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Lutibacter Holmesii Sp. Nov., a Marine Bacterium of the Family

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International Journal of Systematic and Evolutionary Microbiology (2015), 65, 3991–3996 DOI 10.1099/ijsem.0.000525 Lutibacter holmesii sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from the sea urchin Strongylocentrotus intermedius, and emended description of the genus Lutibacter Olga I. Nedashkovskaya,1,2 Stefanie Van Trappen,3 Natalia V. Zhukova2,4 and Paul De Vos3 Correspondence 1G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Olga I. Nedashkovskaya Academy of Sciences, Prospekt 100 Let Vladivostoku 159, 690022 Vladivostok, Russia [email protected] 2Far Easten Federal University, Sukhanova St. 8, 690091 Vladivostok, Russia or 3BCCM/LMG Bacteria Collection, and Laboratory of Microbiology, Ghent University, Ledeganck- [email protected] straat 35, B-9000 Ghent, Belgium 4A.V. Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of Science, Pal’chevskogo St. 17, 690032 Vladivostok, Russia Seven Gram-staining-negative, strictly aerobic, pale-yellow-pigmented, rod-shaped and non- motile strains were isolated from the sea urchin Strongylocentrotus intermedius collected from Troitsa Bay, Sea of Japan. Phylogenetic analyses based on 16S rRNA gene sequences showed that these isolates were affiliated with the family Flavobacteriaceae. The novel isolates showed 99.9–100 % 16S rRNA gene sequence similarity to each other and were closely related to the type strains of the recognized members of the genus Lutibacter with sequence similarities of 95.8–98.4 %. The G+C content of the genomic DNA was 35–36 mol%. DNA–DNA relatedness among the sea urchin isolates was 95–99 % and between strain KMM 6277T and its most closely related type strains, Lutibacter agarilyticus KCTC 23842T and Lutibacter litoralis JCM T 13034 , was 38 and 27 %, respectively. The prevalent fatty acids were iso-C15 : 0, anteiso-C15 : 0, summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1 v7c fatty acids), iso-C15 : 1 and C15 : 0. The polar lipid profile was composed of the phosphatidylethanolamine, one unknown aminolipid and one unknown lipid. The main respiratory isoprenoid quinone was MK-6.The results of phylogenetic, phenotypic and genotypic analyses indicated that the novel strains represent a novel species within the genus Lutibacter, for which the name Lutibacter holmesii sp.nov.is proposed. The type strain is KMM 6277T (5 CCUG 62221T 5 LMG 26737T). The genus Lutibacter was proposed to accommodate het- Choi et al., 2013). The type strains of Lutibacter agarilyticus erotrophic, aerobic, Gram-staining-negative, rod-shaped and Lutibacter oricola were isolated from shallow coastal and oxidase-negative marine bacteria within the family Fla- seawater (Park et al., 2013; Sung et al., 2015). In the vobacteriaceae (Choi & Cho, 2006). At the time of writing, course of a survey of bacterial diversity of the edible sea the genus Lutibacter comprises six species with validly pub- urchin Strongylocentrotus intermedius, seven heterotrophic, lished names. The type strain of the type species, Lutibacter aerobic, pale-yellow-pigmented strains were isolated. The litoralis, and those of Lutibacter maritimus, Lutibacter aes- results of phylogenetic analysis based on 16S rRNA gene tuarii and Lutibacter flavus were recovered from tidal flat sequencing revealed that the novel isolates belonged to sediments collected on the coast of the Korean peninsula the family Flavobacteriaceae and formed a distinct evol- (Choi & Cho, 2006; Park et al., 2010; Lee et al., 2012; utionary lineage within the genus Lutibacter. The present study aimed to report on the isolation and taxonomic identification of the novel strains using a polyphasic The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA approach. T gene sequences of Lutibacter holmesii KMM 6277 , KMM 6302, KMM T 6303, KMM 6305, KMM 6307, KMM 6308 and KMM 6311 are Strains KMM 6277 , KMM 6302, KMM 6303, KMM 6305, JQ241142, JQ241143, JQ241144, JQ241145, JQ241146, KMM 6307, KMM 6308 and KMM 6311 were isolated from JQ241147 and JQ241148, respectively. the sea urchin S. intermedius that was collected in Troitsa Downloaded from www.microbiologyresearch.org by 000525 G 2015 IUMS Printed in Great Britain 3991 IP: 157.193.5.232 On: Mon, 01 Aug 2016 09:46:04 O. I. Nedashkovskaya and others Bay, Gulf of Peter the Great, the Sea of Japan, as described closely related to L. agarilyticus KCTC 23842T and L. litor- previously (Nedashkovskaya et al., 2012). After primary alis JCM 13034T with 98.4 % and 97.3 % 16S rRNA gene isolation and purification, strains were cultivated at 28 8C sequence similarity, respectively, and shared less than on marine agar (Difco) and stored at 280 8C in marine 97 % sequence similarity to the type strains of other recog- broth (Difco) supplemented with 20 % (v/v) glycerol. nized species of the genus Lutibacter (Fig. 1). DNA extraction, PCR and 16S rRNA