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Portibacter Lacus Gen. Nov., Sp. Nov., a New Member of the Family Saprospiraceae Isolated from a Saline Lake

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Portibacter Lacus Gen. Nov., Sp. Nov., a New Member of the Family Saprospiraceae Isolated from a Saline Lake J. Gen. Appl. Microbiol., 58, 191‒197 (2012) Full Paper Portibacter lacus gen. nov., sp. nov., a new member of the family Saprospiraceae 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 bacteria representing a new genus and species, 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 Bacteroidetes 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 taxonomy; 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, Aureispira 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, Aureispira marina 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).
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