J. Gen. Appl. Microbiol., 58, 191‒197 (2012) Full Paper

Portibacter lacus gen. nov., sp. nov., a new member of the family isolated from a saline lake

Jaewoo Yoon,1 Yoshihide Matsuo,2,† Hiroaki Kasai,2,†† and Akira Yokota3,†††

1 College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704‒701, Republic of Korea 2 Marine Biotechnology Institute Co., Ltd., Kamaishi, Iwate 026‒0001, Japan 3 Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113‒0032, Japan

(Received December 13, 2011; Accepted February 2, 2012)

A strictly aerobic, Gram-negative, orange-pigmented, rod-shaped, non-motile and chemo- heterotrophic representing a new genus and , designated YM8-076T, was iso- lated from lake water collected at a harbor on Lake Notoro, Hokkaido, Japan. Preliminary analy- sis based on the 16S rRNA gene sequence revealed that the novel isolate could be affi liated with the family Saprospiraceae of the phylum and that it showed highest sequence similarity (88.5%) to Haliscomenobacter hydrossis ATCC 27775T. The strain could be differenti- ated phenotypically from recognized members of the family Saprospiraceae. The G+C content of DNA was 53.7 mol%, MK-7 was the major menaquinone and iso-C15：0, iso-C15：1 and iso- C17：0 3-OH were the major cellular fatty acids. On the basis of polyphasic taxonomic studies, it was concluded that strain YM8-076T represents a new genus and species of the family Sapros- piraceae. We propose the name Portibacter lacus gen. nov., sp. nov. for this strain; its type strain is YM8-076T (=KCTC 23747T=NBRC 108769T).

Key Words—phylogenetic analysis; polyphasic ; Portibacter lacus gen. nov., sp. nov.; sa- line lake; 16S rRNA gene

Introduction

Bacterial members of the phylum Bacteroidetes (Ludwig and Klenk, 2001) previously known as the * Address reprint requests to: Dr. Jaewoo Yoon, College of Cytophaga-Flavobacterium-Bacteroides (CFB) group, Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo- Gu, Daegu 704‒701, Republic of Korea. are distributed throughout a wide range of ecological Tel: +82‒53‒580‒6648 Fax: +82‒53‒580‒6645 systems such as soil and fresh marine water (Bowman E-mail: [email protected] et al., 1997; DeLong et al., 1993; Glöckner et al., 1999; Present address: O’Sullivan et al., 2002). Representatives of the phylum † Suntory Holdings Limited, R&D Planning Division, 1‒1‒1 Bacteroidetes are characterized by the capability to Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618‒8503, decompose numerous organic biomacromolecules Japan. (Cottrell and Kirchman, 2000) and planktonic blooms †† Marine Biosciences Kamaishi Research Laboratory, Ki- (Brettar et al., 2004); therefore, they are considered to tasato University, 3‒75‒1 Heita, Kamaishi, Iwate 026‒0001, Ja- pan. be important members of the bacterial community in- ††† Department of Biology, Faculty of Mathematics and Natu- volved in ecophysiological activities in a variety of nat- ral Sciences, University of Indonesia, Kampus UI Depok 16424, ural environments. In the second edition of Bergey’s Indonesia. Manual of Systematic Bacteriology (Vol. 4), the family 192 YOON et al. Vol. 58

Saprospiraceae was proposed within this phylum NaCl concentration for growth was determined on MA (Family III. Saprospiraceae fam. nov.). It includes the containing 0‒10% (w/v) NaCl (Atlas, 1993). Gram- genera Saprospira (type genus), Haliscomenobacter, staining was performed as described by Murray et al. Lewinella, and ‘Rubidimonas’. The genera (1994). Spore formation was determined by staining Saprospira, Haliscomenobacter and ‘Rubidimonas’ with malachite green. Anaerobic growth was tested for each accommodate one species, Saprospira grandis up to 2 weeks on MA in a jar containing AnaeroPack- (Reichenbach, 1989), Haliscomenobacter hydrossis Anaero (Mitsubishi Gas Chemical Co., Inc.), which

