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1 Colección Española de Cultivos Tipo (CECT), Universitat de València, Campus de Burjassot, 46100 València, Spain
2 Departamento de Microbiología y Ecología, Universitat de València, Campus de Burjassot, 46100 València, Spain
3 Institut de Ciències del Mar-CMIMA (CSIC), Passeig Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalunya, Spain
4 Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, La Laguna, Tenerife, Spain
5 Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Campus de Burjassot, 46100 València, Spain
6 Marine Microbiology, Department of Biology and Environmental Sciences, University of Kalmar, SE-39182 Kalmar, Sweden
Correspondence
Jarone Pinhassi
jarone.pinhassi{at}hik.se
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ABSTRACT |
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7c/15 iso 2-OH (41.2 % relative amount), 18 : 17c (35.9 %), 16 : 0 (16.1 %), 10 : 0 3-OH (5.0 %) and 18 : 0 (1.0 %). Preferred carbon sources are organic acids and amino acids. The DNA G+C content is 46.6 mol%. Based on a phenotypic, chemotaxonomic and phylogenetic analyses, it is proposed that this marine bacterium represents a novel genus and species, for which the name Neptuniibacter caesariensis gen. nov., sp. nov. is proposed. The type strain is MED92T (=CECT 7075T=CCUG 52065T).
Additional phylogenetic trees constructed using the maximum-parsimony and maximum-likelihood methods and scanning electron micrographs of cells of strain MED92T are available as supplementary figures in IJSEM Online.
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) contains motile and strictly respiratory aquatic organisms of primarily marine origin that cluster in 16S rRNA gene-based phylogenetic trees together with some marine genera currently allocated to the family Alteromonadaceae (e.g. the genera Marinobacter and Marinomicrobium) and in the vicinity of many other members of the class Gammaproteobacteria, most of them from aquatic environments.
In the present study, we describe a novel bacterium, strain MED92T, isolated from a surface seawater sample from the eastern Mediterranean Sea (32° 80' N 34° 93' E) collected on 15 October 2000, offshore from the historic location of Caesarea. The sample was inoculated in 0.2 µm pore-size sterile-filtered seawater (1 part inoculum : 9 parts filtered seawater) and was incubated for 48 h at 28 °C in the dark. Strain MED92T was one of the bacteria that were competitive in seawater cultures with low concentrations of organic carbon and nutrients (for details see Pinhassi & Berman, 2003). For strain isolation, 0.1 ml of a 100x dilution of sample water was spread onto ZoBell agar plates. After primary isolation and purification, strain MED92T was cultivated at room temperature on the same medium and stored at –80 °C in ZoBell medium with 25 % (v/v) glycerol.
Whole-genome sequencing was carried out by the J. Craig Venter Institute (USA) through the Gordon and Betty Moore foundation initiative in Marine Microbiology (https://research.venterinstitute.org/moore/). Genome sequencing indicated that the novel strain had a DNA G+C content of 46.6 mol%, which is markedly different from that of species of the genus Marinobacterium (55–57 mol%). Sequencing of the genome of strain MED92T showed an annotated genome size of approximately 3.92 Mbp (3688 putative open reading frames). The complete 16S rRNA gene sequence of strain MED92T was 1540 nucleotides in length. This sequence was compared with public sequences in EMBL gene databases using the BLAST program (National Center for Biotechnology Information; http://ncbi.nlm.nih.gov/). Related sequences were analysed further using the ARB program package (Ludwig et al., 2004) (http://www.mikro.biologie.tu-muenchen.de). Sequence alignments were corrected manually using the ARB_EDIT sequence editor. Phylogenetic analysis using alternative treeing methods (maximum-parsimony, maximum-likelihood and distance matrix) and data subsets