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Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, PR China
Correspondence
Zongze Shao
shaozz{at}163.com
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ABSTRACT |
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Transmission electron micrographs of cells of strains M-5T and WP0211T and dendrograms showing the phylogenetic positions of the two strains based on 16S rRNA gene sequences are available as supplementary figures in IJSEM Online. Tables detailing the DNA–DNA relatedness values and the cellular fatty acid contents of Thalassospira species are also available.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Amongst these isolates, strains M-5T and WP0211T had similar colony morphologies and shared a high level of 16S rRNA gene sequence similarity (98.5 %). In this study, we present the polyphasic characterization of strains M-5T and WP0211T. Strain M-5T was isolated from the surface water of a waste-oil pool at the oil storage dock in the city of Xiamen, Fujian Province, China, in December 2002. This seawater-based waste-oil pool had suffered long-term pollution with crude oil. Strain WP0211T was retrieved from a deep-sea sediment sample, which was collected by a multi-core sampler from the West Pacific (region 973, station WP02-3; 147° 58' 55'' E 12° 59' 54'' N; water depth 4480 m), during cruise DY105-13 of the vessel HAIYANG Number 4 in 2002. The sediment samples were loaded into sterile falcon tubes aboard the ship and were stored at –20 °C until subsequent analysis on land.
Bacteria were enriched by culturing in artificial seawater medium (ASM; Liu & Shao, 2005a), supplemented with 10 g diesel fuel l–1 (strain M-5T) or 5 g pyrene l–1 (strain WP0211T) as the sole carbon source. HLB medium (modified from Luria–Bertani medium by increasing the NaCl concentration to 30 g l–1; Liu & Shao, 2005a) was used for routine cultivation of the isolates and for most of the phenotypic tests. All cultures were incubated at 28 °C with rotation at 200 r.p.m. unless noted otherwise. As was previously found for Thalassospira lucentensis QMT2T (López-López et al., 2002), both novel strains formed very small colonies in the oligotrophic medium, but showed fairly good growth in the HLB media.
Genomic DNA was prepared according to the method of Ausubel et al. (1995) and the 16S rRNA gene was amplified by PCR using primers that have been described previously (Liu & Shao, 2005a). Sequence data were manually aligned with nucleotide sequences obtained from GenBank using DNAMAN (version 5.1; Lynnon Biosoft). Phylogenetic dendrograms of 16S rRNA gene sequences were constructed using three different algorithms: the neighbour-joining method (Saitou & Nei, 1987) using DNAMAN and the maximum-likelihood (Felsenstein & Churchill, 1996) and the maximum-parsimony (Fitch, 1971) methods using the PHYLIP software package (version 3.6a2.1; Felsenstein, 2004). Bootstrap analysis was used to evaluate the tree topology of the data obtained from the three algorithms based on 1000 resamplings. Regions of 1439 and 1457 nucleotides were obtained of the 16S rRNA gene for strains M-5T and WP0211T, respectively. The sequences of strains M-5T and WP0211T were highly similar (98.5 % similarity) and shared the highest levels of homology with T. lucentensis QMT2T (96.1 % and 96.2 %, respectively). The next highest similarities were to Aquaspirillum itersonii subsp. nipponicum DSM 11590T (90.1 % and 90.2 %, respectively), Terasakiella pusilla IFO 13613T (89.7 % and 89.9 %, respectively) and Aquaspirillum peregrinum subsp. integrum DSM 11589T (89.0 % and 89.0 %, respectively). The 16S rRNA gene sequences shared lower levels of similarity (<90.0 %) with all recognized species of the Alphaproteobacteria. In all the phylogenetic trees, the clade containing strains M-5T, WP0211T and T. lucentensis QMT2T had 100 % bootstrap support (a neighbour-joining tree is shown in Supplementary Fig. S1 in IJSEM Online).
