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1 Center for General Education, Leader University, No. 188, Sec. 5, An-Chung Road, Tainan, Taiwan
2 School of Medicine, China Medical University, No. 91, Shyue-Shyh Road, Taichung, Taiwan
3 Institute of Oceanography, National Taiwan University, PO Box 23-13, Taipei, Taiwan
Correspondence
Wung Yang Shieh
winyang{at}ntu.edu.tw
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ABSTRACT |
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7c and summed feature 3 (C16 : 17c 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).
Fatty acid compositions of strain BD1T and related strains, electron micrographs and growth curves are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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; Jean et al., 2006; Jeon et al., 2005; Kurahashi & Yokota, 2004; Lin & Shieh, 2006; Yi et al., 2004). Only some species in these families and genera are reported to be denitrifying bacteria, e.g. Shewanella denitrificans, Shewanella sediminis, Shewanella decolorationis, Zobellella denitrificans and Zobellella taiwanensis (Brettar et al., 2002; Lin & Shieh, 2006; Xu et al., 2005; Zhao et al., 2005, 2006). A non-fermentative denitrifying isolate has been recovered in our laboratory from a seawater sample collected in the shallow coastal region of An-Ping Harbour, Tainan, Taiwan, during a survey of the diversity of denitrifying bacteria. The polyphasic data obtained in this study indicated that this denitrifying isolate could be classified within a novel genus of the Alteromonas-like gammaproteobacteria.
Polypeptone/yeast extract/nitrate (PYN) broth medium was used for the enrichment cultivation of denitrifying bacteria in the seawater. This medium and others used in this study were described in our recent report (Lin & Shieh, 2006).
An-Ping Harbour is located in the south-west of Taiwan. Seawater samples were collected from the shallow coastal region of this harbour in the morning at low tide. Each sample was decimally diluted with sterile NaCl/Tris buffer (30 g NaCl and 0.24 g Tris base in 1 l deionized water, pH 8.0). A 1 ml aliquot of each dilution (101–103) was transferred to a rimless tube (16 mmx10 cm) containing PYN broth medium (5 ml) into which an inverted Durham tube had been placed. All culture tubes were incubated aerobically at 25 °C in the dark for 3–7 days. Cultures that developed visible turbidity and produced gas (which accumulated in the Durham tubes) were streaked (one loopful) on polypeptone/yeast extract (PY) plate medium. Individual colonies appearing on each plate were picked off and purified by successive streaking on PY plates. Only isolates that exhibited growth and produced gas in PYN broth were maintained in PY stab medium and stored at 25 °C. One of the isolates, designated strain BD1T, was used for the present study.
Growth and other phenotypic properties used for the physiological and morphological characterization of strain BD1T were examined according to established procedures, as described previously (Lin & Shieh, 2006).
Cells grown in PY broth at 30 °C for 2 days were harvested by centrifugation. Polar lipids in the cells were extracted, purified and analysed by the methods described by Lin & Shieh (2006). Isoprenoid quinones were analysed as described by Hu et al. (2001), using HPLC apparatus equipped with a reversed-phase column (4.6x250 mm; Waters model 5C18-AR-II). A mixture of methanol and isopropyl ether (9 : 2, v/v) was used as the mobile phase, and quinones were detected at 270 nm. Fatty acids in whole cells grown on PY plate medium at 30 °C for 2 days were extracted, saponified and esterified; this was followed by GC analysis of the fatty acid methyl esters according to the instructions of the MIDI system (Sasser, 1997). This work, and DNA G+C content determination by HPLC analysis (Shieh & Liu, 1996), was performed at the Bioresources Center for Research and Collection, Food Industry Research and Development Institute, Taiwan.
Strain BD1T was cultivated aerobically in PY broth at 30 °C in the dark for 2 days. The culture was centrifuged to harvest the cells. Extraction and purification of total genomic DNA from the cells and PCR amplification of 16S rRNA gene were performed according to the methods described by Lin & Shieh (2006). The sequencing reaction for the 16S rRNA gene, alignment and comparison of the resulting sequence with reference sequences available in the GenBank database, calculation of distance matrices for the aligned sequences and reconstruction of a phylogenetic tree by the neighbour-joining method were done as described by Shieh et al. (2004). Bootstrap confidence values (Felsenstein, 1985) were obtained using 1000 resamplings with the option of stepwise addition. Phylogenetic trees were also constructed by using the maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods.