were performed using the appropriate ARB tools (Ludwig et al., 1998). In addition to the phylogenetic trees presented in Fig. 1 and Supplementary Figs S1 and S2 (available in IJSEM Online), further trees were generated by applying sequence filters based on sequence conservation profiles (not shown). Phylogenetic analysis revealed that strain MED92T was related, however only distantly, to previously described bacteria, particularly members of the family Oceanospirillaceae but also to some members of the family Alteromonadaceae and some unclassified members of the class Gammaproteobacteria. Consistent clustering across different trees and nodes supported by high bootstrap values were only observed for the genera containing two or more species and the cluster formed by the genera Oceanobacter–Thalassolituus–Oleispira. The rest of the species, including strain MED92T, formed separate lineages for which the ancestry cannot be accurately determined. In such cases, classification in separate genera seems an appropriate solution provided that other discriminating markers can be found. In terms of tree topology, the genera Neptunomonas and Marinobacterium (containing one and four species, respectively) can be considered more closely related to strain MED92T than the rest. In terms of 16S rRNA gene sequence similarity, the highest values found were with Oceanospirillum linum (92.9 %), Marinobacterium stanieri (92.7 %), Neptunomonas naphthovorans (92.5 %), Marinomonas communis (92.3 %), Marinobacterium georgiense (92.2 %), Oceanospirillum multiglobuliferum (92.1 %) and Marinomonas aquimarina (92.0 %). These values are sufficiently low to consider that strain MED92T merits classification in a separate genus. Thus, taken together, the phylogenetic analyses indicate that strain MED92T does not belong to any of the currently recognized genera.
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, 2005). For comparative purposes, the following strains were also included in the study: Neptunomonas naphthovorans CECT 7132T, Marinobacterium georgiense CECT 7200T, Marinobacterium jannaschii CECT 7201T, Marinobacterium stanieri CECT 7202T, Oceanospirillum linum CECT 4190T and Marinomonas communis CECT 5003T. Optical microscopy of bacterial cultures on wet mounts showed that cells of strain MED92T were highly motile. For the characterization of cell morphology, cells were grown at 21 °C in marine broth 2216 (MB; Difco) until early exponential phase (24–48 h incubation), cells were then fixed with glutaraldehyde and filtered onto 0.2 µm-pore-size polycarbonate filters (Nuclepore). Samples were treated by sequential ethanol dehydration steps, critical-point drying with CO2 and coated with silver before being viewed with a scanning electron microscope (S-3500N; Hitachi). As seen in Supplementary Fig. S3 (available in IJSEM Online), cells of strain MED92T appear as straight, or slightly bent, rods of 0.5–0.8 µm in diameter and 1.5–2.3 µm in length; coccoid forms and short chains also occur.
Strain MED92T grew on marine agar 2216 (MA; Difco) as regular, opaque and slightly brownish colonies that did not swarm or luminesce. Strain MED92T required seawater-based media for growth and was unable to grow on salt tolerance agar [1 % (w/v) tryptone, 0.3 % (w/v) yeast extract and 1.5 % (w/v) agar] with the addition of Na+ or K+ chlorides. Negative results were also obtained when Na+ was added together with divalent ions (Mg2+ or Ca2+) or when all four cations were present in the medium. The salinity range supporting growth on diluted MA or in MA supplemented with NaCl, as reported in Macián et al. (2005), was between 1.7 and 6 % (w/v) total salts. Thus, the novel strain is a slight halophile with complex ionic requirements. Strain MED92T is mesophilic, growing from 15 to 37 °C, but not at 4 or 40 °C on MA. All reference strains used in this study differed from strain MED92T at the upper and lower salinity range and most of them also had a different temperature range (Table 1).
