Fulvimarina manganoxydans sp . nov . , isolated from a deep-sea hydrothermal plume in the south-west Indian Ocean

An aerobic, Mn(II)-oxidizing, Gram-negative bacterium, strain 8047, was isolated from a deepsea hydrothermal vent plume in the south-west Indian Ocean. The strain was rod-shaped and motile with a terminal flagellum, and formed yellowish colonies. It produced catalase and oxidase, hydrolysed gelatin and reduced nitrate. 16S rRNA gene sequence analysis showed that strain 8047 belonged to the order Rhizobiales of the class Alphaproteobacteria, and was phylogenetically most closely related to the genus Fulvimarina, sharing 94.4 % sequence identity with the type strain of the type species. The taxonomic affiliation of strain 8047 was supported by phylogenetic analysis of four additional housekeeping genes, gyrB, recA, rpoC and rpoB. The predominant respiratory lipoquinone of strain 8047 was Q-10, the major fatty acid was C18 : 1v7c and the DNA G+C content was 61.7 mol%. On the basis of the phenotypic and genotypic characteristics determined in this study, strain 8047 represents a novel species within the genus Fulvimarina, for which the name Fulvimarina manganoxydans sp. nov. is proposed. The type strain is strain 8047 (5CGMCC1.109725JCM 18890).

As the biggest group in the a2-proteobacteria, the order Rhizobiales comprises more than ten families, and the majority of the members of this order were isolated from terrestrial ecosystems (Cho & Giovannoni, 2003;Weon et al., 2007).Members of only a few genera were retrieved from marine environments, which are distributed primarily in the families Rhodobiaceae (Hiraishi et al., 1995;Brettar et al., 2007) and 'Aurantimonadaceae' (Cho & Giovannoni, 2003;Denner et al., 2003).The latter includes four genera, Aurantimonas (Denner et al., 2003), Fulvimarina (Cho & Giovannoni, 2003), Martelella (Rivas et al., 2005) and Aureimonas (Rathsack et al., 2011).Except for the genus Aureimonas, which was created by division from the genus Aurantimonas (Rathsack et al., 2011), the members of the family 'Aurantimonadaceae' were all derived from marine environments.Furthermore, several strains of this family are capable of Mn(II) oxidation (Anderson et al., 2009).The genus Fulvimarina was proposed by Cho & Giovannoni (2003) and its description was emended by Rathsack et al. (2011).At the time of writing, it comprises only one species, Fulvimarina pelagi, which was isolated from water of the Sargasso Sea by using high-throughput culturing methods (Cho & Giovannoni, 2003).
During a survey of the microbial diversity of a deep-sea hydrothermal plume in the south-west Indian Ocean, where total Fe and dissolved Mn were found in high concentrations (Wang et al., 2012), an aerobic, Gram-negative, yellow bacterium, strain 8047 T , was isolated.Based on 16S rRNA gene sequence analysis, the strain was related phylogenetically to F. pelagi HTCC2506 T and formed a branch within the order Rhizobiales.Here, we describe the characterization of strain 8047 T using a polyphasic taxonomic approach.
Seawater was collected at 37 u 479 S 49 u 399 E from ~2800 m below sea level and concentrated in situ 1000-fold with an integrated biological sampling device (Beijing Pioneer High Technology Development Co.) during cruise DY-115-21 of the DaYang YiHao in January 2010.The geochemical characteristics of the hydrothermal plume at the sampling site were described by Wang et al. (2012).Strain 8047 T was isolated from the concentrated sample following growth on seawater GYM agar (0.4 % yeast extract, 1 % malt extract, 0.4 % glucose, 1.5 % agar and 60 % seawater) at 28 u C for 2-3 days.Individual colonies were picked and transferred to YTSS agar [0.5 % yeast extract, 1 % tryptone, 1.5 % agar and 2 % sea salt (Sigma)] for further purification.Culture purity was checked by microscopic examination and KOHlysis test (Smibert & Krieg, 1994).Colonies of strain 8047 T were smooth, circular, yellow, flat and non-translucent after cultivation at 28 u C for 48 h.