The morphology of cells from 2-day-old cultures of the novel strains grown on HLB agar was studied using an Olympus inverted microscope and by electron microscopy. Cells of both strains were Gram-negative, had a curved-rod shape and varied from 0.8 to 2.3 µm in length and from 0.3 to 0.8 µm in width. Strain M-5T was motile by means of a single polar flagellum. Conversely, strain WP0211T was non-motile and no flagellae were observed (see Supplementary Fig. S2 in IJSEM Online). The optimal growth temperature was determined over the temperature range 4–55 °C. Sodium requirement was examined at 0, 0.5, 1, 5, 7, 10, 15 and 20 % NaCl (w/v). The following physiological and biochemical properties were examined according to standard methods: glucose fermentation, denitrification activities, catalase and oxidase activities, gelatin liquefaction activity, the ability to hydrolyse agar, gelatin, starch, Tweens 40 and 80 and arginine dihydrolase activities.
We also investigated the ability of the novel strains to use various organic substrates as sole carbon sources at a concentration of 0.1 % (w/v) in 5 ml ASM medium. The strains were also characterized using Biolog GN plates as described by Ivanova et al. (1998). Antibiotic activities were assessed as described by Kobayashi et al. (2003). These results are given in the species description.
Cellular fatty acids were analysed by the identification service laboratories of the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany). The fatty acid profiles of both strains are shown in Supplementary Table S1 in IJSEM Online. The predominant fatty acids in both novel isolates were C18 : 1
7c, C16 : 0 and C18 : 0 which accounted for >65 % of the total fatty acids. In T. lucentensis QMT2T, the principal fatty acid components were C18 : 17c, C16 : 17c and C16 : 0. Similar fatty acid components (C18 : 17c, C16 : 0 and C18 : 0) predominate in some species of the genus Rhodospirillum, such as R. salexigens, R. salinarum and R. sodomense. In some other Rhodospirillum species, such as R. rubrum, R. photometricum, R. fulvum and R. molischianum, the main components are C18 : 17c, C16 : 17c and C16 : 0 (Imhoff, 1986, 1998; Imhoff & Bias-Imhoff, 1995). Consequently, no definitive conclusions could be drawn from the major fatty acid profiles of the two novel isolates. There were no significant differences in the minor fatty acid content of strains M-5T, WP0211T and T. lucentensis QMT2T. Notably, in all of these three strains, the fatty acids C17 : 0 cyclo and C19 : 0 cyclo were detected in significant amounts, which may be a characteristic chemotaxonomic marker for the genus Thalassospira. These two components were more abundant in strain WP0211T (accounting for 5.69 and 5.97 %, respectively) than in M-5T (1.37 and 1.16 %) or T. lucentensis QMT2T (4.77 and 1.86 %).
The G+C content of the genomic DNA of the two novel strains was determined by HPLC after digestion with P1 nuclease (Tamaoka & Komagata, 1984; Mesbah & Whitman, 1989). The DNA G+C contents of strains M-5T and WP0211T were determined to be 52.6 mol% and 47.0 mol%, respectively (with the same experimental standard deviation of 0.09 %).
DNA–DNA hybridization experiments were performed with genomic DNA from strains M-5T, WP0211T and T. lucentensis QMT2T using a previously described method (Liu & Shao, 2005a). Genomic DNA from Escherichia coli DH5
was used as an outgroup sample. The results are shown in Supplementary Table S2 in IJSEM Online. Each value reported is the mean from at least two independent hybridization experiments. Results indicated that the two novel isolates shared high levels of DNA–DNA relatedness (62–63 %). Notably, both strains had very low levels of DNA–DNA relatedness with E. coli DH5 (<5 %). However, strains M-5T and WP0211T exhibited relatively low levels of hybridization with T. lucentensis QMT2T (21–23 % and 26 %, respectively). This suggested that the new isolates could be classified as novel species of the genus Thalassospira, according to the criteria for the delineation of bacterial species (Wayne et al., 1987).
