1,2 Hai-Nan Su, 1,2 Qi-Long Qin, 1,2 Bin-Bin Xie 1,2 and Yu-Zhong Zhang 1,2,3 Correspondence Bin-Bin Xie xbb@sdu.edu.cn 1 State Key Laboratory of Microbial Technology, Shandong University, Jinan , PR China 2 Marine Biotechnology Research Center, Shandong University, Jinan , PR China 3 Collaborative Innovation Center of Deep Sea Biology, Shandong University, Jinan , PR China A novel Gram-stain-negative, aerobic, orange-pigmented, non-flagellated, gliding, rod-shaped bacterium, designated strain SM1354 T was isolated from surface seawater of the Atlantic Ocean. The strain hydrolysed gelatin and DNA but did not reduce nitrate. It grew at C and with % (w/v) NaCl. Phylogenetic analysis of the 16S rrna gene sequences revealed that strain SM1354 T belonged to the genus Marivirga with % sequence similarities to known species of the genus Marivirga. The major fatty acids of strain SM1354 T were iso-c 15 : 0, iso-c 15 : 1 G, iso-c 17 : 0 3-OH and summed feature 3 (C 16 : 1 v7c and/or iso-c 15 : 0 2-OH). Polar lipids of strain SM1354 T included phosphatidylethanolamine, three unidentified lipids and one unidentified aminolipid and aminophospholipid. The major respiratory quinone of strain SM1354 T was menaquinone 7 (MK-7). The genomic DNA G+C content of strain SM1354 T was 33.9±0.4 mol%. On the basis of the results of the polyphasic characterization in this study, it is proposed that strain SM1354 T represents a novel species of the genus Marivirga, namely Marivirga atlantica sp. nov. The type strain of Marivirga atlantica is SM1354 T (5CCTCC AB T 5JCM T ). An emended description of the genus Marivirga is also proposed. The genus Marivirga, a member of the family Flammeovirgaceae in the phylum Bacteroidetes, was created by Nedashkovskaya et al. (2010) and currently contains two species: Marivirga tractuosa (which is a type species) and Marivirga sericea ( During a study of the bacterial diversity in Atlantic samples, an orangecolony-forming bacterial strain, SM1354 T, was isolated from the Atlantic surface seawater sample. Analysis of the 16S rrna gene sequence of strain SM1354 T revealed that it was closely related to the genus Marivirga. In this study, strain SM1354 T was further taxonomically characterized using a polyphasic approach and was therefore proposed to represent a novel species of the genus Marivirga for which the name Marivirga atlantica sp. nov. is proposed. The surface seawater samples were collected at the Atlantic Ocean (GPS position; 11.52u S 19.38u W)in 3These authors contributed equally to this work. Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining. The GenBank/EMBL/DDBJ accession number for the16s rrna gene sequence of strain SM1354 T is KM Four supplementary figures are available with the online Supplementary Material. August 2012 during the DY26 cruise of the R/V Da-Yang Yi-Hao. Seawater samples (100 ml) were directly spread onto the TYS agar [0.5 % (w/v) tryptone (Oxoid), 0.1 % (w/v) yeast extract (Oxoid), 1.5 % (w/v) agar and artificial seawater] and incubated at 20 uc for 3 weeks. Differentlooking colonies on the plates were picked and purified by repeated streaking on the same agar plates. The artificial seawater was prepared with Sigma sea salts (3 %, w/v). Strain SM1354 T and its reference strains: M. tractuosa KCTC 2958 T and M. sericea NBRC T were routinely cultivated on TYS agar or in TYS broth [0.5 % (w/v) tryptone, 0.1 % (w/v) yeast extract and artificial seawater] at 30 uc. Strain SM1354 T was stored at 280 uc in TYS broth supplemented with 20 % (w/v) glycerol. Cell morphology and flagella production were observed by transmission electron microscopy (JEM-100CX II) using cells (grown in TYS broth at 30 uc for 2 days) negatively stained with 2 % phosphotungstic acid. Growth at different temperatures (4, 10, 15, 17, 20, 25, 30, 35, 40 and 45 uc) and ph [ph , at 0.5 ph unit, buffered with MES (ph 5 6, 50 mm), MOPS (ph , 50 mm), Tris (ph , 50 mm) and CHES (ph 9 10, 50 mm)] was measured in TYS broth. Growth with different concentrations of NaCl (0, 0.5, 1 15 %, w/v, NaCl at 1 % increments) G 2015 IUMS Printed in Great Britain 1515

