International Journal of Systematic and Evolutionary Microbiology (2006), 56, 2463–2467 DOI 10.1099/ijs.0.64306-0 Bowmanella denitrificans gen. nov., sp. nov., a denitrifying bacterium isolated from seawater from An-Ping Harbour, Taiwan Wen Dar Jean,1 Jwo-Sheng Chen,2 Yu-Te Lin3 and Wung Yang Shieh3 Correspondence 1Center for General Education, Leader University, No. 188, Sec. 5, An-Chung Road, Tainan, Wung Yang Shieh Taiwan [email protected] 2School of Medicine, China Medical University, No. 91, Shyue-Shyh Road, Taichung, Taiwan 3Institute of Oceanography, National Taiwan University, PO Box 23-13, Taipei, Taiwan A heterotrophic, non-fermentative, denitrifying isolate, designated strain BD1T, was obtained from a seawater sample collected in the shallow coastal region of An-Ping Harbour, Tainan, Taiwan. The cells of strain BD1T were Gram-negative. Cells grown in broth cultures were curved rods that were motile by means of a single polar flagellum. Growth occurred between 10 and 40 6C, with an optimum at 30–35 6C. Strain BD1T grew in NaCl levels of 0–10 %, with better growth occurring at 1–3 %. It grew aerobically and could achieve anaerobic growth by adopting a denitrifying metabolism with nitrate or nitrous oxide as the terminal electron acceptor. The major fatty acids were C16 : 0,C18 : 1v7c and summed feature 3 (C16 : 1v7c and/or C15 : 0 iso 2-OH). The polar lipids consisted of phosphatidylethanolamine (56?6 %) and phosphatidylglycerol (43?4 %). The isoprenoid quinones were Q-8 (81?5 %), Q-9 (11?1 %) and Q-10 (7?4 %). The DNA G+C content was 50?0 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain BD1T formed a distinct lineage in the Gammaproteobacteria and that it exhibited the highest level of sequence similarity with species of the genera Alteromonas (92?8–93?7 %), Aestuariibacter (93?0 %), Glaciecola (90?4–92?7 %) and Salinimonas (91?8 %). Strain BD1T was distinguishable from species of these genera by the presence of Q-9 and Q-10. Phenotypically, strain BD1T was also distinguishable from species of these genera in that it did not require NaCl for growth and was capable of denitrification. On the basis of the polyphasic data from this study, the isolate represents a novel species within a novel genus, for which the name Bowmanella denitrificans gen. nov., sp. nov. is proposed. The type strain of Bowmanella denitrificans is BD1T (=BCRC 17491T=JCM 13378T). Alteromonas-like bacteria (Alteromonas-related bacteria) (Pseudoalteromonas and Algicola), the Psychromonadaceae belonging to the class Gammaproteobacteria comprise a (Psychromonas), the Shewanellaceae (Shewanella)and large group of marine, heterotrophic, Gram-negative rods Agarivorans, Oceanimonas, Oceanisphaera and Zobellella that are mainly non-fermentative and polarly flagellated. A (Ivanova et al., 2004; Jean et al., 2006; Jeon et al., 2005; wide variety of families and genera have been classified Kurahashi & Yokota, 2004; Lin & Shieh, 2006; Yi et al., 2004). as belonging to this bacterial group, including the Only some species in these families and genera are reported Alteromonadaceae (Alteromonas, Aestuariibacter, Glaciecola to be denitrifying bacteria, e.g. Shewanella denitrificans, and Salinimonas), the Colwelliaceae (Colwellia and Shewanella sediminis, Shewanella decolorationis, Zobellella Thalassomonas), the Ferrimonadaceae (Ferrimonas), the denitrificans and Zobellella taiwanensis (Brettar et al., 2002; Idiomarinaceae (Idiomarina and Pseudidiomarina), the Lin & Shieh, 2006; Xu et al., 2005; Zhao et al., 2005, 2006). Moritellaceae (Moritella), the Pseudoalteromonadaceae A non-fermentative denitrifying isolate has been recovered in our laboratory from a seawater sample collected in the The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene shallow coastal region of An-Ping Harbour, Tainan, Taiwan, sequence of strain BD1T is DQ343294. during a survey of the diversity of denitrifying bacteria. The Fatty acid compositions of strain BD1T and related strains, electron polyphasic data obtained in this study indicated that this micrographs and growth curves are available as supplementary material denitrifying isolate could be classified within a novel genus in IJSEM Online. of the Alteromonas-like gammaproteobacteria. 64306 G 2006 IUMS Printed in Great Britain 2463 W. D. Jean and others Polypeptone/yeast extract/nitrate (PYN) broth medium was (2004). Bootstrap confidence values (Felsenstein, 1985) used for the enrichment cultivation of denitrifying bacteria were obtained using 1000 resamplings with the option of in the seawater. This medium and others used in this study stepwise addition. Phylogenetic trees were also constructed were described in our recent report (Lin & Shieh, 2006). by using the maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods. An-Ping Harbour is located in the south-west of Taiwan. Seawater samples were collected from the shallow coastal An almost-complete 16S rRNA gene sequence of strain region of this harbour in the morning at low tide. Each BD1T was determined (1459 bp). It was