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Endozoicomonas elysicola gen. nov., sp. nov., a γ-proteobacterium isolated from the sea slug ornata

Article in Systematic and Applied Microbiology · May 2007 DOI: 10.1016/j.syapm.2006.07.003 · Source: PubMed

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Endozoicomonils elysicola gen. nov., sp. nov., a y-proteobacterium isolated from the sea slug Elysia ornata Midori Kurahashi*, Akira Yokota

Institute of Molecular and Cellular Biosciences, The Uniuersity of Tokyo, Yayoi l-l-1, Bunkyo-ku, Tokyo l13-0032, Japan

Received 20 June 2006

Abstract

A Gram-negative, strictly aerobic, rod-formed bacterium, strain MKT110r, was isolated from a mollusk, the sea slug Elysia ornata collected in seawater off the coast of Izu-Miyake Island, Japan at a depth of 1 5 m. Comparative I 65 rRNA gene sequences analysis indicated that the isolate MKT110r constituted a novel lineage in y-proteobacteria related to the genera Zooshikella, Oceanospirillum, Microbulbiftr, Marinobacter, Saccharospirillum and Pseudomonas. The strain MKTll0r was closely related to the clones from marine sponge Halichondria okadai (48054136, 4B054161) and the coral Pocillopora damicornis (AY700600, AY700601). The phylogenetic tree based on the 165 rRNA gene sequences showed that MKTl10r and four clones formed a sub-lineage related to the genus Zooshikella, with a bootstrap value of lOOoA. MKTl10r required salt for its growth and was mesophilic. The bacterium contained l6:1at7c,16:0 and l4:0 as major cellular fatty acids, and 3-OH 14:0, 3-OH 10:0 and 3-OH l2:0 as major hydroxy fatty acids. The DNA base composition of the isolate was 50.4mol% G+ C. The major quinone was Q-9. The bacterium is distinguished from currently recognized bacterial genera based on phylogenetic and phenotypic features and should be classified in a novel genus for which the name Endozoicomonas elysicola gen. nov., sp. nov. is proposed. (type strain MKT1l0r: IAM 15107r: KCTC 12372r GenBank accession no. 4B196667). @ 2006 Elsevier GmbH. All rights reserved.

Keywords: Endozoicomonas elysicola; Phylogeny; ; New genus; Marine bacterium; Sea slug; 165 rRNA gene

Introduction marine or halophilic environments. In this study, we characterized the isolate MKT110r, and showed that it In the last several years much attention has been paid belongs to this cluster. The aim of the present study was to marine bacteria as the promising genetic resources. to isolate, characterize, and identify rrlgLrine bacteria that Most of marine bacteria are Gram-negative, marine were not previously known as genetic resources. We andlor halophilic aerobes and facultative anaerobes focused on marine creatures, particularly , as belonging to the a- or y-subdivision ofthe proteobacter- sources for the isolation of bacteria keeping an eye on ia. In particular, many genera closely related to their ecological diversity. There are several bacteria Oceanospirillum or Marinobacter have been detected in that may have symbiotic or epiphytic relationships with marine creatures and that have not been studied *Corresponding adequately A novel genus, Agariuorans, has been author. Tel.: + 81 3 5841 7828; [6]. fax: -r 81 3 5841 8490. proposed for six isolates from marine creatures, E-mail address: [email protected] jp (M. Kurahashi). and four of these six strains have been isolated from