On the basis of their phenotypic and chemotaxonomic characteristics, their phylogeny based on 16S rRNA gene sequence analysis and their genomic DNA–DNA relatedness, the two new isolates may be categorized as novel members of the genus Thalassospira. We initially considered the possibility that the two strains should be classified as a single species, based on their high levels of 16S rRNA gene sequence similarity (98.5 %). However, their level of DNA–DNA relatedness (62–63 %) falls on the boundary that delineates species (Wayne et al., 1987). Also, the difference in the DNA G+C content between strains M-5T and WP0211T was more than 5 mol%, which is above the levels that indicate heterogeneity within a species (Goodfellow et al., 1997; Rossello-Mora & Amann, 2001). Perhaps more significantly, strains M-5T and WP0211T have a number of distinct physiological and chemotaxonomic properties that allow them to be distinguished from each other, i.e. motility, flagellation, growth at 4 and 40 °C, hydrolysis of Tweens 40 and 80, utilization of 19 different carbon sources (Table 1) and quantitative differences in their fatty acid content (see Supplementary Table S1). Therefore, we propose that these two new strains be designated as representatives of two novel species: Thalassospira xiamenensis sp. nov. for strain M-5T and Thalassospira profundimaris sp. nov. for strain WP0211T.
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Emended description of the genus Thalassospira López-López et al. 2002
Cells are non-motile and non-flagellated or motile by a single polar flagellum. Some species can grow under anaerobic conditions by reducing nitrate. Species are halophilic; require Na+ ions for growth and are able to grow in the presence of up to 10 % NaCl. No requirement for organic growth factors. Carbohydrates are used as the sole carbon source and both nitrate and ammonium are used as sole nitrogen sources. The major hydroxylated fatty acid is C16 : 0 3-OH. The G+C content of the genomic DNA ranges from 47 to 54.7 mol%. On the basis of 16S rRNA gene sequence-based phylogenetic analyses, the genus belongs to the Alphaproteobacteria.
Description of Thalassospira xiamenensis sp. nov.
Thalassospira xiamenensis (xi.a.me.nen'sis. N.L. fem. adj. xiamenensis pertaining to Xiamen, the city where the organism was first isolated).
Cells are 0.8–2.3 µm long and 0.3–0.8 µm wide, non-spore-forming, Gram-negative curved rods that are motile by means of one polar flagellum. Colonies on HLB agar are circular, convex, glistening, viscid, translucent and yellowish. Mesophilic; growth temperature ranges from 4 to 40 °C (optimum 22 °C). NaCl is required for growth, cells grow at 0.5–10 % NaCl (optimum at 2–4 %). Produces acid from D-glucose under aerobic conditions. Positive for catalase and oxidase activities, but negative for arginine dihydrolase, amylase and gelatinase activity. Cannot hydrolyse Tween 40, Tween 80 or agar. Nitrate is reduced to nitrite. Facultatively anaerobic; can grow in anaerobic conditions by reducing nitrate. Sensitive to the following antibiotics (10 µg per 6 mm disc): chloramphenicol, norfloxacin, furazolidone, co-trimoxazole, ofloxacin, midecamycin, ceftriaxone, polymyxin B, doxycycline, tetracycline, neomycin, kanamycin, gentamicin, amikacin, erythromycin, minocycline, carbenicillin, cefalexin, cephradine, ciprofloxacin and cefuroxime, but resistant to: cefoperazone, clindamycin, vancomycin, ceftazidime, cefazolin, penicillin, oxacillin, ampicillin and piperacillin. Among the 95 carbon sources in the Biolog system, the following are utilized: N-acetyl-D-glucosamine, L-arabinose, D-arabitol, D-cellobiose, D-fructose, D-galactose, -D-glucose, myo-inositol, maltose, D-mannitol, D-mannose, sucrose, D-trehalose, turanose, monomethyl succinate, methyl pyruvate, cis-aconitic acid, formic acid, D-gluconic acid, -hydroxybutyric acid, 
-hydroxybutyric acid, 
-hydroxybutyric acid, -ketoglutaric acid, DL-lactic acid, quinic acid, malonic acid, propionic acid, succinic acid, bromosuccinic acid, L-alaninamide, D-alanine, L-alanine, L-alanyl glycine, L-asparagine, L-histidine, hydroxy-L-proline, L-ornithine, L-glutamic acid, glycyl L-glutamic acid, L-proline, L-serine, L-pyroglutamic acid, urocanic acid, thymidine and glycerol. Principal fatty acids are C18 : 17c, C16 : 0, C18 : 0, C16 : 17c and C14 : 0, with minor amounts of C13 : 1, C16 : 0 3-OH, C17 : 0 cyclo and C19 : 0 cyclo and small amounts of 11 methyl C18 : 17c. G+C content of the genomic DNA is 52.6 mol%. Table 1
shows characteristics used to distinguish strain M-5T from other members of the genus Thalassospira.
