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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 was the most abundant fatty acid (32.6–35.7 %). The other major fatty acids included C16 : 17c (27.5–29.4 %) and C16 : 0 (20.1–22.0 %). The two isolates had 16S rRNA gene sequence similarity of 96.8 % and shared 94.1–96.8 % sequence similarity with the most closely related species, Oceanimonas doudoroffii, Oceanimonas baumannii, Oceanimonas smirnovii and Oceanisphaera litoralis. They could be distinguished from these species in that they were capable of fermentative metabolism, had relatively high DNA G+C contents (62.0–64.0 mol%) and contained C18 : 17c instead of C16 : 17c as the most abundant fatty acid. Characterization data accumulated in this study revealed that the two denitrifying isolates could be classified as representatives of two novel species in a new genus, Zobellella gen. nov., with Zobellella denitrificans sp. nov. (type strain ZD1T=BCRC 17493T=JCM 13380T) as the type species and Zobellella taiwanensis sp. nov. (type strain ZT1T=BCRC 17494T=JCM 13381T) as a second species.
Electron micrographs of cells of strains ZD1T and ZT1T are available as supplementary material in IJSEM Online.
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)]. Among these genera, only Ferrimonas, Colwellia, Moritella and Shewanella are known to include fermentative facultative anaerobes (Ivanova et al., 2004).
Denitrification is the dissimilatory reduction of nitrate or nitrite to the gaseous end product(s) nitrous oxide (N2O) or dinitrogen gas. Many Alteromonas-like species classified as Shewanella, Pseudoalteromonas and Ferrimonas have the ability to reduce nitrate to nitrite. However, only some of them, such as Shewanella denitrificans (Brettar et al., 2002), Shewanella sediminis (Zhao et al., 2005) and Shewanella decolorationis (Xu et al., 2005), are capable of denitrification. These bacteria can achieve anaerobic growth using not only nitrate, but also nitrite or nitrous oxide as the electron acceptor.
Two denitrifying bacterial strains were isolated from sediment samples collected from various estuarine mangrove ecosystems during a survey of diversity of denitrifying bacteria in estuarine and coastal environments in Taiwan. Data from this study indicated that the two isolates could achieve anaerobic growth by undertaking either denitrification or fermentation. Evidence presented in this study also showed that the two isolates could be classified as the type strains of two species in a new genus belonging to the Alteromonas-like Gammaproteobacteria.
Polypeptone-yeast extract (PY) broth contained the following constituents (g per l deionized water): polypeptone (Nihon Seiyaku), 3; Bacto yeast extract (Difco), 1; NaCl, 20; MgCl2.6H2O, 2; CaCl2, 0.005; Na2MoO4.7H2O, 0.005; CuCl2.2H2O, 0.005; g FeCl3.6H2O, 0.005; and 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO; Sigma-Aldrich), 4.5. The medium was adjusted to pH 7.0. Bacto agar (Difco) was added to this medium at 3 and 15 g l–1 for the preparation of stab and plate media, respectively. Polypeptone-yeast extract-nitrate (PYN) broth was prepared by adding KNO3 at 2 g l–1 to PY broth. Polypeptone-yeast extract-glucose (PYG) broth was prepared in two parts. The first part contained 3 g polypeptone, 1 g Bacto yeast extract, 20 g NaCl, 2 g MgCl2.6H2O, 0.005 g CaCl2, 0.005 g Na2MoO4.7H2O, 0.005 g CuCl2.2H2O, 0.005 g FeCl3.6H2O and 4.5 g MOPSO dissolved in 900 ml deionized water and adjusted to pH 7.0. The second part contained 5 g glucose dissolved in 100 ml deionized water. The two parts were autoclaved separately and mixed at room temperature. Carbohydrate-mineral (CM) liquid media were made up of two parts. Part I contained 0.54 g NH4Cl, 20 g NaCl, 2 g MgCl2.6H2O, 3 g K2SO4, 0.2 g K2HPO4, 0.01 g CaCl2, 0.005 g FeCl3.6H2O, 0.005 g Na2MoO4.7H2O, 0.005 CuCl2.2H2O and 4.5 g MOPSO dissolved in 900 ml deionized water and adjusted to pH 7.0, and part II contained 5 g glucose or other test carbohydrate (D-arabinose, L-arabinose, cellobiose, galactose, lactose, maltose, mannose, melezitose, melibiose, ribose, starch, sucrose, trehalose, xylose, adonitol, dulcitol, mannitol, myo-inositol, sorbitol) dissolved in 100 ml deionized water. The two parts were autoclaved separately and mixed at room temperature.