0721-20201$-see front matter O 2006 Elsevier GmbH. All rights reserved doi: 1 0. I 0 1 6/j.syapm.2006.07.003 M. Kurahashi, A. Yokota / Systematic and Applied Microbiology 30 (2007) 202-206 203 mollusca [7]. We describe here a novel bacterium that Phylogenetic analysis was isolated from the marine mollusk E. ornata and forms a robust clade with clones from marine sponges or The template DNA for the PCR was prepared coral. following the method of Hiraishi [4]. The 165 rRNA gene was amplified using the universal primers described previously [6]. The PCR product was purified with 20% PEG6000 and 2.5 NaCl. The purified PCR Materials and methods M product was directly sequenced with a BigDye Termi- nator Cycle Sequencing Ready Reaction Kit (Applied Isolation and bacterial strain Biosystems, Foster City, CA). The sequencing reaction mixtures were purified with a CleanSEQ Dye Termi- The strain MKTll0r reported in this study was nator Removal Kit (Agencourt Bioscience, Beverly, obtained from the gastrointestinal tract of a mollusk, MA). The purified sequencing reaction mixture was the sea slrug Elysia ornata which was collected by scuba electrophoresed on an Applied Biosystems model 310 diving off the coast of Izu-Miyake Island, Japan at a sequencer. depth of 15m. The collected sea slug E. ornata was The almost complete 165 rRNA gene sequence was washed several times with sterile seawater. The internal investigated for homology in the DNA databases using organs were excised, homogenized and serially diluted to the BLAST [1] and FASTA algorithms [8,10]. The a 1:10 ratio in sterile seawater. 0.1m1 aliquots of the sequence of the isolate was aligned with the sequences dilution were spread on marine agar (Difco Labora- of reference organisms derived from the databases. tories, Detroit, MI). Plates were then incubated at 25"C Alignment was performed using the program CLUS- for I week. Colonies appearing on the plates were TAL W ver. 1.8 [13]. The evolutionary distance matrix purified by repeated streaking and stored by freezing at was calculated using the Kimura two-parameter method -80'c. [5]. The phylogenetic tree was constructed by the neighbor-joining method [1U. The topology of the Phenotypic characterization phylogenetic tree was evaluated by bootstrap analysis with 1000 sample replications according to the method The phenotypic features of the isolate, i.e., colony of Felsenstein [3]. morphology, pigment production, and cell morphology, were determined by cultivating the isolate on marine General morphology was studied agar at 25'C. cell Results using phase contrast micrography and transmission electron micrography (TEM). Specimens for TEM were Phenotypic characteristics negative-stained with uranium acetate. Biochemical tests were carried out using two microbial identification Cells of strain MKT1l0r were Gram-negative, rods, products, the API 20-E kit and the API 50-CH kit and 0.4-0.6 pm in width and 1.8-2.2 in length. Electron (bioM6rieux, Etoile, France). microscopic analyses revealed the presence of single polar flagella, pili and vesicles released to the cell surface Analyses of fatty acids and isoprenoid quinones (Fig. 1). Colonies on marine agar were 4-5 mm in diameter, circular, convex, and beige, and had entire For the analysis of whole-cell fatty acids, cells were margins after cultivation for 4-5 days at 25"C on grown for 24h at 25"C on marine agar and were marine agar. MKT110r did not grow in temperatures analyzed by using the gas-chromatography-based Mi- above 37 "C or below 4 "C; the optimal temperature crobial Identification System (MIDI). Isoprenoid qui- range was 25 30'C. none was extracted with chloroform/methanol (2:1, v lv) They had an absolute requirement for sodium and purified by thin-layer chromatography (TLC) using chloride. Physiological characteristics that differentiate a mixture of n-hexane and diethyl ether (85:15, v/v) as strain MKTl10r from the closely related genera the solvent. The ubiquinone fraction was analyzed by Zooshikella are shown in Table 1. high-performance liquid chromatography (HPLC). Chemotaxonomic characteristics Genotypic analysis The isoprenoid quinones detected in the strain DNA was extracted using the method of Marmur [9]. MKT110r were Q-9 and Q-8 in the ratio of 9:2. The The G+C content of the DNA was determined by G+C content of the DNA of MKT110r was HPLC [12]. 50.4moloh. The cellular fatty acid composition of M. Kurahashi, A. Yokota / Systematic and Applied Microbiology 30 (2007) 202-206

Table 2. Cellular latty acid proflles of MKT110r and its closest phylogenetic neighbor

Fatty acids (Yo) of: 1 2

IC2o44"l tc2045

Hydroxy 3-OH 10:0 3.1 2.1+0.t 3.2+0.1 3-OH 12:0 2.8 5.1+0.4 5.5 +0.2 3-OH 12:1 Tr 0.8 +0.0 3-OH 14:0 4.1 + Saturated l0:0 1.0 2.6+0.1 3.1+0.1 l2:0 6.5 Tr 1.7 + 0.5 l4:0 9.3 6.0 + 0.1 5.4 + 0.5 15:0 0.7 16:0 18.9 13.9 + 0.9 29.5 + 1.0 18:0 0.8 0.7 +0.2 0.5 + 0.1 Saturated l6:ko7c 54.5 37.1+0.1 36.1 + 1.5 Fig. 1. Transmission electron micrograph showing the general 18:lat1c 5.5 14.5 + 0.5 13.2+0.6 morphology of negatively uranyl acetate-stained cells of strain MKTl10r. Bar,0.5pm. Taxa: 1, MKTl10r; 2, Zooshikella ganghwensis JC2044r and JC2045 (data lrom [14]). -, not detected; Tr, <0.5o/o.