(van Veen et al., 1973) and ‘Rubidimonas crustatorum’ works as an O2 absorber and CO2 generator. Catalase (Yoon et al., 2011) and the genus Lewinella accom- activity was determined by bubble formation in a 3% modates seven species, Lewinella agarilytica (Lee, H2O2 solution. Oxidase activity was determined using 2007), Lewinella antarctica (Oh et al., 2009), Lewinella cytochrome oxidase paper (Nissui Pharmaceutical cohaerens (Khan et al., 2007), Lewinella lutea (Khan et Co., Inc.). Degradation of DNA was tested using DNase al., 2007), Lewinella marina (Khan et al., 2007), Lewi- agar (Scharlau), with DNase activity detected by fl ood- nella nigricans (Khan et al., 2007) and Lewinella per- ing plates with 1 M HCl. Starch hydrolysis were tested sica (Khan et al., 2007). The genus Aureispira (Hosoya as described by Choi et al. (2007). The ability to hy- et al., 2006) comprises two species, drolyze casein, Tween 20, Tween 80 and tyrosine was (Hosoya et al., 2006) and Aureispira maritima (Hosoya determined according to Hansen and Sørheim (1991). et al., 2007). Currently, a classifi cation of members API 20E, API 50CH and API ZYM strips (bioMérieux) within the phylum Bacteroidetes is still in a state of de- were used to determine the biochemical characteris- velopment. For a more comprehensive investigation of tics. All suspension media for the API test strips were the family Saprospiraceae, it is recommended that supplemented with 0.85% (w/v) NaCl solution (fi nal many species of the family Saprospiraceae that thrive concentration). API 20E and API 50CH test strips were in a wide range of terrestrial, aquatic and marine habi- read after 72 h of incubation at 30°C and API ZYM test tats should be fi rst isolated and taxonomically studied. strips were read after 4 h incubation at 37°C. Flexiru- In this study, we performed a polyphasic taxonomic bin-type pigments were investigated by using the characterization of a new marine bacterial member bathochromatic shift test with a 20% (w/v) KOH solu- (strain YM8-076T) isolated from a saline lake in Japan. tion (Bernardet et al., 2002). Determination of G+C content of DNA, 16S rRNA Materials and Methods gene sequencing and phylogenetic analysis. The ge- nomic DNA was prepared according to the method of Isolation of bacterial strain and culture conditions. Marmur (1961) from cells grown on MA and the DNA Strain YM8-076T was isolated from lake water (salinity; base composition was determined by using the HPLC 34‰, pH 6.9) collected at a harbor on Lake Notoro, method of Mesbah et al. (1989). An approximately Hokkaido, Japan. Fifty microliters of the sample was 1,500 bp fragment of the 16S rRNA gene was ampli- plated onto medium ‘P’ (Yoon et al., 2007). After a fi ed from the extracted DNA by using the bacterial uni- 1-month incubation at room temperature, an orange- versal primers: 27F and 1492R (Escherichia coli num- colored colony was subcultured to purify on a marine bering system; Weisburg et al., 1991). To ascertain the agar 2216 (Difco) plate and was used in this study. The phylogenetic position of the novel isolate, the 16S strain was routinely cultured on marine agar 2216 (MA) rRNA gene sequence of strain YM8-076T (GenBank/ at 25°C and maintained in marine broth 2216 supple- EMBL/DDBJ accession number AB675658) was com- mented with 20% (v/v) glycerol at -70°C. pared with sequences obtained from GenBank (Na- Morphological, physiological and biochemical anal- tional Center for Biotechnology Information, http:// ysis. Cell morphology was observed using light mi- www.ncbi.nlm.nih.gov). Multiple alignments of the se- croscopy (BX60; Olympus) and fl agella staining was quences were performed using CLUSTAL_X (version carried out according to Blenden and Goldberg (1965). 