2 C.-Y. Lin and others was determined in TYS broth except that the artificial seawater was replaced with distilled water containing the appropriate concentration of NaCl. Growth under anaerobic conditions was examined in TYS broth supplemented with potassium nitrate (0.1 %, w/v), cysteine hydrochloride (0.05 %, w/v) and sodium sulfide (0.05 %, w/v) in Hungate tubes. Gram-staining was performed following the standard procedure described by Murray et al. (1994). Oxidase activity was examined using Merck oxidase test strips and catalase activity was determined by observing the bubble formation in 3 % (v/v) H 2 O 2 solution. DNA hydrolysis was detected using Oxoid DNase agar. Hydrolysis of casein (0.5 %, w/v), elastin (0.3 %, w/v), starch (1 %, w/v), CMcellulose (1 %, w/v) and Tween 20, 40, 60 and 80 (1 %, v/v) was determined on TYS agar containing the corresponding substrate at 30 uc (Smibert & Krieg, 1994; Bowman, 2000). Hydrolysis of extracellular peptidoglycan was examined as in Jørgensen et al. (2009) on 1/6 TYS agar containing 0.8 % (w/v) peptidoglycan extracted from Staphylococcus warneri MCCC 1A Gliding motility was checked using the hanging drop technique and flexirubin-type pigments were detected with the KOH test (Bernardet et al., 2002). Acid production from carbohydrates was tested using API 50CH strips (biomérieux) with CHB/E medium supplemented with 3 % (w/v) NaCl. Utilization of carbohydrates was assessed by using API 50CH strips (biomérieux) with AUX medium supplemented with 3 % (w/v) NaCl. Enzymic activities and other biochemical properties were determined using API ZYM and API 20NE strips (biomérieux) following the manufacturer s instructions with the exception that the strips were inoculated with cells suspended in artificial seawater. Cells of strain SM1354 T were Gram-stain-negative, nonflagellated, slender rods (Fig. S1, available in the online Supplementary Material). Other morphological, physiological and biochemical characteristics are presented in the species descriptions. Characteristics allowing the differentiation of strain SM1354 T from other known species of the genus Marivirga, such as temperature or NaCl range that supported growth, hydrolysis of elastin and peptidoglycan, API ZYM enzyme activities and genomic DNA G+C content are shown in Table 1. For cellular fatty acid analysis, strain SM1354 T and its reference strains: M. tractuosa KCTC 2958 T and M. sericea NBRC T were cultivated in TYS broth at 30 uc for 48 h. Fatty acid methyl esters were obtained by saponification, methylation and extraction and further analysed using GC (Hewlett Packard 6890) and Sherlock Microbial Identification System software (version 4.5, TSBA40). Polar lipids were extracted as in Komagata & Suzuki (1987) and analysed using two-dimensional TLC with appropriate spraying reagents including: ethanolic molybdophosphoric acid (total lipids), ninhydrin (aminolipids) and Zinzadze reagent (phospholipids). Quinones were extracted as in Komagata & Suzuki (1987) and further identified using an LC-MS system consisting of a Dionex Ultimate 3000 HPLC coupled to a Bruker Impact HD mass spectrometer. Genomic DNA was extracted following the method of Marmur (1961) and the genomic DNA G+C content was determined by the thermal denaturation method (Marmur & Doty, 1962) using a Beckman spectrophotometer (DU800). Five replicate determinations were conducted and the genomic DNA G+C content value of