Table 1. Comparison of phenotypic characteristics of Phylogenetic analysis MKT1 l0r and its closest phylogenetic neighbor A comparison of the 165 rRNA gene sequences of MKT110r and relative strain revealed that the newly Color of colonies Beige Yellowish-red or determined sequence was related to the 7-proteobacter- red ia. In the results of BLAST and FASTA searches, no Cell 0.4-0.6 x 1.8-2.2 0.74.9 x 1.5 2.5 sequence which exhibited more than 92oh similarity was Growth at/in found among the validly published names. The closest 37 "C + relatives of strain MKTll0r were found to be Zooshi- Oxidase + w kella g ang hw ensis J C2044r, O ceano spirillum linum LMG Decomposition of 2864^t, Microbulbifer salipaludis SM-lr, Marinobucter + Gelatin lipolyticus SM19r, Saccharospirillum impatiens DSM Esculin + 12546r, and Pseudomonas gessardi, CIP 105469r with Utilization of: Arginine + 9l.4oA, 90.90 , 90.6oh, 90.0oh, 89.9o/o, and 89.8% Ornithine + similarity, respectively. The phylogenetic position ol Fructose + the novel isolate based on the 165 rRNA gene sequence Ribose + was shown in Fig. 2. The phylogenetic tree indicated G*C content to.o 40-42 that strain MKTl10r constituted a sister clade of genus (mo1%) Zooshikella and the isolate formed a separate cluster Major fatty acids l6:la7c, 16;0, l6:lat7c, 76:0, with four clones (4B054136, ,4.8054161, AY700600, 14:0 l8:lat1c AY700601), as indicated by the 100t/, bootstrap value, creating novel The 165 rRNA gene Taxa: 1, MKT1l0r; 2, Zooshikella aanahwensis IC2044r and JC2045 thus a taxon. (data from [4]). +, positive reaction or growth; -, negative reaction or sequence similarity values between MKT1l0r and the no growth; w, weakly positive reaction. four clones (4B054136, 4.805416l, AY700600, and AY700601) were 96.9o/o, 96.8o , 95.6oh, and 95.2o/o, respectively. MKTll0r was dominated by l6:la7c (54.5%), 16:0 In a previous study, the genus Zooshikellq was (18.9%), and l4:0 (9.3%). The hydroxy fatty acids suggested to be a sister taxon to the gews Hahella detected in MKT110r were 3-OH 14:0 (4.1%),3-OH clade in a neighbor-joining tree based on nearly l0:0 (3.1%), and 3-OH l2:0 (2.8%). Table 2 compares complete 165 rRNA gene sequences [14]. However, this the fatty acid profile of strain MKTl 10r with that of the study showed that Zooshikella constituted a sister clade closely related genera Zooshikella. of strain MKTI l0r and the clade that contained both M. Kurahashi, A. Yokota / Systematic and Applied Microbiology 30 (2007) 202 206 20s

Alteromonas macleodii DSM 6062r (Y1 8228)

Hahella ganghwensis FR 1 050r (AY676463) Hahella chejuensis KCTC 2396r (AF195410) 100 uncultured marine bacterium (AJ31 5452) Zooshikella ganghwensis JC2O44r (AY1 30994)

uncultu red gamma proteobacterium HOC27 (AB054 1 6 1 ) uncultured gamma proteobacterium HOC2 (A8054136) Strain MKT1 10r (A8196667) uncultured bacterium (AY700601 ) uncultured bacterium (AY700600)

Saccharospirillum impatiens DSM 1 2546r (AJ31 5983)

Oceanospirillum beijerinckii IFO 1 5445r (A8006760) 100 Oceanospirillum maris IFO 1 5468r (A8006763) 'Oceanospirillum linum LMG 2864r (M22365)

Marinobacterrinobacter lipolyticus SM1SM 1 9r (AY1 47906) M ari nobacte r litoralis SW-45r (AF479689) Marinobacter hydrocarbonoclasticus ATCC 27 1 32r (A8021372) M icrobulbife r maiti m us TF -17r (AY377986) Microbulbifer salipaludis SM-1r (AF479688) M icrob u I b if e r h yd rol yti cus DSM 1'l 525r (AJ608704) r Pseudomonas argentinensis CH01 (AY691 1 88) Pseudomonas libanensis CIP 1 (AF057645) 0.02 100 05460r Pseudomonas gessardii CIP 1 05469r (AF074384)