Genomic DNA was extracted from strain 8047 T using an AxyPrep bacterial genomic DNA extraction kit (Axygen) and used as the template for amplification of the 16S rRNA gene by PCR with primers 27F and 1492R, as described previously (Cho & Giovannoni, 2003).The amplification product was purified and sequenced at Invitrogen Inc.A sequence search in GenBank with the 16S rRNA gene sequence of strain 8047 T (1449 bp) as a query was performed using the BLAST algorithm.Sequence comparison of the retrieved 16S rRNA gene sequences indicated that strain 8047 T was most closely related to species of the order Rhizobiales of the class Alphaproteobacteria.On the basis of pairwise 16S rRNA gene sequence comparisons using the EzTaxon-e server (Kim et al., 2012), the highest similarity (94.4 %) was found to F. pelagi HTCC2506 T .A phylogenetic tree was reconstructed using the neighbour-joining, maximum-parsimony and maximum-likelihood methods implemented in the software package MEGA version 5.0 (Tamura et al., 2011).Evolutionary distances were calculated by using Kimura's two-parameter model.The resultant tree topologies were evaluated by bootstrap analysis with 1000 resamplings.As revealed by the phylogenetic tree, strain 8047 T formed a stable clade with F. pelagi HTCC2506 T , and the clade was associated closely with the genera Aurantimonas and Martelella, two members of the family 'Aurantimonadaceae' within the order Rhizobiales (Fig. 1).Therefore, F. pelagi HTCC 2506 T , Aurantimonas coralicida DSM 14790 T and Martelella mediterranea DSM 17316 T were used as reference strains for investigation of some properties under the same laboratory conditions.
To learn more about the relationship between strain 8047 T and other strains of the family 'Aurantimonadaceae', we generated a draft genome sequence of strain 8047 T with a 454 Genome Sequencer FLX instrument (Roche Applied Science).The complete sequences of four housekeeping genes, gyrB (2439 bp), recA (1074 bp), rpoC (4197 bp) and rpoB (4140 bp), were obtained from contigs 00036 (66 660 bp), 00085 (130 247 bp) and 00088 (134 521 bp, containing both rpoC and rpoB) in the genome sequence of strain 8047 T .Phylogenetic trees based on the sequences of the housekeeping genes were reconstructed using the same methods as described for the reconstruction of the 16S rRNA gene tree (Fig. S1, available in the online Supplementary Material).The topological structures of the housekeeping gene trees were consistent with that of the 16S rRNA gene tree, and strain 8047 T again clustered with F. pelagi HTCC2506 T .As shown in Table S1, the sequence identities of the housekeeping genes between strain 8047 T and its closest relatives were all below 88.5 %.
Routine cultivation of strain 8047 T was done at 28 u C in YTSS medium.Cell morphology was examined under a light microscope (Olympus BH-2) as well as an electron microscope (Hitachi H-600A) after negative staining with uranyl acetate.Cells of strain 8047 T were rod-shaped (0.5-0.861.8-2.0 mm) after cultivation at 28 u C for 28 h, occurred singly and were motile by polar flagella (Fig. S2).The cells stained Gram-negative after following the procedure described by Doetsch (1981).The growth temperature of strain 8047 T in YTSS broth was determined to be 4-45 u C, with optimum growth 28 u C, as determined by using a water bath.Growth was observed at pH 4.0-9.0,with optimal growth at pH 6.0-7.0, as determined in YTSS medium adjusted to pH 2-12 with KH 2 PO 4 / HCl, KH 2 PO 4 /K 2 HPO 4 and K 2 HPO 4 /NaOH buffer systems (Zhang et al., 2013).The strain grew in sea-salt-free YTSS medium supplemented with 0-10 % (w/v) NaCl, and optimal growth occurred when the medium was supplemented with 2-3 % NaCl.Like F. pelagi HTCC2506 T , NaCl was not essential for growth of strain 8047 T , but the growth rate in the absence of NaCl was much lower than that at the optimum level of NaCl (Cho & Giovannoni, 2003).No growth was detected at 15 % (w/v) NaCl.