1.83) (Thompson et al., 1997). Alignment gaps and Gliding motility was determined by using a semisolid ambiguous bases were not taken into consideration medium as described by Perry (1973). The tempera- when 1,319 bases of the 16S rRNA gene were com- ture (4‒45°C) and pH (5‒10) ranges for growth were pared. Aligned sequences were analyzed by MEGA 4 determined by incubating the isolates on MA. The software (Tamura et al., 2007). The evolutionary dis- 2012 Portibacter lacus gen. nov., sp. nov. 193 tances [distance options according to the Kimura two- has the highest sequence similarity (88.5%) to Halis- parameter model (Kimura, 1983)] and clustering with comenobacter hydrossis ATCC 27775T, followed by the neighbor-joining (Saitou and Nei, 1987) and maxi- Lewinella cohaerens II-2T (86.6%), Lewinella nigricans mum-parsimony (Fitch, 1971) methods were deter- SS-2T (85%), and Lewinella antarctica IMCC3223T mined by using bootstrap values based on 1,000 rep- (84.3%). Sequence similarity was less than 84% with lications (Felsenstein, 1985). The similarity values were all other members of the family Saprospiraceae with calculated using the same software. validly published names. Based on the 16S rRNA gene Chemotaxonomic analysis. Gas chromatography sequences’ divergence, strain YM8-076T is determined analysis of the cellular fatty acid methyl esters was to be related to the family Saprospiraceae. Thus, on performed using a culture grown on MA at 37°C for 4 the basis of phylogenetic data presented, strain YM8- days. Fatty acid methyl esters were extracted and pre- 076T should be classifi ed as a novel genus and new pared according to standard protocols provided by species of the family Saprospiraceae within the phy- the MIDI/Hewlett Packard Microbial Identifi cation Sys- lum Bacteroidetes. tem (MIDI, Inc., 1999; Sasser, 1990). Determination of the respiratory quinone system was carried out as de- Morphological, physiological and biochemical charac- scribed previously (Xie and Yokota, 2003). teristics Nucleotide sequence accession number. The Gen- Cells were mostly straight and rod-shaped. Cells Bank/EMBL/DDBJ accession number for 16S rRNA varied between 0.3 and 0.5 μm in diameter and 3.5 gene sequence of strain YM8-076T is AB675658. and 5.5 μm in length. Gliding motility was not observed and fl exirubin-type pigments were not produced (Table Results and Discussion 1). Strain YM8-076T also showed distinct phenotypic, physiological and biochemical features that discrimi- Molecular phylogenetic analysis nated it from the closest described members in the Phylogenetic analysis based on the 16S rRNA gene family Saprospiraceae as shown in Table 1. sequences revealed that the strain YM8-076T belonged to the family Saprospiraceae within the phylum Bacte- Chemotaxonomic characteristics roidetes (Fig. 1). Analysis of the 16S rRNA gene se- As shown in Table 2, the predominant cellular fatty quences showed that the sequence of strain YM8-076T acids of strain YM8-076T were iso-C15：0 (45.3%), iso-