gene sequencing were DNA was isolated following the method of Marmur (1961) carried out as described by Vancanneyt et al. (2006). The and the DNA G+C content was determined by the thermal alignment of the 16S rRNA gene sequences obtained for denaturation method (Marmur & Doty, 1962). DNA–DNA the novel strains with those retrieved from GenBank, and hybridization experiments were performed spectrophotome- phylogenetic analysis and bootstrap analysis (1000 repli- trically using the initial renaturation rate method described by cates) were performed using the BioNumerics 5.1 software De Ley et al. (1970). The DNA G+C content of strains KMM package (Applied Maths). A phylogenetic tree was recon- 6277T, KMM 6302, KMM 6303, KMM 6305, KMM 6307, structed using the neighbour-joining method (Saitou & KMM 6308 and KMM 6311 ranged from 35.2 to Nei, 1987), a Jukes & Cantor (1969) correction was 36.1 mol%, which is in the range reported for the recognized applied, and unknown bases were discarded for the anal- species of the genus Lutibacter (Choi & Cho, 2006; Park et al., ysis. The phylogenetic position of our strains was deter- 2010; Lee et al., 2012). The strains under study shared mined from their almost-complete 16S rRNA gene 95–99 % DNA–DNA reassociation with each other, indicat- sequences. The phylogenetic analysis revealed that the ing that the sea urchin isolates belong to a single species novel strains were members of the family Flavobacteriaceae, (Wayne et al., 1987). DNA–DNA relatedness between strain phylum Bacteroidetes. They showed 99.9–100 % 16S rRNA KMM 6277T and its closest relatives, L. litoralis JCM 13034T gene sequence similarity to each other, demonstrating their and L. agarilyticus KCTC 23842T, was 27 and 38 %, respect- affiliation to the same species. Strain KMM 6277T was most ively, far below the threshold value of 70 % recommended 1% T Cellulophaga lytica ATCC 23178 (M62796) 100 Salegentibacter salegens DSM 5424T (M92279) 98 Fulvibacter tottoriensis MTT-39T (AB294107) Psychroserpens burtonensis ACAM 188T (U62913) DSW-5T (DQ004686) 100 Polaribacter dokdonensis T 100 Polaribacter filamentus 215 (U73726) 98 Polaribacter irgensii 23-PT (M61002) T 92 Tenacibaculum mesophilum MBIC1140 (AB032501) T 99 Tenacibaculum maritimum NBRC 15946 (AB078057) Maritimimonas rapanae A31T (EU290161) Namhaeicola litoreus DPG-25T (JN033800) 100 T 96 Aestuariicola saemankumensis SMK-142 (EU239499) Actibacter sediminis JC2129T (EF670651) Lutibacter oricola UDC379T (HM031974) Lutibacter holmesii KMM 6277T (JQ241142) 99 99 Lutibacter agarilyticus KYW566T (JN864028) 92 T 86 Lutibacter aestuarii MA-My1 (HM234096) T 81 Lutibacter litoralis CL-TF09 (AY962293) Lutibacter maritimus S7-2T (FJ598048) Lutibacter flavus IMCC 1507T (GU166749) Zobellia galactanivorans DsijT (AF208293) Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of Luti- bacter holmesii sp. nov. within the genus Lutibacter and family Flavobacteriaceae. It was based on comparison of approxi- mately 1374 nt. Bootstrap values ($70 %) based on 1000 replications are shown at the nodes of the tree. Zobellia galactanivorans DsijT (AF208293) was used as the outgroup. Bar, 0.01 substitutions per nucleotide position. Downloaded from www.microbiologyresearch.org by 3992 International Journal of Systematic and Evolutionary Microbiology 65 IP: 157.193.5.232 On: Mon, 01 Aug 2016 09:46:04 Description of Lutibacter holmesii sp. nov. Table 1. Cellular fatty acid content of strain KMM 6277T and In order to determine the fatty acid and polar lipid profiles, allied species of the genus Lutibacter strain KMM 6277T, L. agarilyticus KCTC 23842T, L. litoralis T T T T JCM 13034 , L. aestuarii CCUG 60022 and L. maritimus Species: 1, KMM 6277 ;2,L. litoralis JCM 13034 ;3,L. aestuarii CCUG 57524T were cultivated on marine agar for 3 days CCUG 60022T;4,L. agarilyticus KCTC 23842T;5,L. maritimus 8 T at 28 C, and the cell mass was harvested during the expo- CCUG 57524 . Data are percentages of total fatty acids. Fatty acids nential growth phase. Fatty acid methyl esters of strain , amounting to 1 % of the total fatty acids in all strains are not KMM 6277T and the reference strains were prepared listed. The strains were grown for 3 days on marine agar 2216. TR, according to the methods described by Sasser (1990) , Traces ( 1 %); –, not detected. All data are from this study. using the standard protocol of the Sherlock Microbial Fatty acid 1 2345 Identification System (version 6.0, MIDI) and analysed on a Shimadzu GC-21A chromatograph equipped with a Branched fatty acids fused-silica capillary column (30 m60.25 mm) coated iso-C14 : 0 1.7 3.9 7.9 8.9 1.8 with Supercowax-10 and SPB-5 phases (Supelco) at iso-C15 : 0 26.2 17.3 23.8 15.5 32.2 210 8C. Cellular fatty acids were identified by using equiv- anteiso-C15 : 0 25.5 11.4 4.1 10.6 5.6 alent chain-length measurements and comparing the reten- iso-C15 : 1 5.1 6.7 6.3 2.4 6.3 tion times with those of authentic standards.
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