An almost-complete 16S rRNA gene sequence of strain BD1T was determined (1459 bp). It was aligned and compared with all bacterial sequences available in the GenBank database. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain BD1T was a member of the Alteromonas-like bacteria in the class Gammaproteobacteria. A neighbour-joining tree, showing the phylogenetic relationship between strain BD1T and selected representatives of Alteromonas-like bacteria, is presented in Fig. 1; similar results were obtained using the maximum-parsimony and maximum-likelihood algorithms (not shown). The phylogenetic analyses showed that strain BD1T formed a robust cluster at sequence similarity levels of 90.4–93.7 % (91–140 differences out of 1259–1459 nucleotide sites) with species of the genera Alteromonas (92.8–93.7 %), Aestuariibacter (93.0 %), Glaciecola (90.4–92.7 %) and Salinimonas (91.8 %) in the family Alteromonadaceae. No other bacterial species shared more than 90 % sequence similarity with the strain. The low levels of sequence similarity with other bacteria indicated that strain BD1T could be assigned to a novel genus. Phylogenetically, the strain could be considered to represent a novel family, since it was clearly an outgroup with respect to species of the family Alteromonadaceae. Moreover, the 16S rRNA gene sequences of Alteromonadaceae species have been defined as possessing signature nucleotides, 737 (A), 770 (T) and 809 (A) (Ivanova et al., 2004), whereas strain BD1T has nucleotides G, C and G, respectively, at these sequence positions.
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, 2005). Phosphatidic acid, bis-phosphatidic acid and lysophosphatidylethanolamine were detected as minor constituents of polar lipids in these species (Ivanova et al., 2000, 2005). However, none of these phospholipids has been detected in strain BD1T. The isoprenoid quinones of strain BD1T consisted of Q-8 (81.5 %), Q-9 (11.1 %) and Q-10 (7.4 %). Q-8 was also found to be the predominant isoprenoid quinone in species of the genera Alteromonas, Aestuariibacter and Salinimonas (Ivanova et al., 2000, 2005; Jeon et al., 2005; Martínez-Checa et al., 2005; Yi et al., 2004; Yoon et al., 2003, 2004), but these species have never been shown to contain Q-9 or Q-10. The data from the analysis of isoprenoid quinones supported the assignment of strain BD1T to a novel genus. Strain BD1T, like species belonging to the Alteromonadaceae, contained C16 : 0 (21.7 %), C18 : 17c (19.4 %) and summed feature 3 (C16 : 17c and/or C15 : 0 iso 2-OH) (24.1 %; the two fatty acids could not be differentiated in the MIDI system) as the major cellular fatty acids. Other cellular fatty acids present at levels greater than 3 % included C16 : 0 N alcohol (3.7 %), C17 : 18c (3.8 %), C12 : 0 3-OH (7.1 %) and C17 : 0 10-methyl (3.5 %). Quantitative differences in the cellular fatty acids might serve to differentiate strain BD1T from all species in the Alteromonadaceae, although the relevant species were not cultivated under the same conditions (see Supplementary Table S1 available in IJSEM Online). The DNA G+C content of strain BD1T (50.0 mol%) fell within the range for Aestuariibacter (48.0–54.0 mol%) but was greater than those of Alteromonas (43.0–46.3 mol%), Glaciecola (40.0–46 mol%) and Salinimonas (48.0 mol%) (Table 1).
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). Other characteristics useful for differentiating strain BD1T from species belonging to the family Alteromonadaceae are listed in Table 1. The phylogenetic, chemotaxonomic and physiological data obtained in this study indicate that strain BD1T should be assigned to a novel genus within the category of Alteromonas-like bacteria. Therefore the novel isolate represents a novel genus and species, for which the name Bowmanella denitrificans gen. nov., sp. nov. is proposed.
Description of Bowmanella gen. nov.
Bowmanella (Bow.man.el'la. L. dim ending -ella; N.L. fem. n. Bowmanella named after John P. Bowman, to honour his work in marine microbiology).
Members are Gram-negative rods belonging to the class Gammaproteobacteria. Cells grown in broth cultures are motile by means of a single, polar flagellum. Chemo-organotrophs capable of respiratory but not fermentative metabolism. Mesophilic, growing well at 25–35 °C; no growth occurs at 4 or 45 °C. NaCl stimulates growth but is not an absolute requirement. Oxidase- and catalase-positive. Major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. Isoprenoid quinones consist of Q-8 (most abundant), Q-9 and Q-10. Major cellular fatty acids are C16 : 0, C18 : 17c and summed feature 3 (C16 : 17c and/or C15 : 0 iso 2-OH). The DNA G+C content of the type strain of the type species is 50.0 mol%. The type species is Bowmanella denitrificans.