Fig. 2. Neighbor-joining tree showing the phylogenetic positions of strain MKT110r and representatives of y-proteobacteria inferred from 165 rRNA gene sequence analyses. The percentage of 1000 bootstrap resamplings that support branching points above 50% confldence is indicated. Bar, 0.02 substitution per nucleotide position.

genera was more closely related to Oceanospirillum, yellowish-red or red. MKT110r cannot grow at a Microbulbifer, Marinobacter, Saccharospirillum and temperature of more than 37 "C, but Zooshikella can Pseudomonas than to the genus Hahella. The 165 rRNA grow at 45'C. The DNA G + C content of strain gene sequence similarity values between MKT1l0r and MKTl10r was 50.5 molo/o, while that of Zooshikellawas Hahella chejuensis KCTC 2396r or H. ganghwensis 4042moloh. 3-OH 14:0 of hydroxy fatty acid was FR1050r were only 88.8% and89.loh, respectively. present in the isolate MKTll0r but not in genus Zooshikella. These characteristics clearly distinguish the isolate and Zooshikella. IdKTllOr is significantly distinguishable from Oceanospirillum, Microbulbifer, Discussion Marinobacter, Saccharospirillum and Pseudomonas by the difference in fatty acid profile, the very low 165 The isolate MKTIl0r had <92o similarity with the rRNA gene sequence similarity, and the formation of a species that have validly published names. Neighbor- distinctive phylogenetic position. On the basis of the joining analyses placed MKT1l0r in the y-subclass of above data, MKTl10T can be assigned to a novel genus the proteobacteia, and the closest cultured bacterial in the y-proteobacteria from the point of view of both relatives were Zooshikella, O ceanospirillum, M icrobulbi- phenotype and genotype. We propose that straln fer, M ar ino b a c t e r, S a c c har o sp ir illum, arrd P s e udomonas. MKTllOr should be classified in a novel genus and Among them, Zooshikella was the most closely related species as Endozoicomonqs elysicola. with the isolate on the basis of phylogenetic analysis. Four clones (AB054136, AB054161, AY700600, The similarity between the 165 rRNA gene sequences of AY700601) showed a more thar,95.20/" 165 rRNA gene strain MKTll0r and Zooshikellawasgl.4o/o, which was sequence similarity value to MKTl10r and constituted a higher than the similarity between MKTl10r and any of novel lineage with MKTIl0r. The sequences of two of the other validly published names. The sister relation- the clones (AB054161, AB054136) were obtained from ship of strain MKTllOr to genus Zooshikella is the marine sponge Halichondoria okadai, and the supported with a bootstrap value of 88%o. However, sequences of the other two clones (AY700600, MKTll0r was clearly different from Zooshikella in AY700601) [2] were obtained from coral Pocillopora colony color, maximum growth temperature, G * C damicornis. MKT110r was also isolated from the marine content and hydroxy fatty acid profile. The colony color slug .E'. ornata. All the 165 rRNA gene sequences of MKTll0r is beige, while that of Zooshikella is constituted a new lineage derived from marine . 206 M. Kurahashi, A. Yokota / Systematic and Applied Microbiology 30 (2007) 202 206

It is interesting that almost all of the species of the Acknowledgments related genera Zooshikella, Oceanospirillum, Microbul- b ife r, M ar ino b ac t er, and S a c c har o sp ir illum were isolated This work was supported in part by a Grant-in-Aid from seawater, hypersaline lakes, ar,df or sea sediment - for Scientific Research (No. 17310135) from the i.e., from marine environments rather than marine Ministry of Education, Culture, Sports, Science, and animals. Technology of Japan.

