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Pseudidiomarina Taiwanensis Gen. Nov., Sp. Nov., a Marine Bacterium

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Pseudidiomarina Taiwanensis Gen. Nov., Sp. Nov., a Marine Bacterium International Journal of Systematic and Evolutionary Microbiology (2006), 56, 899–905 DOI 10.1099/ijs.0.64048-0 Pseudidiomarina taiwanensis gen. nov., sp. nov., a marine bacterium isolated from shallow coastal water of An-Ping Harbour, Taiwan, and emended description of the family Idiomarinaceae Wen Dar Jean,1 Wung Yang Shieh2 and Hsiu-Hui Chiu2 Correspondence 1Center for General Education, Leader University, No. 188, Sec. 5, An-Chung Rd, Tainan, Wung Yang Shieh Taiwan [email protected] 2Institute of Oceanography, National Taiwan University, PO Box 23-13, Taipei, Taiwan Two strains of heterotrophic, aerobic, marine bacteria, designated strains PIT1T and PIT2, were isolated from sea-water samples collected at the shallow coastal region of An-Ping Harbour, Tainan, Taiwan. Both strains were Gram-negative. Cells grown in broth cultures were straight rods that were non-motile, lacking flagella. Both strains required NaCl for growth and exhibited optimal growth at 30–35 6C, 1–4 % NaCl and pH 8. They grew aerobically and were incapable of anaerobic growth by fermentation of glucose or other carbohydrates. Cellular fatty acids were predominantly iso-branched, with C15 : 0 iso and C17 : 0 iso representing the most abundant components. The DNA G+C contents of strains PIT1T and PIT2 were 49?3 and 48?6 mol%, respectively. Phylogeny based on 16S rRNA gene sequences, together with data from phenotypic and chemotaxonomic characterization, revealed that the two isolates could be assigned to a novel genus in the family Idiomarinaceae, for which the name Pseudidiomarina gen. nov. is proposed. Pseudidiomarina taiwanensis sp. nov. is the type species of the novel genus (type strain PIT1T=BCRC 17465T=JCM 13360T). Alteromonas-like bacteria belonging to the class Gamma- iso-branched cellular fatty acids (Ivanova et al., 2004). They proteobacteria comprise a large group of marine, hetero- are also distinguished from other Alteromonas-like bacteria trophic, Gram-negative rods that are mainly strictly aerobic by their poor ability to use carbohydrates as sole sources and motile by means of a single polar flagellum. Identifi- of carbon and energy (Ivanova et al., 2004). The genus cation of these bacteria is impeded at the species level and Idiomarina, the only genus of the family Idiomarinaceae,was even at the genus level, due to similar phenotypic char- originally established to accommodate two species, Idioma- acteristics. A recent phylogenetic study based on 16S rRNA rina abyssalis (the type species) and Idiomarina zobellii gene sequences, however, revealed that the Alteromonas-like (Ivanova et al., 2000). Both species were recovered from bacteria could be divided into the families Alteromonad- deep-sea water samples taken from the north-western Pacific aceae, Pseudoalteromonadaceae, Psychromonadaceae, Col- Ocean. Five more species have been described in recent welliaceae, Ferrimonadaceae, Idiomarinaceae, Moritellaceae years, including Idiomarina baltica (Brettar et al., 2003), and Shewanellaceae and the genera Oceanimonas and Ocean- Idiomarina loihiensis (Donachie et al., 2003), Idiomarina isphaera (Ivanova et al., 2004). The taxonomic affiliation of fontislapidosi (Martı´nez-Ca´novas et al., 2004), Idiomarina the genera Oceanimonas and Oceanisphaera at the family ramblicola (Martı´nez-Ca´novas et al., 2004) and Idiomarina level remains undetermined and the recognition of a family seosinensis (Choi & Cho, 2005). These species were isolated must await more sequence data (Ivanova et al., 2004). from surface water of the central Baltic Sea, hydrothermal fluid from a submarine volcano in Hawaii, soil from a Members of the family Idiomarinaceae are unique among hypersaline wetland in Spain, water from a hypersaline the Alteromonas-like bacteria in possessing a high content of rambla in Spain and water from a solar saltern in Korea, respectively. Published online ahead of print on 16 December 2005 as DOI During a survey of the diversity of heterotrophic marine 10.1099/ijs.0.64048-0. bacteria, two bacterial strains were recovered from sea-water Abbreviation: PHB, poly-b-hydroxybutyrate. samples collected at the shallow coastal region of An-Ping The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene Harbour, Tainan, Taiwan. The data presented in this study sequence of strain PITT is DQ118948. show that the two isolates can be classified as representatives 64048 G 2006 IUMS Printed in Great Britain 899 W. D. Jean, W. Y. Shieh and H.-H. Chiu of a novel species in the family Idiomarinaceae and deserve a the distance matrices for the aligned sequences and recon- novel genus rank. struction of a phylogenetic tree by the neighbour-joining method were performed as described previously by Shieh An-Ping Harbour is located in the south-west of Taiwan. et al. (2004). Phylogenetic trees were also reconstructed by Sea-water samples were collected from a shallow coastal using maximum-parsimony (Fitch, 1971) and maximum- region of the harbour in the morning at low tide. Each likelihood (Felsenstein, 1981) methods. The GenBank acces- sample was decimally diluted with sterile NaCl/Tris buffer sion numbers for the sequences used to reconstruct the (30 g NaCl and 0?24 g Tris in 1 l deionized water, pH 8?0). 