Sediment samples collected from various estuarine mangrove ecosystems were processed within a few hours. Some wet mass (approx. 5 g) of each sample was vigorously shaken in 95 ml sterile NaCl-MOPSO buffer (20 g NaCl and 0.45 g MOPSO in 1 l deionized water, pH 7.0). The shaken solutions were decimally diluted with the same buffer and a volume (1 ml) of each dilution (103–105 times) was transferred to a rimless tube (16 mmx10 cm) containing PYN broth medium (5 ml) in which an inverted Durham insert 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 (accumulated in Durham inserts) were streaked (one loopful) on PY plate medium. Individual colonies appearing on each plate were picked and purified by successive streaking on PY plates. More than 100 strains that exhibited growth and produced gas in PYN broth were isolated using this enrichment cultivation method. These isolates were maintained in PY stab medium and stored at 25 °C. Two of these isolates, strains ZD1T and ZT1T, collected from the estuarine mangrove ecosystems of Chungkang, Miaoli County, and Kuantu, Taipei, respectively, were used for the present study.
Strains ZD1T and ZT1T were cultivated aerobically in PY broth at 30 °C in the dark for 2 days. The cultures were centrifuged to harvest the cells. Total genomic DNA was extracted and purified from cells using a Puregene DNA isolation kit (Gentra Systems) in accordance with the manufacturer's instructions. DNA hydration solutions of about 500 µg ml–1 were used for PCR amplification. PCR amplification of the bacterial 16S rRNA gene was conducted using a universal primer pair at positions 8–27 and 1488–1510 [Escherichia coli numbering system (Shieh et al., 2003a)]. Accessory PCR amplification was performed using either of the primer pairs at positions 8–27 and 685–704 (Lane, 1991) or positions 907–926 and 1488–1511 (Lane, 1991). The PCR mixture contained 2.5 µl DNA hydration solution, 2 µl mixture of one of the primer pairs (5 µM of each primer), 1 µl mixture of the four dNTPs (Gene Teks Bioscience) (2.5 mM each), 5 µl 10x Taq buffer (Gene Teks Bioscience), 5 µl 10x BSA (Promega) and 0.5 µl (2 U) Taq DNA polymerase (Gene Teks Bioscience). Each sample was made up to 50 µl with sterile distilled water. PCR amplification was performed in a GeneAmp PCR System 2700 (Applied Biosystems) with the following temperature profile: initial denaturation at 94 °C for 10 min, 35 cycles of denaturation (1 min at 92 °C), annealing (1.5 min at 52 °C) and extension (1.5 min at 72 °C) and a final extension at 72 °C for 4 min. Aliquots (2 µl) of the PCR products were checked for size and purity by electrophoresis at 100 V for 30 min on 1 % agarose gels in TAE buffer (MDBio). Gels were stained with ethidium bromide (1 µg ml–1) for 5 min in TAE buffer and DNA bands appearing on the gels were examined under an image analysing system consisting of a UV transilluminator (Spectroline), a dark box (Kodak EDAS 290) and a zoom digital camera (Kodak DC290).
Sequencing of the 16S rRNA genes, alignment and comparison of the resulting sequences and 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 carried out as described by Shieh et al. (2004). Bootstrap confidence values (Felsenstein, 1985) were obtained with 100 resamplings with an option of stepwise addition. Phylogenetic trees were also reconstructed using maximum-parsimony (Fitch, 1971) and maximum-likelihood (Felsenstein, 1981) methods.
