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1 Marine Microbiology, Department of Biology and Environmental Sciences, University of Kalmar, SE-39182 Kalmar, Sweden
2 Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Campus de Burjassot, 46100 València, Spain
3 Departamento de Microbiología y Ecología, Universitat de València, Campus de Burjassot, 46100 València, Spain
4 Colección Española de Cultivos Tipo (CECT), Universitat de València, Campus de Burjassot, 46100 València, Spain
5 Institut de Ciències del Mar-CMIMA (CSIC), Passeig Marítim de la Barceloneta 37-49, ES-08003 Barcelona, Catalunya, Spain
6 Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, La Laguna, Tenerife, Spain
Correspondence
David R. Arahal
arahal{at}uv.es
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ABSTRACT |
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Maximum-parsimony and maximum-likelihood phylogenetic trees, based on almost-complete 16S rRNA gene sequences of strain MED297T and closely related species, are available as supplementary material with the online version of this paper.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Furthermore, the phylogenetic diversity of cultured and uncultured gammaproteobacteria accounts for 25–44 % of the global marine bacterioplankton diversity, as revealed by analysis of 16S rRNA gene sequences reported in public databases (i.e. GenBank) (Hagström et al., 2002). In Blanes Bay in the north-western Mediterranean Sea, gammaproteobacteria account for up to 8 % (and occasionally 50 %) of the total bacterial community, as shown by catalysed reporter deposition fluorescence in situ hybridization (Alonso-Sáez et al., 2007). Phylogenetic analysis of cultured isolates and phylotypes detected by molecular biological techniques revealed that the gammaproteobacteria in Blanes Bay consist of a diverse array of bacteria belonging to genera such as Alteromonas, Pseudoalteromonas, Vibrio, Marinomonas and Marinobacter, and to the SAR86 and NOR5 clades (Alonso-Sáez et al., 2007; I. Lekunberri and C. Pedrós-Alió, unpublished). As a result of attempts to characterize the culture collection comprising marine bacteria isolated at the Blanes Bay Microbial Observatory (Pinhassi et al., 2006; Arahal et al., 2007), a number of novel gammaproteobacteria were found. One among them, strain MED297T, was subsequently selected for complete sequencing of its genome as part of a survey of phylogenetically and phenotypically distinct bacteria thriving in the marine environment. In the present study, we describe a novel bacterium, strain MED297T, isolated from a surface seawater sample from the Blanes Bay Microbial Observatory in the north-western Mediterranean Sea (4 ° 40' N ° 48' E) collected on 13 November 2001. The sample was incubated for 72 h at 17 °C in the dark. For strain isolation, 0.1 ml aliquots of a 100x dilution of sampled seawater (representing an abundance of approximately 1x103 c.f.u. ml–1) were spread onto ZoBell agar plates. After primary isolation and purification, strain MED297T was cultivated at room temperature on the same medium and stored at –80 °C in ZoBell's medium with 25 % (v/v) glycerol.
Whole-genome sequencing was carried out at the J. Craig Venter Institute through the Gordon and Betty Moore Foundation Initiative in Marine Microbiology (https://research.venterinstitute.org/moore/). Genome sequencing showed the DNA G+C content of strain MED297T to be 52.4 mol%. Sequencing of the genome produced an annotated genome size of approximately 4.51 Mbp (4301 putative open reading frames). The complete 16S rRNA gene sequence of strain MED297T was 1530 nt in length. This sequence was compared with public sequences in EMBL by using the BLAST program (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). Related sequences were further analysed using the program package ARB (Ludwig et al., 2004; http://www.arb-home.de/). Sequence alignments were corrected manually using the sequence editor ARB_EDIT. Phylogenetic analyses using alternative treeing methods (neighbour joining, maximum parsimony and maximum likelihood) and data subsets were performed using the appropriate ARB tools (Ludwig et al., 1998). The tree derived using the neighbour-joining method with Jukes–Cantor corrections is shown in Fig. 1. Phylogenetic analysis revealed that strain MED297T was related to the species Reinekea marinisedimentorum and Saccharospirillum impatiens. However, the low levels of sequence similarity between strain MED297T and the type strains of these two species (95.0 and 94.0 %, respectively) indicate that they are not related at the species level. Other genera in the Gammaproteobacteria are clearly more distantly related (<90 % similarity). The high bootstrap percentages (Fig. 1) and the comparison of local topologies obtained using different treeing methods (see Supplementary Figs S1 and S2, available in IJSEM Online) both confirm that strain MED297T clusters consistently with R. marinisedimentorum in the first instance, and thereafter with S. impatiens.
