International Journal of Systematic and Evolutionary Microbiology (2013), 63, 1143–1148 DOI 10.1099/ijs.0.030254-0 Phaeocystidibacter luteus gen. nov., sp. nov., a member of the family Cryomorphaceae isolated from the marine alga Phaeocystis globosa, and emended description of Owenweeksia hongkongensis Yanyan Zhou,13 Jianqiang Su,1,23 Qiliang Lai,1,3 Xinyi Li,1 Xiaoru Yang,1,2 Peiyan Dong1 and Tianling Zheng1 Correspondence 1State Key Laboratory of Marine Environmental Science and Key Laboratory of the MOE for Tianling Zheng Coastal and Wetland Ecosystems, School of Life Sciences, Xiamen University, Xiamen 361005, [email protected] PR China 2The Institute of Urban Environment (IUE), Chinese Academy of Sciences, Xiamen 361005, PR China 3Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, PR China A taxonomic study was carried out on strain PG2S01T, isolated from a culture of Phaeocystis globosa, a haemolytic, toxin-producing, harmful marine alga. Cells of strain PG2S01T were Gram- negative, strictly aerobic, non-motile, non-fermentative, orange-pigmented, moderately halophilic rods. Growth was observed in the presence of 0.25–7.5 % NaCl and at 10–40 6C. The dominant fatty acids were iso-C15 : 0, summed feature 3 (C16 : 1v7c and/or C16 : 1v6c), iso-C17 : 0 3-OH, iso-C16 : 0 3-OH, iso-C15 : 1 G and iso-C15 : 0 3-OH. The major polar lipids were phosphatidylethanolamine and several unidentified aminolipids, phospholipids and other lipids. The G+C content of the chromosomal DNA was 44.6 mol%. 16S rRNA gene sequence comparison showed that strain PG2S01T was most closely related to Owenweeksia hongkongensis UST20020801T (88.9 % 16S rRNA gene sequence similarity); levels of similarity between strain PG2S01T and the type strains of recognized representatives of genera in the family Cryomorphaceae were ,88 %. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PG2S01T formed a distinct evolutionary lineage within the family Cryomorphaceae. Strain PG2S01T was distinguishable from members of phylogenetically related genera by differences in several phenotypic properties. On the basis of phenotypic data and phylogenetic inference, strain PG2S01T represents a novel species in a new genus in the family Cryomorphaceae, for which the name Phaeocystidibacter luteus gen. nov., sp. nov. is proposed. The type strain of the type species is PG2S01T (5CCTCC AB 209288T 5LMG 25704T 5MCCC 1F01079T). An emended description of O. hongkongensis Lau et al. 2005 is also proposed. Members of the family Cryomorphaceae, phylum and terrestrial, from tropical to polar regions (Bowman, Bacteroidetes, are widespread in a wide range of marine 2011). There are seven genera in the family Cryomor- and terrestrial habitats. Most cultured species have been phaceae, six of them of marine origin – Brumimicrobium, isolated from low temperature ecosystems. Molecular Cryomorpha and Crocinitomix (Bowman et al., 2003), studies, however, have suggested that members of the Owenweeksia (Lau et al., 2005), Lishizhenia (Lau et al., family are present in a wide range of habitats, both marine 2006) and Wandonia (Lee et al., 2010) – and a freshwater genus Fluviicola (O’Sullivan et al., 2005). Members of the 3These authors contributed equally to this work. family Cryomorphaceae are Gram-negative, strictly aerobic The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene or facultatively anaerobic, chemo-organotrophic bacteria. sequence of strain PG2S01T is HQ434766. Most of them require seawater salts for growth. They have A supplementary figure is available with the online version of this paper. limited versatility in regard to nutritional and catabolic Downloaded from www.microbiologyresearch.org by 030254 G 2013 Chinese Government Printed in Great Britain 1143 IP: 137.108.70.7 On: Mon, 04 Jul 2016 03:50:22 Y. Zhou and others activities and are generally unreactive in most standard strain PG2S01T formed a distinct lineage within the family phenotypic tests (Bowman, 2011). They cannot utilize Cryomorphaceae, most closely linked to Cryomorpha ignava carbohydrates and require complex organic compounds for 1-22T (Fig. 1). The two algorithms yielded essentially the growth (Bowman et al., 2003; Lau et al., 2005; O’Sullivan et same tree topology (data not shown). al., 2005). Molecular phylogenetic studies show that Cell morphology of strain PG2S01T was studied under an phylotypes related to the family Cryomorphaceae are Olympus inverted microscope using 5-day-old cultures on associated with phytoplankton blooms (Pinhassi et al., T MA. For electron microscopy, strain PG2S01 was 2004). Blooms of the alga Phaeocystis globosa can cause collected from the agar, dyed with 2 % phosphotungstic marine fish mortality by producing haemolysin and acid and examined with a JEM2100 transmission electron blocking gills. In 1997, a P. globosa bloom occurred in 2 microscope (Fig. S1, available