Cells grown in PY broth at 30 °C for 2 days were harvested by centrifugation. Total lipids in the cells were extracted by the method of Bligh & Dyer (1959). Individual lipids were separated by two-dimensional TLC in solvent systems described by Vaskovsky & Terekhova (1979). They were detected on the TLC using 10 % H2SO4 in methanol with subsequent heating to 180 °C and using 1.3 % molybdenum blue spray reagent (Sigma) for phospholipids, 2 % ninhydrin (Fluka) in acetone for amino-containing lipids and Dragendorf's reagent (Fluka) for choline lipids. Phospholipids were quantified by the method of Vaskovsky et al. (1975). Fatty acids in whole cells grown on PY plate medium at 30 °C for 2 days were extracted, saponified and esterified, followed by GC analysis of the fatty acid methyl esters according to the instructions of the MIDI system (Sasser, 1997). This work was performed at the Bioresources Center for Research and Collection (BCRC), Food Industry Research and Development Institute, Taiwan. The DNA G+C content was determined by HPLC analysis (Shieh & Liu, 1996), which was also performed at the BCRC. DNA–DNA relatedness between strains ZD1T and ZT1T was determined as described by Shieh et al. (2003a).
Growth and other phenotypic characteristics of strains ZD1T and ZT1T were examined by the methods of Shieh et al. (2000) with modifications and additional tests as described below. The ability to grow at different temperatures was determined in PY broth and recorded daily for up to 7 days at 15, 20, 25, 30, 35, 40, 45 and 50 °C and for 20 days at 4 and 10 °C, unless significant growth had been observed. Growth in different NaCl concentrations was determined in PY broth containing 0–15 % (w/v) NaCl. Anaerobic growth in PY, PYN and PYG broth media in the presence or absence of N2O was examined as described by Shieh et al. (2004). Growth on various carbohydrates as sole carbon and energy sources was determined in CM media. Denitrifying activity was determined by C2H2 blockage and N2O reduction procedures (Shieh & Liu, 1996). H2S production from thiosulfate was tested as described by Shieh et al. (2004). Urease was determined with modified Christensen urea agar (Smibert & Krieg, 1994) containing 25 g NaCl l–1. Tests for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase were performed in broth media containing Bacto decarboxylase base Moeller (10.5 g l–1; Difco), NaCl (20 g l–1), MgCl2.6H2O (2 g l–1) and the appropriate L-amino acid (10 g l–1). Other constitutive enzyme activities were detected using the API ZYM system (bioMérieux Vitek). Cell suspensions used for these tests were prepared in a mineral medium (0.54 g NH4Cl, 20 g NaCl, 2 g MgCl2.6H2O, 3 g K2SO4, 0.2 g K2HPO4, 0.01 g CaCl2, 0.005 g FeCl3.6H2O, 0.005 g Na2MoO4.7H2O, 0.005 g CuCl2.2H2O and 4.5 g MOPSO, dissolved in 1000 ml deionized water and adjusted to pH 7.0). Antibiotic susceptibility tests were performed by disc diffusion methods as described previously (Shieh et al., 2003a, b). All test cultures were incubated aerobically at 30 °C in the dark for 7 days, unless stated otherwise.
Effects of pH, temperature and NaCl on growth were determined in PY broth under aerobic conditions. Strains ZD1T and ZT1T grew at pH 6–10, with optimum growth at pH 7–8. No growth was observed at pH 5.0. Growth was observed at 10–45 °C, with optimum growth at 30–35 °C. Both strains grew in 0–12 % NaCl, but only strain ZT1T grew in 13–14 % NaCl. Although NaCl was not indispensable for growth, optimal growth was observed at 1–3 % NaCl.
Strains ZD1T and ZT1T exhibited good growth in PYN (maximum OD600 1.02–1.05) and PYG (maximal OD600 0.40–0.44) broth media under anaerobic conditions, whereas anaerobic growth in PY broth was relatively weak (maximal OD600 0.14–0.17) unless N2O was present in the culture systems (maximum OD600 0.34–0.73) (Fig. 1). This indicated a lack of sufficient 
and fermentable substrates for the strains in PY broth cultures. The results also suggested that the strains were capable of anaerobic growth by carrying out denitrifying metabolism with 
or N2O as the terminal electron acceptor. Anaerobic growth in PYG broth was accompanied by a remarkable decrease in the pH of the medium, from pH 7.0 to 5.4 within 72 h (data not shown), regardless of the large buffer content (approx. 20 mM MOPSO) in the medium. The strains could have achieved anaerobic growth in PYG by fermenting glucose, with considerable production of organic acids. In addition to glucose, both strains also showed anaerobic growth by fermenting sucrose or mannitol as substrates (data not shown).