Cellular pigments were extracted with acetone from a culture of strain 8047 T grown on YTSS agar, and the UVvisible absorption spectrum was determined by HPLC (1200 Infinity Series with diode array detector; Agilent Technologies) scanning from 350 to 600 nm with a linear gradient of 10-100 % aqueous acetonitrile from 0 to 30 min, 100 % acetonitrile from 30 to 50 min and 10 % aqueous acetonitrile from 50 to 55 min [Phenomenex Luna 5U C8(2) 25064.6 mm column; flow rate, 1 ml min 21 ].
Strain 8047 T was found to produce carotenoid pigments with absorption peaks at 450-460 and 480-490 nm.There were no differences between light-and dark-grown cultures of 8047 T in the absorption spectrum, and no bacteriochlorophyll was detected in cultures of strain 8047 T .
Although the identities of the fatty acids of strain 8047 T were similar to those of its phylogenetic neighbours, their proportions were different, as shown in Table 2.
The genomic DNA G+C content of strain 8047 T was 61.7 mol%, as determined by HPLC (Mesbah et al., 1989) with the modifications of Lee et al. (2005).
Taken together, strain 8047 T is most closely related to F. pelagi HTCC2506 T , and the two strains share similar chemotaxonomic properties.However, several differences exist between the two strains in morphological and physiological characteristics, in addition to their low similarities in 16S rRNA and housekeeping gene sequences.Unlike F. pelagi, strain 8047 T is motile with polar flagella, capable of gelatin hydrolysis and nitrate reduction and not able to utilize glycerol, D-arabinose, D-fructose, D-mannose, D- mannitol or sucrose as sole carbon sources (Table 1).Therefore, it is evident that strain 8047 T represents a novel Cells are Gram-negative, aerobic, rod-shaped and 0.5-0.861.8-2.0 mm.Cells are motile by means of polar flagella.No endospores are observed.Colonies on YTSS medium are circular, smooth, yellowish and usually 1-3 mm in diameter within 2 days at 28 uC.Grows in the presence of 0-10 % (w/v) NaCl, with an optimum at 2-3 % (w/v).The temperature range for growth is 4-45 u C, with optimal growth at 28 u C. Grows at pH 4-9, with optimal growth at pH 6-7.Tests for oxidase, catalase and urease are positive, but tests for agarase, amylase, arginine dihydrolase, caseinase and ornithine and lysine decarboxylases are negative.Nitrate is reduced to nitrite.H 2 S is not produced from thiosulphate.Indole is not produced from tryptophan.Gelatin is liquefied.Tweens 20 and 80 are not hydrolysed.The Voges-Proskauer test is negative.Oxidizes Mn 2+ .Utilizes L-arabinose, cellobiose, D-fucose, D-galactose, D-glucose, geraniol, lactose, D-lyxose, maltose, melibiose, D-ribose, trehalose and D-and L-xylose as sole carbon sources, but not N-acetylglucosamine, amygdalin, arbutin, caproate, aesculin, D-fructose, inositol, mannitol, mannose, L-rhamnose or salicin, according to Biolog GN2 tests and the API 20E system.Ubiquinone 10 (Q-10) is the predominant respiratory lipoquinone.C 18 : 1 v7c is the dominant fatty acid.The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylcholine, an unidentified phospholipid and an unidentified glycolipid.
The type strain is 8047 T (5CGMCC 1.10972 T 5JCM 18890 T ), isolated from a hydrothermal plume in the south-west Indian Ocean.The DNA G+C content of the type strain is 61.7 mol%.

Fig. 1 .
Fig.1.Phylogenetic dendrogram of strain 8047 T and related species based on 16S rRNA gene sequences.The tree was reconstructed using the neighbour-joining method, and Rhodobacter capsulatus ATCC 11166 T (GenBank accession no.D16428) was used as an outgroup (not shown).Numbers at nodes represent percentage levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values .50% are shown.GenBank accession numbers are given in parentheses.Filled circles indicate branches also recovered using the maximum-likelihood and maximum-parsimony methods.Bar, 2 % sequence divergence.

Table 2 .
Cellular fatty acid profiles of strain 8047 T and its phylogenetic relatives species within the genus Fulvimarina, for which the name Fulvimarina manganoxydans sp.nov. is proposed.