aligned and sample was decimally diluted with sterile NaCl/Tris buffer compared with all bacterial sequences available in the (30 g NaCl and 0?24 g Tris base in 1 l deionized water, GenBank database. A phylogenetic analysis based on 16S pH 8?0). A 1 ml aliquot of each dilution (101–103)was rRNA gene sequences showed that strain BD1T was a transferred to a rimless tube (16 mm610 cm) containing member of the Alteromonas-like bacteria in the class PYN broth medium (5 ml) into which an inverted Durham Gammaproteobacteria. A neighbour-joining tree, showing tube had been placed. All culture tubes were incubated the phylogenetic relationship between strain BD1T and aerobically at 25 uC in the dark for 3–7 days. Cultures that selected representatives of Alteromonas-like bacteria, is developed visible turbidity and produced gas (which presented in Fig. 1; similar results were obtained using the accumulated in the Durham tubes) were streaked (one maximum-parsimony and maximum-likelihood algorithms loopful) on polypeptone/yeast extract (PY) plate medium. (not shown). The phylogenetic analyses showed that strain Individual colonies appearing on each plate were picked off BD1T formed a robust cluster at sequence similarity levels and purified by successive streaking on PY plates. Only of 90?4–93?7 % (91–140 differences out of 1259–1459 nuc- isolates that exhibited growth and produced gas in PYN leotide sites) with species of the genera Altero- broth were maintained in PY stab medium and stored at monas (92?8–93?7 %), Aestuariibacter (93?0 %), Glaciecola T 25 uC. One of the isolates, designated strain BD1 , was used (90?4–92?7 %) and Salinimonas (91?8 %) in the family for the present study. Alteromonadaceae. No other bacterial species shared more than 90 % sequence similarity with the strain. The low Growth and other phenotypic properties used for the levels of sequence similarity with other bacteria indicated physiological and morphological characterization of strain that strain BD1T could be assigned to a novel genus. Phy- BD1T were examined according to established procedures, logenetically, the strain could be considered to represent a as described previously (Lin & Shieh, 2006). novel family, since it was clearly an outgroup with respect to Cells grown in PY broth at 30 uC for 2 days were harvested species of the family Alteromonadaceae. Moreover, the 16S by centrifugation. Polar lipids in the cells were extracted, rRNA gene sequences of Alteromonadaceae species have purified and analysed by the methods described by Lin & been defined as possessing signature nucleotides, 737 (A), Shieh (2006). Isoprenoid quinones were analysed as 770 (T) and 809 (A) (Ivanova et al., 2004), whereas strain T described by Hu et al. (2001), using HPLC apparatus BD1 has nucleotides G, C and G, respectively, at these equipped with a reversed-phase column (4?66250 mm; sequence positions. Waters model 5C -AR-II). A mixture of methanol and 18 The polar lipids of strain BD1T consisted of phosphatidyl- isopropyl ether (9 : 2, v/v) was used as the mobile phase, and ethanolamine (56?6 %) and phosphatidylglycerol (43?4 %). quinones were detected at 270 nm. Fatty acids in whole cells These two phospholipids have also been reported to be the grown on PY plate medium at 30 uC for 2 days were extracted, saponified and esterified; this was followed by GC major constituents of the polar lipids in some Alteromonas analysis of the fatty acid methyl esters according to the species, such as Alteromonas addita and Alteromonas instructions of the MIDI system (Sasser, 1997). This work, macleodii (Ivanova et al., 2000, 2005). Phosphatidic acid, and DNA G+C content determination by HPLC analysis bis-phosphatidic acid and lysophosphatidylethanolamine (Shieh & Liu, 1996), was performed at the Bioresources were detected as minor constituents of polar lipids in these species (Ivanova et al., 2000, 2005). However, none of these Center for Research and Collection, Food Industry Research T and Development Institute, Taiwan. phospholipids has been detected in strain BD1 . The isoprenoid quinones of strain BD1T consisted of Q-8 Strain BD1T was cultivated aerobically in PY broth at 30 uC (81?5 %), Q-9 (11?1 %) and Q-10 (7?4 %). Q-8 was also in the dark for 2 days. The culture was centrifuged to harvest found to be the predominant isoprenoid quinone in species the cells. Extraction and purification of total genomic DNA of the genera Alteromonas, Aestuariibacter and Salinimonas from the cells and PCR amplification of 16S rRNA gene were (Ivanova et al., 2000, 2005; Jeon et al., 2005; Martı´nez-Checa performed according to the methods described by Lin & et al., 2005; Yi et al., 2004; Yoon et al., 2003, 2004), but these Shieh (2006). The sequencing reaction for the 16S rRNA species have never been shown to contain Q-9 or Q-10. The gene, alignment and comparison of the resulting sequence data from the analysis of isoprenoid quinones supported the with reference sequences available in the GenBank database, assignment of strain BD1T to a novel genus.