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sea salts solution before addition to the API ZYM strips and the strips were incubated for 20 h at 26 °C. Ten (out of nineteen) enzyme activities were negative for all strains tested and two activities were positive for all (although with differences in intensity). Neptunomonas naphthovorans CECT 7132T and Marinomonas communis CECT 5003T were the strains showing fewer and more enzyme activities (three and nine positives, respectively) on this system. The differences between the strains are shown in Table 1
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Utilization of sugars, alcohols and organic acids as sole carbon and energy sources was analysed in basal medium agar [BMA; 50 mM Tris/HCl, pH 7.5, 19 mM NH4Cl, 0.33 mM K2HPO4.3H2O, 0.1 mM FeSO4.7H2O on half strength ASW solidified with 1.3 % (w/v) purified agar (Oxoid); Baumann & Baumann, 1981]. Amino acids and amines were tested as sole carbon, nitrogen and energy sources on BMA without NH4Cl. Compounds were added at 2 g l–1. Positive control plates and tubes were prepared with 5 g yeast extract l–1, while negative control media consisted of BMA. Growth was monitored for 16 days. For strain MED92T, the only compounds yielding growth were organic acids and, to a lesser extent, amino acids. Among the reference strains, Oceanospirillum linum CECT 4190T and Marinomonas communis CECT 5003T were the least and most versatile, respectively (Table 1). Only two strains, Marinomonas communis CECT 5003T and Neptunomonas naphthovorans CECT 7132T, were able to use at least some sugars and sugar alcohols. Of the three strains that grew on L-tyrosine, Marinobacterium georgiense CECT 7200T and Marinobacterium stanieri CECT 7202T produced a brown diffusible pigment, whereas strain MED92T did not.
The cellular fatty acid content of strain MED92T was determined by GLC at the DSMZ, Braunschweig, Germany, as previously described (Kämpfer & Kroppenstedt, 1996). In total, only five fatty acids with percentages above 1 % were detected. The major fatty acid was 16 : 17c/15 iso 2-OH (41.2 %), followed by 18 : 17c (35.9 %), 16 : 0 (16.1 %), 10 : 0 3-OH (5.0 %) and 18 : 0 (1.0 %). In addition, traces (<1 %) of the following fatty acids were also detected: 14 : 0, 20 : 17c, and 10 : 0. According to the chemotaxonomic study of Sakane & Yokota (1994) on heterotrophic spirilla, the major fatty acids (in order of abundance) of Oceanospirillum linum, Marinobacterium jannaschii and Oceanobacter kriegii are 16 : 1, 18 : 1 and 16 : 0, whereas the order in Oceanospirillum maris, Oceanospirillum beijerinckii, Oceanospirillum multiglobuliferum and Pseudospirillum japonicum is 16 : 1, 16 : 0 and 18 : 1. Other combinations of the order of abundance of the same major fatty acids are found in Marinospirillum minutulum (16 : 0, 18 : 1, 16 : 1) (Sakane & Yokota, 1994) or Marinobacterium stanieri, Marinomonas communis and Marinomonas aquimarina (18 : 1, 16 : 1, 16 : 0) (Lau et al., 2006). Minor constituents of the fatty acid profiles enhanced the differences between species, however a clear pattern for generic circumscription could not be found.
Analyses of polar lipids and respiratory quinones from strain MED92T were carried out by the Identification Service of the DSMZ and by Dr B. J. Tindall, DSMZ. The results of the polar lipid analysis are presented in the species description. The major phospholipids, phosphatidylethanolamine and phosphatidylglycerol, also occur in many related marine isolates (Ivanova et al., 2000, 2005; Gupta et al., 2006). Analyses of quinones revealed that the novel strain has a major peak corresponding to Q8 (95 %) and two minor peaks of about the same size corresponding to unidentified components. Ubiquinone Q8 is a common characteristic among marine Gammaproteobacteria (Sakane & Yokota, 1994; Romanenko et al., 2004).
Considering that strain MED92T can be readily differentiated from neighbouring bacteria in different genera based on a combination of phylogenetic, genotypic, phenotypic and chemotaxonomic characteristics, we conclude that this marine bacterium represents a novel genus and species, for which the name Neptuniibacter caesariensis gen. nov., sp. nov. is proposed.