strain SM1354 T was expressed as the mean±sd of the five replicate values obtained. The cellular fatty acids of strain SM1354 T contained predominantly (.5 %) iso-c 15 : 0 (47.3 %), iso-c 15 : 1 G (22.2 %), iso-c 17 : 0 3-OH (8.9 %) and summed feature 3 (C 16 : 1 v7c and/or iso-c 15 : 0 2-OH, 7.9 %), showing a profile basically similar to those in M. tractuosa and M. sericea (Table 2), supporting the affiliation of the strain to the genus Marivirga. Summed feature 3 (C 16 : 1 v7c and/or iso- C 15 : 0 2-OH) was not detected in M. tractuosa KCTC 2958 T, in accordance with a previous report that only trace amounts of summed feature 3 were detected in this strain (Nedashkovskaya et al., 2010). Polar lipids of strain SM1354 T included major amounts of phosphatidylethanolamine and one unidentified lipid, moderate amounts of two unidentified lipids and one unidentified aminolipid and minor amounts of one unidentified aminophospholipid, in perfect accordance with the other two species of the genus Marivirga (Fig. S2). The major respiratory quinone of strain SM1354 T was menaquinone 7 (MK-7), same as those of M. tractuosa and M. sericea (Nedashkovskaya et al., 2010). The genomic DNA G+C content of strain SM1354 T was 33.9±0.4 mol%, lower than those reported for type strains of M. tractuosa and M. sericea (36.1 and 37.1 mol%, respectively) (Nedashkovskaya et al., 2010). Genomic DNA of strain SM1534 T was extracted using a bacterial DNA extraction kit (BioTeke). The 16S rrna gene sequence of strain SM1534 T was PCR-amplified with 27F and 1492R primers (Lane, 1991) and then sequenced using an Applied Biosystems model 3730 DNA sequencer at Biosune. Sequence similarity searches against GenBank and Eztaxon-e databases (Kim et al., 2012) were performed using BLASTN (Altschul et al., 1997). Pairwise sequence similarity values were obtained through the EzTaxon-e server ( Kim et al., 2012). Sequence alignment and phylogenetic analysis were performed using MEGA version 5 (Tamura et al., 2011). Phylogenetic trees were reconstructed using the neighbourjoining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971) methods with bootstrap analyses based on 1000 replications. Evolutionary distances were calculated using the Kimura two-parameter model (Kimura, 1980). The almost-complete 16S rrna gene sequence of strain SM1354 T (1482 bp) was obtained. Sequence comparison revealed the strain displayed the highest 16S rrna gene sequence similarity with type strain of M. tractuosa (96.2 %), followed by that of M. sericea (96.0 %) and less 1516 International Journal of Systematic and Evolutionary Microbiology 65

3 Marivirga atlantica sp. nov. Table 1. Differential characteristics of strain SM1354 T and other species of the genus Marivirga Strains: 1, SM1354 T ;2,M. tractuosa KCTC 2958 T (this study); 3, M. sericea NBRC T (this study). +, Positive; 2, negative; W, weakly positive. Characteristic Temp. range for growth (uc) NaCl range for growth (%, w/v) Hydrolysis of: Elastin + W W Peptidoglycan API ZYM Lipase (C14) W 2 2 b-galactosidase + W W N-Acetyl-b-glucosaminidase W 2 2 Trypsin DNA G+C content (mol%) * 37.1* Source Surface seawater Beach sand Marine aquarium outflow *Data from Nedashkovskaya et al. (2010). than 89.6 % sequence similarities to other recognized species in the phylum Bacteroidetes. As shown in the phylogenetic trees (Figs 1, S3 and S4), strain SM1354 T clustered tightly with M. tractuosa and M. sericea and formed a separate subbranch with very high bootstrap support (100 %), suggesting that it belongs to the genus and represents a putative novel species. Based on the polyphasic characterization for strain SM1354 T in this study, including phylogenetic analysis of the 16S rrna gene sequences and determination of cellular fatty acid