2 4 phylogenetic tree are shown in Fig. 1. Bootstrap confidence Aliquots (0?1 ml) of the decimal dilutions (10 –10 times) values (Felsenstein, 1985) were obtained with 1000 resamp- were spread on polypeptone-yeast extract (PY) plate medium lings with an option of stepwise addition. (Shieh et al., 2000). The plates were incubated at 25 uCin the dark for 7 days under aerobic conditions. Individual Fatty acids in whole cells grown on PY plate medium at 30 uC colonies appearing on the plates were picked off and purified for 3 days were extracted, saponified and esterified, followed by successive streaking on PY plates. PY stab cultures of the by GC analysis of the fatty acid methyl esters according to isolates were maintained at 25 uC under aerobic conditions. the instructions of the MIDI system (Sasser, 1997). This T Two of the isolates, designated strains PIT1 and PIT2, were work was performed at the Bioresources Center for Research used for the present study. and Collection (BCRC), Food Industry Research and Development Institute, Taiwan. Genomic DNA G+C PY broth cultures of strains PIT1T and PIT2 were incubated content was determined by HPLC analysis (Shieh & Liu, aerobically at 30 uC in the dark for 3 days. The cultures were 1996), which was also performed at the BCRC. centrifuged to harvest the cells. Total genomic DNA was extracted and purified from the cells by using a Puregene Growth and phenotypic characteristics of strains PIT1T and DNA isolation kit (Gentra Systems) in accordance with the PIT2 were examined according to the methods of Shieh et al. manufacturer’s instructions. Solutions of the purified DNA 21 (2000), with modifications and additional tests as described samples were prepared at concentrations of 200–700 mgml below. The ability to grow at different temperatures was in sterile distilled water. The DNA solutions were used for determined in PY broth and recorded daily for up to 7 days PCR amplification. PCR amplification of each bacterial 16S at 20–45 uC and for 20 days at 4–15 uC, unless significant rRNA gene was conducted using a universal primer pair growth was observed. The ability to grow at various NaCl at positions 8–27 and 1488–1511 of the Escherichia coli concentrations was determined in PY broth containing numbering system (Shieh et al., 2003a). Accessory PCR 0–15 % NaCl. Utilization of various carbohydrates as sole amplification was performed using either of the primer pairs carbon and energy sources for growth was tested in carbo- at positions 8–27 and 518–534 (Muyzer et al., 1993) or hydrate/mineral (CM) media (Shieh et al., 2004). Anaerobic positions 907–926 and 1488–1511 (Muyzer & Smalla, 1998). growth in PY, polypeptone-yeast extract-glucose (PYG) and The PCR mixture contained 2?5 ml DNA solution, 1 ml polypeptone-yeast extract-nitrate (PYN) broth media under m mixture of one of the primer pairs (10 M each primer), argon gas was analysed as described by Shieh et al. (2004). 1 ml mixture of the four deoxyribonucleoside 59-triphos- Poly-b-hydroxybutyrate (PHB) accumulation and H S pro- phates (2?5 mM; Gene Teks Bioscience), 5 ml106 Taq 2 duction from thiosulphate were tested according to the buffer (Gene Teks Bioscience), 5 ml106 BSA (Promega) methods of Shieh et al. (1988) and Shieh et al. (2004), and 0?5 ml(2U)Taq DNA polymerase (Gene Teks respectively. Tests for endospore formation and for activities Bioscience). Each sample was made up to 50 ml with sterile of arginine dihydrolase and lysine and ornithine decarboxy- distilled water. PCR amplification was performed in a lases essentially followed the methods of Shieh et al. (2003b). GeneAmp PCR System 2700 (Applied Biosystems) with the Biolog GN2 microplates were used to determine the oxida- following temperature profile: an initial denaturation at tion of carbohydrates, alcohols, organic acids, amino acids 94 uC for 10 min; 35 cycles of denaturation (1 min at 92 uC), and nucleosides presented as single carbon sources. The API annealing (1?5 min at 52 uC) and extension (1?5 min at 50CH system (bioMe´rieux) was used to test either oxidation 72 uC); and a final extension at 72 uC for 4 min. Amplified DNAs were checked for size and purity by electrophoresis at or fermentation of alcohols and carbohydrates. Constitutive 100 V for 30 min on 1 % agarose gels in Tris/acetate/EDTA enzyme activities were detected by using the API ZYM ´ (TAE) buffer (MDBio) with 5 ml aliquots of PCR products. system (bioMerieux). Cell suspensions used for the API 21 50CH tests were prepared in PY broth supplemented The gels were stained with ethidium bromide (1 mgml ) 21 for 5 min in TAE buffer and the DNA bands that appeared with 0?18 g phenol red l .
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