Description of Neptuniibacter gen. nov.
Neptuniibacter (Nep.tu.ni.i.bac'ter. L. adj. Neptunius Neptunian, pertaining to Neptune, Roman god of the sea; N.L. masc. n. bacter a rod; N.L. masc. n. Neptuniibacter a Neptunian rod, referring to the habitat of the bacteria).
Gram-negative, strictly aerobic, chemoorganotrophic bacteria. Oxidase- and catalase-positive. Cells are rod-shaped and motile. Gas vesicles not observed. Produce poly-
-hydroxybutyrate granules. Slightly halophilic; no growth can be obtained without seawater or the addition of combined marine salts to the medium. Mesophilic. Do not ferment carbohydrates. Preferred carbon sources are organic acids and amino acids. Possess ubiquinone Q8 as a respiratory quinone. DNA G+C content is around 47 mol%. The genus is affiliated to the Class Gammaproteobacteria. The type species is Neptuniibacter caesariensis.
Description of Neptuniibacter caesariensis sp. nov.
Neptuniibacter caesariensis (ca.e.sa.ri.en'sis. L. masc. adj. caesariensis pertaining to Caesaria, as the isolate was found close to the Roman city Caesaria maritima, south of Haifa in present day Israel).
Has the following characteristics in addition to those given in the genus description. Cells are straight or slightly bent rods. Cells range from 0.5–0.8 µm in diameter and from 1.5–2.3 µm in length. Vigorous motility can be observed on fresh mounts. Does not ferment carbohydrates. Does not reduce nitrate to nitrite or gas. Requires at least 1.7 % (w/v) marine salts and tolerates up to 6 % (w/v) salts, failing to grow at 7 %. Positive growth at temperatures from 15 to 37 °C. No growth detected at 4 or 40 °C. Does not hydrolyse casein, gelatin (no growth in medium), starch, lecithin, alginate, agar, Tween-80 or DNA. Negative for arginine dihydrolase, ornithine decarboxylase and indole production from tryptophan. According to the API ZYM gallery (bioMérieux), produces acid and alkaline phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidase and naphthol-AS-BI-phosphohydrolase, but not lipase (C14), valine and cystine arylamidases, trypsin, 
-chymotrypsin, - and -galactosidases, -glucuronidase, - and -glucosidases, N-acetyl--glucosaminidase, -mannosidase or -fucosidase. Utilizes the following compounds as a carbon and energy source: propionate, pyruvate, citrate, succinate, butyrate, fumarate, malate, acetate, lactate, DL--hydroxybutyrate, L-tyrosine (without production of a brown water-soluble pigment), L-glutamate, L-alanine, 
-aminobutyric acid, L-aspartate and putrescine. Growth is negative on: D-ribose, L-arabinose, D-xylose, D-glucose, D-fructose, D-trehalose, D-galactose, D-mannose, L-rhamnose, maltose, cellobiose, sucrose, lactose, melibiose, amygdalin, salicin, D-gluconate, D-saccharate, D-glycerate, 2-oxoglutarate, trans-aconitate, N-acetyl-D-glucosamine, glycerol, D-mannitol, myo-inositol, D-sorbitol, glycine, L-leucine, L-serine, L-threonine, L-arginine, L-ornithine, L-citrulline, L-histidine, L-lysine and L-sarcosine. Cellular fatty acids are, in order of abundance: 16 : 17c/15 iso 2-OH, 18 : 17c, 16 : 0, 10 : 0 3-OH and 18 : 0. The polar lipid profile consists of the major compounds phosphatidylethanolamine and phosphatidylglycerol, a moderate amount of an unknown aminophospholipid and a minor amount of diphosphatidylglycerol. The DNA G+C content of the type strain is 46.6 mol%.
The type strain, MED92T (=CECT 7075T=CCUG 52065T), was isolated from Mediterranean Sea surface water.
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ACKNOWLEDGEMENTS |
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