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Department of Molecular Biosciences and Bioengineering, University of Hawai‘i at Manoa, 1955 East West Road, Honolulu, HI 96822, USA
Correspondence
Qing X. Li
qingl{at}hawaii.edu
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ABSTRACT |
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9c, C16 : 0 and C17 : 18c. On the basis of this polyphasic study, strain P2S11T (=ATCC BAA-1480T=DSM 18821T) represents a novel species of the genus Ferrimonas, for which the name Ferrimonas senticii sp. nov. is proposed.
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The genus Ferrimonas was established in 1995 with the description of the type species Ferrimonas balearica (Rosselló-Mora et al., 1995
). To date, three other species, Ferrimonas marina (Katsuta et al., 2005), Ferrimonas futtsuensis and Ferrimonas kyonanensis (Nakagawa et al., 2006), have been isolated, characterized and described.
The male puffer fish weighed 306 g. It was dissected into separate organs and body parts. Subsamples of each organ, as well as 5–10 g samples of the mucus, were homogenized with sterile artificial seawater (50–100 ml; Coralife) and serial dilutions were plated onto ORI agar (Simidu & Tsukamoto, 1985) and 1/2 MA (Difco). The media were incubated at 30 °C for 1–5 days. Genomic DNA was isolated from pure cultures using a phenol/chloroform mixture (Marmur, 1961). Amplification of the 16S rRNA gene from genomic DNA was performed by PCR using primers 27F and 1492R (Lane, 1991) and Mastermix Taq DNA polymerase (Eppendorf). Amplification of the gyrase subunit B gene (gyrB) was achieved using the UP1Ei and UP2ri primers as described in Katsuta et al. (2005). All PCR products were purified using the Ultraclean PCR purification kit (Mo Bio Lab) and sequenced by the Advanced Studies of Genomics, Proteomics and Bioinformatics Center at the University of Hawai'i at Manoa. For gyrB gene sequencing, a different set of primers was used from the amplification primers: UP1s and UP2rs were used as described in Katsuta et al. (2005). Sequences were edited manually and assembled using Seqman II (Lasergene) and subjected to a BLAST search for comparison with sequences in the public domain (Altschul et al., 1997). Sequence sizes were 1405 and 1063 bp for the 16S rRNA and gyrB genes, respectively.
The phylogenetic relationship of strain P2S11T to other species of the genus Ferrimonas was determined based on comparative 16S rRNA and gyrB gene sequence analyses. These analyses were performed with programs in the PHYLIP 3.63 package (Felsenstein, 2004). Pairwise similarities were calculated using CLUSTAL W multiple sequence alignment software (Chenna et al., 2003). Evolutionary distances were calculated by the maximum-likelihood method with DNADIST. Bootstrap analyses were based on 100 replicates using SEQBOOT, DNADIST and CONSENSE. Phylogenetic trees were constructed with jumbled orders of sequences and the neighbour-joining method (Saitou & Nei, 1987).
Fig. 1 shows the phylogenetic trees based on the 16S rRNA (Fig. 1a) and gyrB (Fig. 1b) gene sequences. The 16S rRNA gene sequence of strain P2S11T was most closely related to that of F. marina DSM 16917T (Katsuta et al., 2005) with 93.5 % similarity. For the gyrB gene sequence, strain P2S11T was most closely related to F. balearica DSM 9799T with 82.9 % similarity (Rosselló-Mora et al., 1995). Based on these low sequence similarities (<97 %), strain P2S11T should be considered distinct from other species of the genus Ferrimonas (Stackebrandt & Goebel, 1994).
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; Tamaoka & Komagata, 1984). The DNA G+C content of strain P2S11T was 55 mol%, a value lower than that of all other species of the genus Ferrimonas, for which the DNA G+C content varies from 57 to 60 mol%. Fatty acid methyl ester analysis for strain P2S11T was performed by MIDI Laboratories using whole cells grown on MA for 24 h at 28 °C (Sasser, 1997). The fatty acid content of strain P2S11T and that of other species of the genus Ferrimonas that are available are summarized in Table 1. The major fatty acids for F. balearica DSM 9799T and F. marina DSM 16917T were C18 : 19c (17.6 and 16.3 %, respectively), C16 : 0 (13.4 and 13.9 %, respectively) and C15 : 0 iso (9.8 and 11.5 %, respectively). The major fatty acids for F. futtsuensis FUT3661T and F. kyonanensis Asr22-7T were C16 : 19c (20.4 and 28.5 %, respectively), C16 : 0 (19.4 and 15.6 %, respectively) and C18 : 19c (15.9 and 10.9 %, respectively). For strain P2S11T, the main three fatty acids were C16 : 19c (16.9 %), C16 : 0 (13.2 %) and C17 : 18c (10.6 %). Fatty acid C16 : 19c was not detected in the analysis of F. balearica and F. marina.