Fig. 1. Neighbor-joining tree of 16S rRNA gene sequence similarity, showing the phyloge- netic position of strain YM8-076T and representatives of the family Saprospiraceae. The sequence of Flammeovirga aprica NBRC 15941T was used as an outgroup. The sequence determined in this study is shown in bold. Bootstrap values for both neighbor-joining (above nodes) and maximum-parsimony (below nodes) are shown. Bar, 2% sequence divergence. 194 YOON et al. Vol. 58

Table 1. Differential characteristics of strain YM8-078T and other closely related taxa.

Characterisitic 1 2 34567 Short to Short to Short to Flexible rods Short to Cell shape Rods Short rods elongated rods elongated rods elongated rods or fi laments elongated rods Colony color Orange Reddish orange Light orange Black Dark orange Light orange Orange Gliding motility -----+ - Oxidase + - ++++- Flexirubin-type pigments ------NaCl range for growth (%) 1‒81‒72‒42‒42‒4 0.5‒1 0.5‒5 Temperature range for growth (°C) 15‒40 10‒40 10‒30 15‒37 4‒30 4‒37 3‒25 Hydrolysis of: Casein --++++- DNA --++++ND Starch - ++++++ DNA G+C content (mol%) 53.7 55.3 44.9 53.1 52.6 51.3 50.3

Characterisitic 8 9 10 11 12 13 Short to Short to Flexible helical Flexible helical Flexible helical Cell shape Thin rods elongated rods elongated rods fi laments fi laments fi laments Colony color Dark orange Dull orange Pink Yellowish orange Yellow Orange-red Gliding motility --++++ Oxidase + + ND v v ND Flexirubin-type pigments --+ --- NaCl range for growth (%) 2‒41‒7NDNDNDND Temperature range for growth (°C) 10‒35 4‒42 8‒30 25‒30 25‒30 30‒37 Hydrolysis of: Casein + + ND + + + DNA + + ND - +ND Starch + + + --- DNA G+C content (mol%) 56 61 49 38‒39 38.7 46‒48

Strains: 1, YM8-076T (Portibacter lacus gen. nov., sp. nov.; present study); 2, ‘Rubidimonas crustatorum’ AK17-053T (Yoon et al., 2011); 3, Lewinella cohaerens II-2T (Khan et al., 2007); 4, Lewinella nigricans SS-2T (Khan et al., 2007); 5, Lewinella persica T- 3 T (Khan et al., 2007); 6, Lewinella agarilytica SST-19T (Lee, 2007); 7, Lewinella antarctica IMCC3223T (Oh et al., 2009); 8, Lewinella lutea FYK2402M69T (Khan et al., 2007); 9, Lewinella marina MKG-38T (Khan et al., 2007); 10, Haliscomenobacter hydrossis ATCC 27775T (van Veen et al., 1973; Mulder, 1989); 11, Aureispira marina 24T (Hosoya et al., 2006); 12, Aureispira maritima 59SAT (Hosoya et al., 2007); 13, Saprospira grandis ATCC 23119T (Reichenbach, 1989). Symbols: +, positive; -, negative; v, variable; ND, no data.

C15：1 (11.9%) and iso-C17：0 3-OH (17.2%). On the as a novel species within a new genus, Portibacter basis of the fatty acid composition, strain YM8-076T lacus gen. nov., sp. nov. can be differentiated from recognized species of the genera Saprospira, Haliscomenobacter, Lewinella, Au- Description of Portibacter gen. nov. reispira and ‘Rubidimonas’ as shown in Table 2. These Portibacter (Por.ti.bac’ter. L. n. portus, a harbor, ha- results also suggest that strain YM8-076T represents ven, port; N.L. masc. n. bacter, a rod; N.L. masc. n. an independent genus of the family Saprospiraceae. Portibacter, a rod isolated from a harbor). Cells are rod-shaped, Gram-negative and strictly Polyphasic taxonomic conclusion aerobic. Do not form endospores. Catalase- and oxi- From the distinct phylogenetic position and combi- dase-positive. Flexirubin-type pigments are absent. nations of genotypic and phenotypic characteristics, The major respiratory quinone is menaquinone 7 (MK- strain YM8-076T cannot be assigned to any previously 7). The G+C content of the genomic DNA of the type recognized bacterial genus and thus can be described strain is 53.7 mol%. The predominant cellular fatty ac- 2012 Portibacter lacus gen. nov., sp. nov. 195

Table 2. Comparison of cellular fatty acids for strain YM8-078T and other closely related taxa.