in IJSEM Online). The the East China Sea in a range of about 3000 km causing following tests were performed on strain PG2S01T and O. great economic loss (Shen et al., 2000). Similar blooms hongkongensis UST20020801T. Gram-reaction, catalase and have also been reported in Denmark, the Netherlands, oxidase activities and hydrolysis of casein, chitin, Tween Norway and the Arabian Sea (Baumann et al., 1994). In a 80, DNA and starch were carried out according to Dong study aimed at obtaining axenic cultures of P. globosa, et al. (2001). Growth in MB was evaluated at 0, 4, 10, 16, many bacterial strains were isolated and characterized 20, 25, 28, 30, 37, 40 and 45 uC and at pH 3.0–10.0 (at taxonomically. This study focuses on one of these isolates, T intervals of one pH unit). The pH of MB was adjusted designated PG2S01 . Comparative 16S rRNA gene prior to sterilization using the following buffers: citric acid/ sequence analysis indicated that strain PG2S01T formed a sodium citrate (pH 3.0–6.0), Na2HPO4/citric acid deep branch within the family Cryomorphaceae. (pH 7.0–8.0) and lysine/NaOH (pH 9.0–10.0). Verifi- Accordingly, the aim of the present work was to determine T cation of the pH values after autoclaving revealed only the exact taxonomic position of strain PG2S01 . minor changes. Requirement for and growth with 0, 0.25, Strain PG2S01T was isolated from a culture of P. globosa by 0.5, 1, 2, 3.5, 5, 6.5, 7.5, 10, 15 and 20 % (w/v) NaCl was inoculating serial dilutions of crushed alga on laboratory- tested using ASW supplemented with 0.5 % tryptone and 2+ 2+ prepared marine agar (MA; l21 aged seawater: 5.0 g 0.1 % yeast extract. Requirement for Mg and Ca was tryptone, 1.0 g yeast extract, 10 g agar; pH 7.6–7.8) examined by evaluating growth in MB prepared with ASW containing 0, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04 and 0.05 M incubated at 28 uC for 5 days. Aged seawater could be + + Mg2 or Ca2 . Anaerobic growth was examined using the replaced by artificial seawater (ASW; 0.1 % CaCl2 .2H2O, Shell Lab anaerobic system. Presence of gliding motility 0.1 % KCl, 0.5 % MgSO4 .7H2O, 2.5 % NaCl; Lau et al., 2005). Marine agar 2216E (Difco) was also used for and flexirubin-type pigments was assessed according to morphological and biochemical characterization. The Bernardet et al. (2002). As no flexirubin-type pigment was strain was stored at 280 uC in marine broth (MB) detected using the simple KOH test, the pigments were supplemented with 20 % (v/v) glycerol. extracted with 100 % ethanol and the absorption spectrum was recorded at 300–700 nm with a Thermo Spectronic T Strain PG2S01 was grown on MA and DNA was prepared UV300 spectrophotometer. Other biochemical tests were according to Su et al. (2007). The 16S rRNA gene was carried out using API 20 E (37 uC), API 20 NE (28 uC), API amplified using primers 27F and 1492R (DeLong, 1992). 50 CH (28 uC) and API ZYM (28 uC) strips (bioMe´rieux) Sequences of related taxa were obtained from the according to the manufacturer’s instructions except that GenBank database. Phylogenetic analysis was performed ASW was used to prepare the bacterial suspensions. using MEGA version 4 (Tamura et al., 2007) after multiple Antibiotic susceptibility was tested on MA at 28 uC for alignment of data by DNAMAN version 5.1 (Lynnon 5–10 days using the disc-diffusion method (Oxoid; Drew et Biosoft). Distances or distance options were determined al., 1972). The phenotypic characteristics of strain PG2S01T according to the Kimura two-parameter model. Clustering are given in the genus and species descriptions and in was performed with the neighbour-joining method Table 1. (Saitou & Nei, 1987) and the minimum-evolution method The fatty acids, polar lipids and respiratory quinones of (Rzhetsky & Nei, 1993) using bootstrap values based on T T 1000 replications. strain PG2S01 and O. hongkongensis UST20020801 were analysed. Following growth on MA at 28 uC for 5 days, The 16S rRNA gene sequence of strain PG2S01T (1480 nt fatty acids were extracted, saponified and esterified and the positions) showed 97 % similarity to ‘Coccinistipes vermicola’ fatty acid methyl esters were analysed by GC (7890A GC; IMCC1411 (GenBank no. EF108212), which was isolated Agilent Technologies). The TBS6 database was used from a marine environment in 2006 but is still unpublished, according to the instructions of the MIDI system version and 88.9 % to Owenweeksia hongkongensis UST20020801T 6.0 (MIDI; Sasser, 1997). Polar lipid analysis was (Lau et al., 2005). Sequence similarities to other members of carried out by the Identification Service of the DSMZ the family Cryomorphaceae were ,87 % (Wandonia haliotis (Braunschweig, Germany). Respiratory quinones were also Haldis-1T, 86.2 %; Cryomorpha ignava 1-22T, 83.1 %; and analysed at the DSMZ on a LDC Analytical HPLC (Thermo Crocinitomix catalasitica IFO 15977T, 85.5 %).