compositions and phenotypic characteristics, strain SM1354 T represents a novel species in the genus Marivirga, for which the name Marivirga atlantica sp. nov. is proposed. Description of Marivirga atlantica sp. nov. Marivirga atlantica (at.lan9ti.ca. L. fem. adj. atlantica of or pertaining to the Atlantic Ocean, the origin of the type strain) Cells of the type strain are Gram-stain-negative, aerobic, oxidase- and catalase-positive, non-flagellated rods, 2 8 mm in length and mm in diameter. Cells are motile by gliding and do not produce flexirubin-type pigments. Table 2. Cellular fatty acid compositions (%) of strain SM1354 T and other species of the genus Marivirga Strains: 1, SM1354 T ;2,M. tractuosa KCTC 2958 T ;3,M. sericea NBRC T. Fatty acids,1 % in all strains are not shown. Predominant fatty acids (.5 %) in each strain are shown in bold. TR, Trace amount (less than 1 %); ND, not detected. Fatty acid This study Nedashkovskaya et al. (2010) This study Nedashkovskaya et al. (2010) iso-c 11 : 0 TR TR TR TR 2.7 iso-c 13 : 0 TR TR iso-c 14 : 0 ND ND TR iso-c 15 : 1 G D D iso-c 15 : anteiso-c 15 : 0 TR TR TR C 15 : 0 TR iso-c 16 : 1 G TR TR iso-c 16 : 0 TR TR iso-c 15 : 0 3-OH iso-c 16 : 0 3-OH TR TR iso-c 17 : iso-c 17 : 0 3-OH Summed feature 3* 7.9 ND TR *Summed feature 3, comprising C 16 : 1 v7c and/or iso-c 15 : 0 2-OH. Diso-C 15 :

4 C.-Y. Lin and others /-/74 93/85/- 97/98/98 93/92/79 85/87/83 100/99/98 90/84/- 100/99/98 93/87/76 91/93/87 95/97/98 100/100/99 100/100/99 99/99/99 100/99/99 Fulvivirga kasyanovii KMM 6220 T (DQ836305) Fulvivirga imtechensis AK7 T (FR687203) Reichenbachiella faecimaris PCP11 T (GU143096) Reichenbachiella agariperforans KMM 3525 T (AB058919) Roseivirga ehrenbergii KMM 6017 T (AY608410) Roseivirga echinicomitans KMM 6058 T (AY753206) Fabibacter pacificus DY53 T (KC005305) Fabibacter halotolerans UST T (DQ080995) Marinoscillum furvescens NBRC T (AB078079) Marinoscillum luteum SJP7 T (HM161878) Marinoscillum pacificum MRN461 T (DQ660388) Marivirga tractuosa DSM 4126 T (CP002349) Marivirga sericea LMG T (AB078081) SM1354 T (KM117235) Nafulsella turpanensis ZLM-10 T (ANNU ) Cesiribacter andamanensis AMV16 T (AODQ ) Cesiribacter roseus 311 T (HM775387) Fulvitalea axinellae VI.18 T (JN699063) Aureibacter tunicatorum A5Q-118 T (AB572584) Persicobacter psychrovividus NBRC T (AB260934) Persicobacter diffluens NBRC T (AB260929) Flexithrix dorotheae IFO T (AB078077) Rapidithrix thailandica TISTR 1750 T (AB265192) 100/99/99 Limibacter armeniacum YM T (AB359907) Perexilibacter aurantiacus Shu-F-UV2-2 T (AB276355) Flammeovirga kamogawensis YS10 T (AB251933) Flammeovirga pacifica WPAGA1 T (HQ412594) Flammeovirga yaeyamensis NBRC T (AB247554) Thermonema lapsum DSM 5718 T (HE582775) Flavobacterium algicola TC2 T (AB455265) Fig. 1. Neighbour-joining (NJ) phylogenetic tree based on 16S rrna gene sequences showing the phylogenetic positions of strain SM1354 T (in bold) and related species. Filled circles indicate clades that were recovered in all the trees inferred with NJ, maximum-likelihood (ML) and maximum-parsimony (MP) methods. Bootstrap values (.70 %, NJ/MP/ML), based on 1000 replications, are shown at nodes. Bar, 0.01 substitutions per nucleotide position. Colonies incubated on TYS agar at 30 uc for 2 5 days are orange-pigmented, circular (1 3 mm in diameter) and convex. The type strain hydrolyses casein, DNA, elastin, peptidoglycan and Tween 20, 40, 60 and 80 but does not hydrolyse starch, urea and CM-cellulose. It grows at 4 40 uc (optimum at 30 uc), with the presence of % (w/v) NaCl (optimum in 3 %) and at ph (optimum at ph 6 7). In API 20NE tests, cells of the type strain are positive for b-galactosidase, hydrolysis of aesculin and gelatin and assimilation of glucose, maltose and malate. In the API