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) shows the phylogenetic positions between strains ZD1T and ZT1T and species of Oceanimonas and Oceanisphaera and other related taxa belonging to the Alteromonas-like Gammaproteobacteria. Similar results were obtained from maximum-likelihood and maximum-parsimony algorithms (not shown). The 16S rRNA gene-based phylogeny revealed that the two isolates could be classified either as different species in a novel genus or as novel species of Oceanimonas (Fig. 2).
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) and Oceanisphaera (56.4 mol%; Romanenko et al., 2003). Both Oceanimonas doudoroffii and Oceanimonas baumannii had a G+C content of 54.0 mol% according to Brown et al. (2001), whereas the G+C contents of the two determined by Ivanova et al. (2005) were 59.0 and 57.0 mol%, respectively; this discrepancy between the G+C contents remains to be clarified. Strains ZD1T and ZT1T, like species of Oceanimonas and Oceanisphaera, contained C16 : 0, C16 : 17c and C18 : 17c as the major cellular fatty acids (Table 1). However, the most abundant fatty acid of the isolates was C18 : 17c (32.6–35.7 %), whereas it was C16 : 17c (40.1–45 %) in species of Oceanimonas and Oceanisphaera. Moreover, only the novel isolates contained C14 : 0 3-OH (6.4–6.6 %). Both isolates contained phosphatidylethanolamine (47.2–51.0 %), phosphatidylglycerol (40.8–41.7 %) and diphosphatidylglycerol (8.2–11.1 %) as the major polar lipids. These polar lipids also accounted for the total phospholipids in Oceanimonas (Brown et al., 2001; Ivanova et al., 2005) and Oceanisphaera (Romanenko et al., 2003). However, relatively low levels of diphosphatidylglycerol (trace to 3.1 %) were detected in Oceanimonas doudoroffii and Oceanimonas baumannii (Brown et al., 2001).
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Strains ZD1T and ZT1T were Gram-negative and oxidase- and catalase-positive. Colonies produced on PY plate medium were circular, off-white and non-luminescent. Cells grown in PY broth, which appeared to be straight, motile rods, normally possessed a single polar flagellum, as revealed by TEM (see electron micrographs available as Supplementary Fig. S1 in IJSEM Online). Both strains were facultative anaerobes capable of fermenting glucose, sucrose, ribose, maltose, melezitose, starch, mannitol and myo-inositol. Their activity to reduce 
to N2O and further to N2 was detected by the C2H2 blockage and N2O reduction procedures. Additional phenotypic characterization data are given below in the species descriptions.
Phenotypically, strains ZD1T and ZT1T could be differentiated from each other by different reactions in several phenotypic tests, including growth in 14 % NaCl, fermentation of galactose, melibiose and trehalose and utilization of galactose, melibiose, trehalose and adonitol as sole carbon and energy sources. Both strains could be distinguished from species of Oceanimonas and Oceanisphaera in that they were capable of fermentative metabolism. The ability to grow in the absence of NaCl also distinguished the strains from these species, with the exception of Oceanimonas smirnovii. Other phenotypic characteristics useful for differentiating strains ZD1T and ZT1T from species of Oceanimonas and Oceanisphaera are listed in Table 2.
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Zobellella denitrificans and Zobellella taiwanensis are the only species in the Alteromonas-like Gammaproteobacteria that possess DNA G+C contents greater than 60 mol%. They have so far been found only in the sediment of estuarine mangrove forests. However, their tolerance of rather wide ranges of temperatures and salinities and ability to grow in the absence of oxygen and organic growth factors suggest that these bacteria may also occur in other habitats, including saline and non-saline environments.
Description of Zobellella gen. nov.
Zobellella (Zo.bell.el'la. N.L. dim. ending -ella; N.L. fem. n. Zobellella named after C. E. ZoBell, a pioneer marine microbiologist).
Members are heterotrophic, Gram-negative rods belonging to the Gammaproteobacteria. Cells grown in broth cultures are motile by means of a single, polar flagellum. NaCl stimulates growth, but is not an absolute requirement. Facultative anaerobes capable of both respiratory and fermentative metabolism. Mesophilic; grow optimally at 30–35 °C, but do not grow at 4 or 50 °C. Oxidase- and catalase-positive. C18 : 17c is the most abundant fatty acid; C16 : 17c and C16 : 0 are the next most abundant fatty acids. Major constituents of polar lipids are the phospholipids phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The DNA G+C contents of the strains so far examined are 62.0–64.0 mol%. The type species is Zobellella denitrificans.