Fatty acid 1 2 3 4 5 6 7 8 9 10 11 12 13 C14：0 ̶ 1.3 ̶̶̶ 1.3 tr ̶̶ tr ̶̶̶ C15：0 2.2 1.113 6trtrtrtrtrtr̶̶̶ C16：0 tr 4.3 tr 7 2 10.9 2.3 3 4 5.7 16.9 25.6 ̶ C16：1 ̶̶̶̶̶̶̶̶̶ 17.3 ̶̶̶ C18：0 ̶ 6.2 ̶̶̶13.5 ̶̶̶ 6.9 ̶̶̶ iso-C13：0 ̶ tr ̶̶̶̶̶̶̶ tr ̶̶ 1.4 iso-C15：0 45.3 37.1 30 32 28 11.1 15.9 16 22 21.1 12.2 3 43.3 iso-C15：1 11.9 1.7 29 19 4 ̶̶ 2 3̶̶̶̶ iso-C16：0 ̶̶ 3 3 tr 3.2̶̶̶ 2.8 8.8 3.7 ̶ iso-C16：1 ̶̶ 1 ̶ tr ̶̶ 1 ̶̶̶̶̶ iso-C17：0 ̶̶ 2trtr̶̶ 1 4 1 25.8 7.1 ̶ anteiso-C15：0 ̶ 3.5 ̶̶̶ tr ̶̶̶̶̶̶̶ C15：1ω6c 1.5 7.7 ̶̶ 2 2.4 8.9 2 1 ̶̶̶̶ C15：1ω8c ̶̶̶̶ 2 ̶ 2 ̶̶̶̶̶̶ C16：1ω5c ̶ tr 2 ̶ tr ̶̶̶̶̶̶̶̶ C16：1ω7c / iso-C15：0 2-OH ̶ 18.7 tr 9 30 24.1 39.4 53 ̶ 15.5 ̶̶ 4.1 C17：1ω6c ̶̶ tr tr 5 2.6 ̶ 2 3̶̶̶̶ C17：1ω7c ̶̶̶̶̶̶ 10.5 ̶̶̶̶̶̶ C20：4ω6c ̶̶̶̶̶̶̶̶̶̶46.3 43.6 ̶ iso-C17：1ω9c ̶̶ tr 2 10 tr 2.6 9 23 ̶̶̶̶ C16：0 3-OH ̶ 2.1 ̶̶̶ 1.3 tr ̶̶̶̶ tr 3.8 iso-C11：0 3-OH ̶ 2.7 3 ̶̶̶̶̶̶̶̶̶̶ iso-C15：0 3-OH 2.7 tr 3 2 2 ̶̶ 4 2 22.8 ̶̶̶ iso-C16：0 3-OH 2.6 ̶̶̶̶̶̶̶̶̶ 1.5 ̶̶ iso-C17：0 3-OH 17.2 7.1 3 6 5 ̶̶ 4 6 tr 4.3 1.5 24.8

Strains: 1, YM8-076T (Portibacter lacus gen. nov., sp. nov.; present study); 2, ‘Rubidimonas crustatorum’ AK17-053T (Yoon et al., 2011); 3, Lewinella cohaerens II-2T (Khan et al., 2007); 4, Lewinella nigricans SS-2T (Khan et al., 2007); 5, Lewinella persica T- 3 T (Khan et al., 2007); 6, Lewinella agarilytica SST-19T (Lee, 2007); 7, Lewinella antarctica IMCC3223T (Oh et al., 2009); 8, Lewinella lutea FYK2402M69T (Khan et al., 2007); 9, Lewinella marina MKG-38T (Khan et al., 2007); 10, Haliscomenobacter hydrossis ATCC 27775T (Kämpfer, 1995); 11, Aureispira marina 24T (Hosoya et al., 2006); 12, Aureispira maritima 59SAT (Hosoya et al., 2007); 13, Saprospira grandis ATCC 23119T (Reichenbach, 1989). Values are percentages of total fatty acids; -, not detected; tr, traces (<1%).