Description of Bowmanella denitrificans sp. nov.
Bowmanella denitrificans (de.ni.tri'fi.cans. N.L. v. denitrifico to denitrify; N.L. part. adj. denitrificans denitrifying).
Description is as for the genus, with the following additional characteristics. Cells during late-exponential to early stationary phase of growth in broth cultures are polarly flagellated, curved rods (see Supplementary Fig. S1 in IJSEM Online), approximately 3.7–5.3 µm long by 1.2–1.6 µm wide. Cells form flake-like aggregations at late growth stages. Colonies produced on agar plates are circular, off-white in colour and non-luminescent. Swarming does not occur. Growth occurs at temperatures between 10 and 40 °C, with an optimum at 30–35 °C; no growth occurs at 43 °C. Growth occurs at pH values in the range 6–10, with an optimum at pH 8. Growth occurs at NaCl levels of 0–10 %, with an optimum at 1–3 %; no growth occurs at 12 % NaCl. Unable to ferment D-glucose, D-arabinose, L-arabinose, D-cellobiose, D-galactose, D-mannose, sucrose, D-trehalose, D-xylose, dulcitol, inositol or D-mannitol. Molecular oxygen is a universal electron acceptor while anaerobic growth can be achieved by carrying out denitrification with 
or N2O as the terminal electron acceptor (see Supplementary Fig. S2 in IJSEM Online). Indole is produced from tryptophan. H2S is not produced from thiosulfate. Aesculin, casein, DNA, gelatin, lecithin, starch, Tween 80 and urea are hydrolysed, but agar is not. Arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities are not detected. D-Cellobiose, D-fructose, D-glucose, D-galactose, D-lactose, D-mannose, sucrose, D-trehalose, D-xylose, acetate and 
-hydroxybutyrate can be utilized as sole carbon and energy sources, but D-arabinose, L-arabinose, adonitol, dulcitol, inositol, D-mannitol, D-melezitose, D-melibiose, D-sorbitol and L-lactate cannot. The following constitutive enzyme activities are detected in API ZYM tests: leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), N-acetyl--glucosaminidase, naphthol-AS-BI-phosphohydrolase, acid phosphatase and alkaline phosphatase. Resistant to the vibriostatic agent O/129 at 10–150 µg. Susceptible to chloramphenicol (30 µg), colistin (10 µg), nalidixic acid (30 µg), neomycin (30 µg), polymyxin B (300 U) and tetracycline (30 µg) and intermediately susceptible to gentamicin (10 µg) and kanamycin (30 µg). Resistant to ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), clindamycin (2 µg), erythromycin (15 µg), lincomycin (2 µg), novobiocin (30 µg), oxacillin (1 µg), penicillin G (10 µg), streptomycin (10 µg) and vancomycin (30 µg). Polar lipids consist of phosphatidylethanolamine (56.6 %) and phosphatidylglycerol (43.4 %). Isoprenoid quinones consist of Q-8 (81.5 %), Q-9 (11.1 %) and Q-10 (7.4 %). Cellular fatty acids consist of C16 : 0 (21.7 %), C18 : 17c (19.4 %), summed feature 3 (C16 : 17c and/or C15 : 0 iso 2-OH) (24.1 %), C12 : 0 3-OH (7.1 %), C17 : 18c (3.8 %), C16 : 0 N alcohol (3.7 %), C17 : 0 10-methyl (3.5 %), C12 : 0 (2.5 %), C14 : 0 (2.1 %), C16 : 17c alcohol (2.1 %), C17 : 0 (1.6 %) C18 : 0 (1.5 %), C19 : 0 10-methyl (1.2 %), C11 : 0 3-OH (1.0 %), C13 : 1 (1.0 %), C10 : 0 3-OH (0.8 %), C16 : 0 3-OH (0.8 %) and C10 : 0 (0.6 %).
The type strain, BD1T (=BCRC 17491T=JCM 13378T), was isolated from shallow coastal water from An-Ping Harbour, Tainan, Taiwan.
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ACKNOWLEDGEMENTS |
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