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, 2005). For comparative purposes, R. marinisedimentorum CECT 7208T and S. impatiens CECT 5721T were also included in the study.
Optical microscopy of bacterial cultures on wet mounts showed that cells were highly motile. To determine the cell morphology, strain MED297T was grown at 21 °C in marine broth 2216 (Difco) until early exponential phase (24–48 h incubation), at which point cells were fixed with glutaraldehyde and filtered onto polycarbonate filters with a 0.2 µm pore size (Nuclepore). Samples were treated using sequential ethanol dehydration steps, critical-point drying with CO2 and silver coating before being viewed in a scanning electron microscope (S-3500N; Hitachi). As seen in Fig. 2, cells of strain MED297T appear as single curved rods, 0.3–0.7 µm in diameter and 1.2–2.8 µm in length. In some cells, the curvature resembles a gentle spiral. Budding can also be seen at the tips of some cells.
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, was between 0.3 and 12 % (w/v) total salts. Thus, strain MED297T is a slight halophile with complex ionic requirements. In contrast, R. marinisedimentorum CECT 7208T and S. impatiens CECT 5721T only require sodium ions and exhibit different salinity ranges (Table 1). Strain MED297T is mesophilic, growing at 15–42 °C, but not at 4 or 45 °C on MA. Again, R. marinisedimentorum CECT 7208T and S. impatiens CECT 5721T have narrower ranges.
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). Strain MED297T was negative for arginine dihydrolase, ornithine decarboxylase and indole production from tryptophan. It was able to hydrolyse DNA, casein, starch and Tween 80, but not alginate, agar or lecithin. The strain was unable to grow on marine broth 2216 with 12 % (w/v) gelatin. The API ZYM (bioMérieux) gallery was used to determine the enzymic activities of the strain; the gallery was used according to the manufacturer's instructions, except that the cells were suspended in a 3.5 % sea salts solution prior to addition to the API ZYM strips, and the strips were incubated for 20 h at 26 °C. The range of enzymic activities for strain MED297T was broader than that for R. marinisedimentorum CECT 7208T, but similar to that for S. impatiens CECT 5721T (Table 1
).
The 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 and 0.1 mM FeSO4 . 7H2O on half-strength artificial seawater 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 were prepared with 5 g yeast extract l–1, while negative-control medium consisted of BMA. Growth was monitored for 12 days. About half of the compounds tested enabled growth of strain MED297T. The preferred sources were as follows, in decreasing order of preference: organic acids, sugars, alcohols and then amino acids. Compared to strain MED297T, S. impatiens and R. marinisedimentorum used much narrower ranges of utilizable resources and showed different substrate preferences (Table 1).
The cellular fatty acid composition of strain MED297T was determined by GLC at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany), as described previously (Kämpfer & Kroppenstedt, 1996). Analyses of the polar lipids and respiratory quinones of strain MED297T were carried out by the Identification Service of the DSMZ and Dr B. J. Tindall (DSMZ). The results are presented in the species description and in Table 2. Although some differences can be found between the cellular fatty acid and polar lipid profiles of strain MED297T and those of R. marinisedimentorum and S. impatiens, they all exhibited the same major quinone, Q-8, and the polar lipids phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain MED297T and R. marinisedimentorum, but not S. impatiens, contained phosphatidylinositol. In general, it seems that the chemotaxonomic data are in agreement with the phylogenetic results in the sense that strain MED297T is more closely related to R. marinisedimentorum than to S. impatiens.
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Description of Reinekea blandensis sp. nov.
Reinekea blandensis (blan.den'sis. L. fem. adj. blandensis pertaining to Blande or Blanda, the name the Romans used for the city of Blanes, which has given its name to the Bay of Blanes, where the type strain was isolated).