ids are iso-C15：0, iso-C15：1 and iso-C17：0 3-OH. ter, circular, shiny with entire edges and orange-pig- A member of the family Saprospiraceae, phylum Bacte- mented, becoming reddish-orange after 1 week of roidetes, according to 16S rRNA gene sequence anal- incubation. The temperature range for growth is yses. 15‒40°C, the optimal temperature is between 30 and The type species is Portibacter lacus. 37°C but no growth occurs at 4 or 45°C. The pH range for growth is pH 6‒10 (optimum, pH 7), no growth is observed below pH 5 or above pH 11. NaCl is required Description of Portibacter lacus sp. nov. for growth and can be tolerated at a concentration of Portibacter lacus (la’cus. L. gen. n. lacus, of/from a up to 8% (w/v). No growth occurs in the presence of lake). 9% (w/v) NaCl. Nitrate and nitrite reduction are nega- The main characteristics are the same as those giv- tive. Agar, casein, DNA, tyrosine, Tweens 20 and 80, en for the genus. In addition, cells are rods 0.3‒0.5 μm starch and urea are not hydrolyzed but gelatin is hydro- in diameter and 3.5‒5.5 μm in length. Cells lack fl a- lyzed. The o-nitrophenyl-β-D-galactosidase (ONPG), gella and are non-motile. Gliding motility is not ob- arginine dihydrolase and lysine decarboxylase test are served. Colonies grown on MA are 1‒2 mm in diame- positive but the reactions for citrate utilization, Voges- 196 YOON et al. Vol. 58