Description of Zobellella denitrificans sp. nov.
Zobellella 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 straight rods, approximately 1.6–2.6 µm long by 0.6–0.8 µm wide. Colonies produced on PY agar plates at 30 °C for 48–60 h are approximately 1.5–4.0 mm in diameter, circular, off-white and non-luminescent, with an entire edge. Swarming does not occur. Capable of complete denitrification, i.e. capable of reducing 
to N2 via 
and N2O. Able to ferment glucose, cellobiose, galactose, maltose, mannose, melezitose, melibiose, ribose, sucrose, trehalose, starch, mannitol, myo-inositol and sorbitol with production of acid, but no gas. Unable to ferment D-arabinose, L-arabinose, lactose, xylose or dulcitol. Growth occurs at 10–45 °C, with optimum growth at 30–35 °C. Growth occurs at pH 6–10, with optimum growth at pH 7–8. Growth occurs in 0–12 % NaCl, with optimum growth in 1–3 %; no growth is observed in 13–14 % NaCl. Weakly positive for urease. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, agarase, amylase, DNase, gelatinase and lipase. H2S is not produced from thiosulfate. Indole is not produced from tryptophan. Cellobiose, glucose, galactose, maltose, mannose, mannitol, melezitose, melibiose, myo-inositol, ribose, sorbitol, starch, sucrose and trehalose can be utilized as sole carbon and energy sources, but D-arabinose, L-arabinose, lactose, xylose, adonitol and dulcitol cannot. The following constitutive enzyme activities are detected in API ZYM tests: acid phosphatase, alkaline phosphatase, esterase (C4), esterase lipase (C8), 
-galactosidase, -glucosidase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase. Susceptible to ampicillin (10 µg), carbenicillin (100 µg), cephalothin (30 µg), chloramphenicol (30 µg), colistin (10 µg), gentamicin (10 µg), nalidixic acid (30 µg), neomycin (30 µg), penicillin G (10 U), polymyxin B (300 U) and tetracycline (30 µg); intermediate susceptibility to kanamycin (30 µg); resistant to clindamycin (2 µg), erythromycin (15 µg), lincomycin (2 µg), novobiocin (30 µg), oxacillin (1 µg), streptomycin (10 µg) and vancomycin (30 µg).
The type strain is ZD1T (=BCRC 17493T=JCM 13380T), isolated from a sediment sample collected from the estuarine mangrove ecosystem of Chungkang, Miaoli County, Taiwan. It has a DNA G+C content of 64.0 mol%.
Description of Zobellella taiwanensis sp. nov.
Zobellella taiwanensis (tai.wan.en'sis. N.L. fem. adj. taiwanensis pertaining to Taiwan, where the type strain was isolated).
Description is as for the genus and the species description of Zobellella denitrificans with the following modifications. Growth occurs in 0–14 % NaCl, with optimum growth at 1–3 %. Cells are 1.0–1.9 µm long by 0.6–0.7 µm wide. Positive for urease test. Unable to ferment galactose, melibiose or trehalose. Adonitol can be utilized as a sole carbon and energy source, but not galactose, melibiose or trehalose. In API ZYM tests, -galactosidase, esterase lipase (C8), naphthol-AS-BI-phosphohydrolase and trypsin activities are not detected. Resistant to ampicillin (10 µg), carbenicillin (100 µg) and penicillin G (10 U).
The type strain is ZT1T (=BCRC 17494T=JCM 13381T), isolated from a sediment sample collected from the estuarine mangrove ecosystem of Kuantu, Taipei, Taiwan. It has a DNA G+C content of 62.0 mol%.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Brettar, I., Christen, R. & Höfle, M. G. (2002). Shewanella denitrificans sp. nov., a vigorously denitrifying bacterium isolated from the oxic–anoxic interface of the Gotland Deep in the central Baltic Sea. Int J Syst Evol Microbiol 52, 2211–2217.[Abstract]
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, 
), PYN (
) and PYG (
) broth media under argon in the presence (