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). After filtration and dilution in acetone, the extract was analysed by LC-MS using a HPLC (1100 series; Agilent) coupled to a diode array detector and a single quadrupole mass spectrometer (model SL; Agilent). The HPLC column used was a Vydac C18 Mass Spec column. The most abundant quinone found in F. balearica and F. marina was menaquinone MK7 (Rosselló-Mora et al., 1995; Katsuta et al., 2005), similar to F. futtsuensis and F. kyonanensis (Nakagawa et al., 2006). MK7 was also the most abundant menaquinone in strain P2S11T.
Cells of strain P2S11T were Gram-negative rods and non-motile. Motility was checked by the hanging drop method using light microscopy under a 100x objective with oil immersion and by stab inoculation into modified motility test agar (l–1: 10 g tryptone, 20 g NaCl and 5 g agar). After two days growth on MA at 30 °C, cells were around 1.7 µm in diameter. Salt was required for growth and the range of salt tolerance was 1.5–7.0 % (w/v), with an optimum value of 2.0 %. The cells grew within a temperature range of 20 °C (slow growth, 2–3 days) to 37 °C. The temperatures tested were 5, 20, 25, 30, 37 and 42 °C on MA plates over 1 week. Other strains of the genus Ferrimonas are facultative anaerobes capable of Fe(III) reduction. Strain P2S11T grew anaerobically on MA (in a Gas-Pak Pouch; BD). The iron-reducing capability of strain P2S11T was tested anaerobically with lactate and iron (III) citrate (Lovley & Phillips, 1986), but the novel strain did not grow under these conditions.
Physiological and biochemical tests performed are summarized in Table 2. The presence or absence of constitutive enzymes was determined using API ZYM (bioMérieux) and the profile of substrates utilized was obtained with API 20NE (bioMérieux). The oxidation of carbon substrates was determined with Biolog GN microplates (Table 2). Hydrogen sulfide production was tested with modified triple-sugar iron (TSI) agar slants (EMD). Since TSI contains only 0.5 % NaCl, 2 % NaCl (w/v) was added to the medium for optimal growth. The TSI tubes were incubated at 30 °C. After 24 h, a black precipitate indicating the presence of H2S was observed. No fermentation occurred. In comparison, F. marina does not produce H2S and F. balearica does.
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Description of Ferrimonas senticii sp. nov.
Ferrimonas senticii (sen.ti.ci.i. N.L. gen. n. senticii of Sentic, named in honour of Sinisa Sentic for scientific and philosophical discussions).
Aerobic, facultatively anaerobic. Off-white colonies. Gram-negative rods, cell size around 1.7 µm in length and non-motile. Circular colonies form on MA at 30 °C after 1–2 days. Optimum temperature range is 20–37 °C. NaCl is required for growth in a range of 1.7–7.0 % (w/v), with an optimum at 2.0 %. Strain P2S11T utilizes DL-lactic acid, acetic acid, Tween 80, inosine, uridine, thymidine and pyruvic acid methyl ester as carbon sources in the Biolog GN2 test. Produces H2S on TSI+2 % NaCl agar. The DNA G+C content is 54.9 mol%. The predominant fatty acids are C16 : 19c, C16 : 0 and C17 : 18c.
The type strain, P2S11T (=ATCC BAA-1480T=DSM 18821T), was isolated from the slime of a puffer fish, Arothron hispidus, caught off the coast of the windward side of the island of O'ahu, Hawai'i.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. (2003). Multiple sequence alignment with the CLUSTAL series of programs. Nucleic Acids Res 31, 3497–3500.
Felsenstein, J. (2004). PHYLIP (phylogeny inference package), version 3.63. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
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Katsuta, A., Adachi, K., Matsuda, S., Shizuri, Y. & Kasai, H. (2005). Ferrimonas marina sp. nov. Int J Syst Evol Microbiol 55, 1851–1855.
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Nakagawa, T., Iino, T., Suzuki, K. & Harayama, S. (2006). Ferrimonas futtsuensis sp. nov. and Ferrimonas kyonanensis sp. nov., selenate-reducing bacteria belonging to the Gammaproteobacteria isolated from Tokyo Bay. Int J Syst Evol Microbiol 56, 2639–2645.
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