Proskauer, ornithine decarboxylase, hydrogen sulfi de cum gen. nov., sp. nov., a novel marine bacterium of the and indole production are negative (API 20E). In the Cytophaga-Flavobacterium-Bacteroides group isolated API 50CH strip, acid is produced from esculin ferric from surface water of the central Baltic Sea. Int. J. Syst. Evol. Microbiol., 54, 2335‒ 2341. citrate, D-arabinose, L-arabinose, D-xylose, L-xylose, Choi, J. H., Im, W. T., Liu, Q. M., Yoo, J. S., Shin, J. H., Rhee, S. methyl-β-D-xylopyranoside, galactose, glucose, and K., and Roh, D. H. (2007) Planococcus donghaensis sp. α fructose but not from mannose, rhamnose, methyl- - nov., a starch-degrading bacterium isolated from the East α D-mannopyranoside, methyl- -D-glucopyranoside, N- Sea, South Korea. Int. J. Syst. Evol. Microbiol., 57, 2645‒ acetyl-glucosamine, amygdalin, arbutin, salicin, cello- 2650. biose, maltose, lactose, melibiose, sucrose, trehalose, Cottrell, M. T. and Kirchman, D. L. (2000) Community composi- melezitose, D-turanose, D-lyxose, D-tagatose, D-fucose, tion of marine bacterioplankton determined by 16S rRNA L-fucose, inulin, ribose, raffi nose, gentiobiose, glycer- gene clone libraries and fl uorescence in situ hybridization. ol, erythritol, adonitol, sorbose, dulcitol, inositol, man- Appl. Environ. Microbiol., 66, 5116‒ 5122. DeLong, E. F., Franks, D. G., and Alldredge, A. L. (1993) Phylo- nitol, sorbitol, starch, glycogen, xylitol, D-arabitol, L- genetic diversity of aggregate-attached vs. free-living ma- arabitol, gluconate, 2-keto-gluconate or 5-keto- rine bacterial assemblages. Limnol. Oceanogr., 38, 924‒ gluconate. In the API ZYM strip, alkaline phosphatase, 934. leucine arylamidase, valine arylamidase, trypsin, and Felsenstein, J. (1985) Confi dence limits on phylogenies: An ap- naphthol-AS-BI-phosphohydrolase are positive but proach using the bootstrap. Evolution, 39, 783‒ 791. acid phosphatase, esterase (C4), esterase lipase (C8), Fitch, W. M. (1971) Towards defi ning the course of evolution: lipase (C4), cystein arylamidase, chymotrypsin, Minimum change for a specifi c tree topology. Syst. Zool., α-galactosidase, β-galactosidase, β-glucuronidase, 20, 406‒ 416. Glöckner, F. O., Fuchs, B. M., and Amann, R. (1999) Bacterio- α-glucosidase, β-glucosidase, N-acetyl-β-glucosa- plankton composition of lakes and oceans: A fi rst compari- minidase, α-mannosidase and α-fucosidase are nega- son based on fl uorescence in situ hybridization. Appl. Envi- tive. The major (＞5.0%) fatty acids are iso-C15：0, ron. Microbiol., 65, 3721‒ 3726. iso-C15：1 and iso-C17：0 3-OH. The G+C of the ge- Hansen, G. H. and Sørheim, R. (1991) Improved method for nomic DNA is 53.7 mol%. phenotypical characterization of marine bacteria. J. Micro- The type strain is YM8-076T (=KCTC 23747T=NBRC biol. Methods, 13, 231‒ 241. 108769T), which was isolated from lake water collected Hosoya, S., Arunpairojana, V., Suwannachart, C., Kanjana- at a harbor on Lake Notoro, Hokkaido, Japan. Opas, A., and Yokota A. (2006) Aureispira marina gen. nov., sp. nov., a gliding, arachidonic acid-containing bacterium isolated from the southern coastline of Thailand. Int. J. Syst. Acknowledgments Evol. Microbiol., 56, 2931‒ 2935. Hosoya, S., Arunpairojana, V., Suwannachart, C., Kanjana- The present research has been conducted by the Settlement Opas, A., and Yokota, A. (2007) Aureispira maritima sp. Research Grant of Keimyung University in 2011. nov., isolated from marine barnacle debris. Int. J. Syst. Evol. Microbiol., 57, 1948‒ 1951. References Kämpfer, P. (1995) Physiological and chemotaxonomic charac- terization of filamentous bacteria belonging to the genus Atlas, R. M. (1993) Handbook of Microbiological Media, ed. by Haliscomenobacter. Syst. Appl. Microbiol., 181, 363‒ 367. Parks, L.C., CRC, Boca Raton. Khan, S. T., Fukunaga, Y., Nakagawa, Y., and Harayama, S. Bernardet, J. F., Nakagawa, Y., and Holmes, B. (2002) Proposed (2007) Emended descriptions of the genus Lewinella and minimal standards for describing new taxa of the family Fla- of Lewinella cohaerens, Lewinella nigricans and Lewinella vobacteriaceae and emended description of the family. Int. persica, and description of Lewinella lutea sp. nov. and J. Syst. Evol. Microbiol., 52, 1049‒ 1070. Lewinella marina sp. nov. Int. J. Syst. Evol. Microbiol., 57, Blenden, D. C. and Goldberg, H. S. (1965) Silver impregnation 2946‒ 2951. stain for Leptospira and fl agella. J. Bacteriol., 89, 899‒ Kimura, M. (1983) The Neutral Theory of Molecular Evolution, 900. Cambridge University Press, Cambridge. Bowman, J. P., McCammon, S. A., Brown, M. V., Nichols, D. S., Lee, S. D. (2007) Lewinella agarilytica sp. nov., a novel marine and McMeekin, T. A. (1997) Diversity and association of bacterium of the phylum Bacteroidetes, isolated from psychrophilic bacteria in Antarctic sea ice. Appl. Environ. beach sediment. Int. J. Syst. Evol. Microbiol., 57, 2814‒ Microbiol., 63, 3068‒ 3078. 2818. Brettar, I., Christen, R., and Höfl e, M. G. (2004) Aquifl exum balti- Ludwig, W. and Klenk, H. P. (2001) Overview: A phylogenetic 2012 Portibacter lacus gen. nov., sp. nov. 197