ZYM tests, cells of the type strain are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14) (weakly), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase, b-galactosidase, a-glucosidase, b-glucosidase and N-acetyl-b-glucosaminidase (weakly). With the API 50CH kits, the type strain produces acid from arbutin, aesculin and potassium 5- ketogluconate and utilizes D-glucose, amygdalin, cellobiose, maltose, lactose and D-gentiobiose as the sole carbon sources. The major fatty acids (.5 %) of the type strain are iso-c 15 : 0, iso-c 15 : 1 G, iso-c 17 : 0 3-OH and summed feature 3 (C 16 : 1 v7c and/or iso-c 15 : 0 2-OH). The predominant polar lipids of the type strain are phosphatidylethanolamine and one unidentified lipid. The major respiratory quinone of the type strain is menaquinone 7 (MK-7). The type strain is SM1354 T (5CCTCC AB T 5JCM T ), isolated from surface seawater of the Atlantic Ocean. The genomic DNA G+C content of the type strain is 33.9±0.4 mol%. Emended description of the genus Marivirga Nedashkovskaya et al. (2010) The description is as given by Nedashkovskaya et al. (2010) with the following amendments. The DNA G+C content is mol% International Journal of Systematic and Evolutionary Microbiology 65

5 Marivirga atlantica sp. nov. Acknowledgements The work was supported by the China Ocean Mineral Resources R & D Association (COMRA) Special Foundation (grant nos DY R-03 and DY T-05), the National Natural Science Foundation of China (grant nos , and ), the Hi-Tech Research and Development Program of China (grant no. 2012AA092103), the Independent Innovation Foundation of Shandong University (grant nos 2012ZD028, 2012TB004 and 2012GN019) and the Fundamental Research Funds of Shandong University (grant no. 2014QY006). References Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, Bernardet, J.-F., Nakagawa, Y., Holmes, B. & Subcommittee on the taxonomy of Flavobacterium and Cytophaga-like bacteria of the International Committee on Systematics of Prokaryotes (2002). Proposed minimal standards for describing new taxa of the family Flavobacteriaceae and emended description of the family. Int J Syst Evol Microbiol 52, Bowman, J. P.(2000). Description of Cellulophaga algicola sp. nov., isolated from the surfaces of Antarctic algae, and reclassification of Cytophaga uliginosa (ZoBell and Upham 1944) Reichenbach 1989 as Cellulophaga uliginosa comb. nov. Int J Syst Evol Microbiol 50, Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, Jørgensen, N. O. G., Brandt, K. K., Nybroe, O. & Hansen, M. (2009). Delftia lacustris sp. nov., a peptidoglycan-degrading bacterium from fresh water, and emended description of Delftia tsuruhatensis as a peptidoglycan-degrading bacterium. Int J Syst Evol Microbiol 59, Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y. S., Lee, J.-H. & other authors (2012). Introducing EzTaxon-e: a prokaryotic 16S rrna gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62, Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods Microbiol 19, Lane, D. J. (1991). 16S/23S rrna sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, Marmur, J. & Doty, P. (1962). Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol 5, Murray, R. G. E., Doetsch, R. N. & Robinow, C. F. (1994). Determinative and cytological light microscopy. In Methods for General and Molecular Bacteriology, pp Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Nedashkovskaya, O. I., Vancanneyt, M., Kim, S. B. & Bae, K. S. (2010). Reclassification of Flexibacter tractuosus (Lewin 1969) Leadbetter 1974 and Microscilla sericea Lewin 1969 in the genus Marivirga gen. nov. as Marivirga tractuosa comb. nov. and Marivirga sericea nom. rev., comb. nov. Int J Syst Evol Microbiol 60, Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28,