The type strain, M-5T (=DSM 17429T=CGMCC 1.3998T), was isolated from surface water collected from a waste-oil pool at an oil storage dock in the city of Xiamen on the Eastern coast of China.
Description of Thalassospira profundimaris sp. nov.
Thalassospira profundimaris (pro.fun.di.ma'ris. L. adj. profundus deep; L. gen. n. maris from/of the sea; N.L. gen. n. profundimaris from/of the deep-sea, reflecting from where the type strain was isolated).
Cells are Gram-negative curved rods that are 0.8–2.3 µm long and 0.3–0.8 µm wide. Cells are non-motile, non-flagellated and non-spore-forming. Colonies on HLB agar are circular, convex, glistening, viscid, translucent and opalescent. Mesophilic; grows between 10 and 37 °C (optimum of 22 °C). No growth occurs at 4 or 40 °C. NaCl is required for growth; cells grow in 2–8 % NaCl (optimum 3–4 %). Does not produce acid from D-glucose under aerobic conditions. Actively hydrolyses Tween 40 and Tween 80, but not agar. Catalase- and oxidase-positive, but negative for arginine dihydrolase, amylase and gelatinase activities. Nitrate is reduced to nitrite. Facultatively anaerobic; grows in anaerobic conditions by reducing nitrate. Sensitive to the following antibiotics (10 µg per 6 mm disc): chloramphenicol, norfloxacin, furazolidone, co-trimoxazole, ofloxacin, midecamycin, ceftriaxone, polymyxin B, doxycycline, tetracycline, neomycin, kanamycin, gentamicin, amikacin, erythromycin, minocycline, carbenicillin, cefalexin, ciprofloxacin, cefoperazone, vancomycin, ceftazidime, cefazolin, penicillin, oxacillin, ampicillin and piperacillin. Resistant to the following antibiotics: clindamycin, cefuroxime and cephradine. Among the 95 carbon sources in the Biolog system, the following are utilized: Tween 40, Tween 80, D-arabitol, D-cellobiose, D-fructose, -D-glucose, D-mannitol, D-mannose, monomethyl succinate, methyl pyruvate, formic acid, -hydroxybutyric acid, -hydroxybutyric acid, -ketoglutaric acid, DL-lactic acid, quinic acid, propionic acid, succinic acid, D-gluconic acid, L-alanine, L-alanyl glycine, L-glutamic acid, glycyl L-glutamic acid, L-proline, L-pyroglutamic acid and glycerol. Principal fatty acids are C18 : 17c, C16 : 0, C18 : 0, C19 : 0 cyclo and C17 : 0 cyclo; with minor amounts of C14 : 0, C16 : 17c, C16 : 0 3-OH and C13 : 1 and small amounts of 11 methyl C18 : 17c. G+C content of the genomic DNA is 47 mol%. Table 1 shows characteristics used to distinguish strain WP0211T from other members of the genus Thalassospira.
The type strain, WP0211T (=DSM 17430T=CGMCC 1.3997T), was isolated from a deep-sea sediment from the West Pacific Ocean.
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ACKNOWLEDGEMENTS |
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