backbone and taxonomic framework for procaryotic sys- Saitou, N. and Nei, M. (1987) The neighbor-joining method: A tematics. In Bergey’s Manual of Systematic Bacteriology, new method for reconstructing phylogenetic trees. Mol. 2nd ed., Vol. 1, ed. by Boone, D. R., Castenholz, R. W., and Biol. Evol., 4, 406‒ 425. Garrity, G. M.. Springer, New York, pp. 49‒ 66. Sasser, M. (1990) Identifi cation of bacteria by gas chromatogra- Marmur, J. (1961) A procedure for the isolation of deoxyribo- phy of cellular fatty acids. MIDI Technical Note 101, MIDI, nucleic acid from micro-organisms. J. Mol. Biol., 3, 208‒ Inc., Newark, DE. 218. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Mesbah, M., Premachandran, U., and Whitman, W. B. (1989) Molecular evolutionary genetics analysis (MEGA) software Precise measurement of the G+C content of deoxyribo- version 4.0. Mol. Biol. Evol., 24, 1596‒ 1599. nucleic acid by high-performance liquid chromatography. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Int. J. Syst. Bacteriol., 39, 159‒ 167. and Higgins, D. G. (1997) The CLUSTAL_X windows inter- MIDI, Inc. (1999) Sherlock Microbial Identifi cation System, Op- face: Flexible strategies for multiple sequence alignment erating Manual, version 3.0, MIDI, Inc., Newark, DE. aided by quality analysis tools. Nucleic Acids Res., 25, Mulder, E. G. (1989) Genus Haliscomenobacter van Veen, van 4876‒ 4882. der Kooy, Geuze and van der Vlies 1973. In Bergey’s Man- van Veen, W. L., van der Kooij, D., Geuze, E. C. W. A., and van ual of Systematic Bacteriology, Vol. 3, ed. by Staley, J. T., der Vlies, A. W. (1973) Investigations on the sheathed bac- Bryant, M. P., Pfennig, N., Holt, J. G., Williams & Wilkins, terium Haliscomenobacter hydrossis gen. n., sp. n., isolat- Baltimore, pp. 2003‒ 2004. ed from activated sludge. Antonie van Leeuwenhoek, 39, Murray, R. G. E., Doetsch, R. N., and Robinow, C. F. (1994) De- 207‒ 216. terminative and cytological light microscopy. In Methods Weisburg, W. G., Barns, S. M., Pelletier, D. A., and Lane, D. J. for General and Molecular Bacteriology, ed. by Gerhardt, (1991) 16S ribosomal DNA amplifi cation for phylogenetic P., Murray, R. G. E., Wood, W. A., and Krieg, N. R., Ameri- study. J. Bacteriol., 173, 697‒ 703. can Society for Microbiology, Washington, DC, pp. 21‒ 41. Xie, C. and Yokota, A. (2003) Phylogenetic analysis of Lampro- Oh, H. M., Lee, K., and Cho, J. C. (2009) Lewinella antarctica pedia hyalina based on the 16S rRNA gene sequence. J. sp. nov., a marine bacterium isolated from Antarctic seawa- Gen. Appl. Microbiol., 49, 345‒ 349. ter. Int. J. Syst. Evol. Microbiol., 59, 65‒ 68. Yoon, J., Katsuta, A., and Kasai, H. (2011) Rubidimonas crusta- O’Sullivan, L. A., Weightman, A. J., and Fry, J. C. (2002) New torum gen. nov., sp. nov., a novel member of the family degenerate Cytophaga-Flexibacter-Bacteroides-specific Saprospiraceae isolated from a marine crustacean. Antonie 16S ribosomal DNA-targeted oligonucleotide probes re- van Leeuwenhoek, DOI 10.1007/s10482-011-9653-3. veal high bacterial diversity in River Taff epilithon. Appl. En- Yoon, J., Yasumoto-Hirose, M., Matsuo, Y., Nozawa, M., Matsu- viron. Microbiol., 68, 201‒ 210. da, S., Kasai, H., and Yokota, A. (2007) Pelagicoccus mo- Perry, L. B. (1973) Gliding motility in some non-spreading fl exi- bilis gen. nov., sp. nov., Pelagicoccus albus sp. nov. and bacteria. J. Appl. Microbiol., 36, 227‒ 232. Pelagicoccus litoralis sp. nov., three novel members of Reichenbach, H. (1989) Genus Saprospira Gross 1911. In subdivision 4 within the phylum ‘Verrucomicrobia,’ isolated Bergey’s Manual of Systematic Bacteriology, Vol. 3, ed. by from seawater by in situ cultivation. Int. J. Syst. Evol. Micro- Staley, J. T., Bryant, M. P., Pfennig, N., and Holt, J. G.. Wil- biol., 57, 1377‒ 1385. liams & Wilkins, Baltimore, pp. 2078‒ 2082.