Gram-negative, strictly aerobic, chemo-organotrophic bacterium. Oxidase- and catalase-positive. Cells are straight or slightly bent motile rods, 0.3–0.7 µm in diameter and 1.2–2.8 µm long. Gas vesicles are not observed. Poly-
-hydroxybutyrate granules are produced. Carbohydrates are not fermented. Nitrate is not reduced to nitrite or N2. At least 0.3 % (w/v) marine salts is required; up to 12 % (w/v) salts is tolerated. Positive for growth at 15–42 °C. No growth detected at 4 or 45 °C. Casein, starch, Tween 80 and DNA are hydrolysed. Does not hydrolyse gelatin (no growth in medium), alginate, agar or lecithin. Negative for arginine dihydrolase, ornithine decarboxylase and indole production from tryptophan. Utilizes the following compounds as carbon and energy sources: L-arabinose, D-glucose, D-fructose, D-mannose, maltose, cellobiose, sucrose, lactose, N-acetyl-D-glucosamine, glycerol, D-mannitol, D-gluconate, propionate, pyruvate, acetate, citrate, 2-ketoglutarate, succinate, fumarate, lactate, DL--hydroxybutyrate, L-leucine, L-tyrosine and L-arginine. Weakly positive results are obtained on D-galactose, melibiose, salicin, butyrate, malate, L-glutamate and L-ornithine. Growth is negative on D-ribose, D-xylose, trehalose, L-rhamnose, amygdalin, D-glucuronate, D-galacturonate, myo-inositol, D-sorbitol, D-saccharate, D-glycerate, glycine, L-serine, L-threonine, L-alanine, trans-aconitate, 
-aminobutyric acid, L-citrulline, L-histidine, L-aspartate, L-lysine, L-sarcosine, betaine and putrescine. Major cellular fatty acids are, in decreasing order of abundance, 16 : 1
7c/15 : 0 iso 2-OH, 16 : 0 iso, 18 : 17c and 16 : 0. The whole pattern and relative abundances are given in Table 2. Polar lipids consist of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, three unidentified phospholipids, an unknown aminolipid and a lipid. Main respiratory quinone is Q-8 (92 %); Q-9 is present in minor amounts. The DNA G+C content of the type strain is 52.4 mol%.
The type strain, MED297T (=CECT 7120T =CCUG 52066T), was isolated from surface water from the Mediterranean Sea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arahal, D. R., Lekunberri, I., González, J. M., Pascual, J., Pujalte, M. J., Pedrós-Alió, C. & Pinhassi, J. (2007). Neptuniibacter caesariensis gen. nov., sp. nov., a novel marine genome-sequenced gammaproteobacterium. Int J Syst Evol Microbiol 57, 1000–1006.
Baumann, P. & Baumann, L. (1981). The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In The Prokaryotes, vol. 2, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. Schleger. Berlin: Springer-Verlag.
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Kämpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005.
Labrenz, M., Lawson, P. A., Tindall, B. J., Collins, M. D. & Hirsch, P. (2003). Saccharospirillum impatiens gen. nov., sp. nov., a novel -Proteobacterium isolated from hypersaline Ekho Lake (East Antarctica). Int J Syst Evol Microbiol 53, 653–660.
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Macián, M. C., Ludwig, W., Schleifer, K.-H., Garay, E. & Pujalte, M. J. (2001). Thalassomonas viridans gen. nov., sp. nov., a novel marine -proteobacterium. Int J Syst Evol Microbiol 51, 1283–1289.[Abstract]
Macián, M. C., Arahal, D. R., Garay, E., Ludwig, W., Schleifer, K.-H. & Pujalte, M. J. (2005). Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine 
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Pinhassi, J., Bowman, J. P., Nedashkovskaya, O. I., Lekunberri, I., Gómez-Consarnau, L. & Pedrós-Alió, C. (2006). Leeuwenhoekiella blandensis sp. nov., a genome-sequenced marine member of the family Flavobacteriaceae. Int J Syst Evol Microbiol 56, 1489–1493.
Romanenko, L. A., Schumann, P., Rohde, M., Mikhailov, V. V. & Stackebrandt, E. (2004). Reinekea marinisedimentorum gen. nov., sp. nov., a novel gammaproteobacterium from marine coastal sediments. Int J Syst Evol Microbiol 54, 669–673.
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