Description of Endozoicomonas gen. noy. References

Endozoicomonas (En.do.zo.i.co.mo'na s. Gr. adj. endo, [1] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhatg, Z. inside; Gr. adj. zoicos, ; Gr. n. monas, a unit, Zharg, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: new generation of protein database search monad; N. L. fem. n. Endozoicomonas, monad living programs, Nucleic Acids Res. 25 (1997) 3389-3402. inside an animal). [2] D.G. Bourne, C.B. Munn, Diversity of bacteria asso- The colonies on marine agar medium are beige, ciated with the coral Pocillopora damicornis from circular, convex, smooth, and shiny. The cells are rod, the Great Barrier Reef, Environ. Microbiol. 7 (2005) Gram-negative, and motile. On marine agar, the strain tt62-n74. develops colonies with 3 days, and the colonies grow [3] J. Felsenstein, Confidence limits on phylogenies: an aerobically but not anaerobically. In media devoid of approach using the bootstrap, Evolution 39 (1985) sodium chloride, no growth is observed. They do not 783-791. form spores. The cells have numerous vesicles derived [4] A. Hiraishi, Direct automated sequencing of 165 rDNA from the cell surface. The optimal growth temperature is amplified by polymerase chain reaction from bacterial 25-30"C. Oxidase- and catalase-positive. The ubiqui- cultures without DNA purification, Lett. Appl. Micro- biol. 15 (1992) 210213. none system is The major cellular fatty acid Q-9. M. Kimura, A simple method for estimating evolutionary composition is l6:la7c, 16:0, 14:0 and the major [5] rates ofbase substitutions through comparative studies of acids 10:0, 3-OH hydroxy fatty are 3-OH l4:0, 3-OH nucleotide sequences, J. Mol. Evol. 16 (1980) 111 120. genus y-proteobacteria 12:0. The is affiliated with and [6] M. Kurahashi, A. Yokota, A preliminary report of contains one species, E. elysicola. The type species is phylogenetic diversity of bacterial strains isolated from E. elysicola. marine creatures, J. Gen. Appl. Microbiol. 48 (2002) 251 259. [7] M. Kurahashi, A. Yokota, Agariuorans albus gen nov., Description of E. elysicola sp. nov. sp. nov., a y-proteobacterium isolated from marine animals, Int. J. Syst. Evol. Microbiol. 54 (2004) 693 697. E. elysicola (e.ly.si.i.co'la. N. L. n. elysia, Elysia, [8] D.J. Lipman, W.R. Pearson, Rapid and sensitive protein name of zoological genus; L. suff. cola, dweller; N. L. n. similarity searches, Science 227 (1985) 1435-1441. elysiicola, Elysia dweller). [9] J. Marmur, A procedure for the isolation of deoxyribo- J. Mol. Biol. 3 (1961) In addition to the properties given in the genus nucleic acid from micro-organisms, 208118. description, the colonies on marine agar are 4-5 mm in W.R. Pearson, D.J. Lipman, Improved tools for biologi- diameter after 3-5 days incubation at 25 "C. No growth [0] cal sequence comparison, Proc. Natl. Acad. Sci. USA 85 occurs at temperatures above 37 "C or below 4 "C. (t988) 244+2448. Hydrolysis esculin is observed, and hydrolysis of of U U N. Saitou, M. Nei, The neighbor-joining method: a new gelatin is not observed. Nitrate is reduced to nitrite. method for reconstructing phylogenetic trees, Mol. Biol. B-Galactosidase, urease, acetoin, H2S, and indole are Evol. 4 (1987) 406/.25. not produced. Utilization of citrate is not observed. [12] J. Tamaoka, K. Komagata, Determination of DNA base Acids are produced from the following compounds: composition by reversed-phase high-.performance liquid glucose, galactose, N-acetyl-o-glucosamine, maltose, chromatography, FEMS Microbiol. Lett. 25 (1984) and gluconate. Acids are not produced from the 125-128. Higgins, T.J. Gibson, CLUSTAL following compounds: arginine, ornithine, arabinose, [13] J.D. Thompson, D.G. W: improving the sensitivity of progressive multiple ribose, rhamnose, mannose, mannitol, sorbitol, lactose, sequence aligment through sequence weighting, position- melibiose, saccharose, xylose, fructose, and gluconate. speciflc gap penalties and weight matrix choice, Nucleic The G+C content is 50.4mol%. The type strain is Acids Res. 22 (1994) 46734680. MKT110r (: IAM 15107r: KCTC 12372r), which U4l H. Yi, Y.H. Chang, H.W. Oh, K.S. Bae, J. Chun, was isolated from the gastrointestinal tract of the Zooshikella ganghwensis gen. nov., sp. nov., isolated from mollusk sea slug E. ornata at Miyake Island, Tokyo, tidal flat sediments,, Int. J. Syst. Evol. Microbiol. 53

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