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Thalassobacter stenotrophicus Macián et al. 2005 is a later synonym of Jannaschia cystaugens Adachi et al. 2004, with emended description of the genus Thalassobacter

¹ Instituto Cavanilles de Biodiversidad y Biología Evolutiva, 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 Colección Española de Cultivos Tipo (CECT), Facultad de Biología, Universitat de València, Campus de Burjassot, 46100 València, Spain

Correspondence
M. J. Pujalte
maria.j.pujalte{at}uv.es

	ABSTRACT

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The type strains of Jannaschia cystaugens (LMG 22015^T) and Thalassobacter stenotrophicus (CECT 5294^T) were analysed by means of genomic DNA–DNA hybridization, comparison of 16S rRNA gene sequences and phenotypic properties determined under the same methodological conditions. J. cystaugens LMG 22015^T showed DNA–DNA relatedness levels of 72 % when hybridized with the genomic DNA of T. stenotrophicus CECT 5294^T. Sequence comparisons revealed that the 16S rRNA genes of the two strains had a similarity of 99·8 %. The cellular fatty acid and polar lipid compositions of the two strains and their DNA mol% G+C contents were almost identical. Bacteriochlorophyll a (Bchl a) and polyhydroxybutyrate were produced by both strains under the same culture conditions. Their closest phylogenetic neighbours were Jannaschia helgolandensis and Jannaschia rubra; however, the low sequence similarity values (95·7–95·9 %) and several important differences in phenotypic traits (ionic requirements, Bchl a production and polar lipids) support the distinction between the genera Thalassobacter and Jannaschia. Thus, we propose the unification of J. cystaugens (LMG 22015^T) and T. stenotrophicus (CECT 5294^T) as Thalassobacter stenotrophicus (type strain, CECT 5294^T=DSM 16310^T). An emended description of the genus Thalassobacter is also presented.

Published online ahead of print on 6 May 2005 as DOI 10.1099/ijs.0.63617-0.

	MAIN TEXT

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The continuous increase in the number of taxa within the Roseobacter group, or marine alpha-proteobacterial group, in recent years has extended our knowledge of this essential and ubiquitous fraction of marine bacterioplankton. They are currently recognized as belonging to the order ‘Rhodobacterales’, within the class ‘Alphaproteobacteria’ (Garrity et al., 2004). An important number of genera/species belonging to this group are able to synthesize bacteriochlorophyll a (Bchl a) under aerobic conditions. These include Dinoroseobacter (Biebl et al., 2005), Roseivivax (Suzuki et al., 1999b), Roseobacter (Shiba, 1991), Roseisalinus (Labrenz et al., 2005), Roseovarius (Labrenz et al., 1999), Rubrimonas (Suzuki et al., 1999a), Staleya (Labrenz et al., 2000) and Thalassobacter (Macián et al., 2005a).

The genus Thalassobacter was recently described as comprising a single species, Thalassobacter stenotrophicus, represented by a single strain. The closest phylogenetic neighbour of Thalassobacter was the genus Jannaschia, from which it could be distinguished by phenotypic traits and phylogenetic distance. However, the recently described species Jannaschia cystaugens (Adachi et al., 2004) was not included in the comparison, because the two descriptions originated from separate studies and the dates of publication overlapped. Nevertheless, when the 16S rRNA gene sequences of both organisms became available, it was noted that T. stenotrophicus and J. cystaugens had almost identical sequences, in spite of the lack of close similarity in the described phenotypes. Thus, we undertook a study to compare the two species by using a polyphasic approach, with the aim of determining their relatedness by using the same methodology of phenotypic characterization and by comparing their genomic DNA similarity through DNA–DNA hybridization experiments. We also included in the study the type strain (CECT 7023^T) of the recently described species Roseisalinus antarcticus, in order to compare this novel Bchl a-producing species with T. stenotrophicus.

The methods used for the phenotypic characterization and DNA–DNA hybridization have been described in detail previously (Macián et al., 2005a, b). Phylogenetic analysis of 16S rRNA gene sequences was performed as described by Ludwig et al. (1998) using the software package ARB (Ludwig et al., 2004). The strains used were T. stenotrophicus CECT 5294^T, J. cystaugens LMG 22015^T and R. antarcticus CECT 7023^T. All three strains were routinely cultured on marine agar and in marine broth (MA and MB; Scharlab) at 22–25 °C, in the dark. The following phenotypic characteristics were determined in parallel: the ability to grow on MA diluted to 0·34, 0·85, 1·7 and 2·55 % (w/v) total salinity (dilution factors 0·1, 0·25, 0·50 and 0·75, respectively); the ability to grow on MA supplemented with NaCl to a total salinity of 6, 7, 8, 9 and 10 % (w/v); specific ionic requirements for Na⁺, Mg²⁺, Ca²⁺ and K⁺ cations in salt-tolerance agar (STA) containing 1 % (w/v) tryptone, 0·3 % (w/v) yeast extract and 1·5 % (w/v) agar; the ability to grow at 4, 28, 37 and 40 °C on MA plates; extracellular hydrolytic activities on casein, starch, alginate, agar, Tween 80, lecithin and DNA; substrates used as sole carbon and energy sources for growth in basal medium agar (BMA; Baumann & Baumann, 1981); polyhydroxybutyrate (PHB) accumulation determined after staining cells grown on MA and on BMA plus acetate with Nile blue A (Smibert & Krieg, 1994); and Bchl a production determined in acetone extracts obtained from cells grown on MA, BMA plus lactate and BMA plus acetate using a Beckman DU-600 spectrophotometer, as described by Takaichi et al. (1991).

Cellular morphology was reported to be irregular rods for both J. cystaugens and T. stenotrophicus, but in the latter ovoid cells were also observed. The mode of division and absence of rosette formation were common to both strains.

The flagellar position of J. cystaugens was not stated in its original description, although the cells were reported as being motile. Very young cultures of T. stenotrophicus have one polar flagellum, but this trait was difficult to determine. Similar difficulties were found with J. cystaugens, which, in our hands, was non-motile, even in very young cultures, and showed no flagella.

Pigmentation was observed under various culture conditions, and was very similar in the two strains. It varied from light cream to dark brown, including salmon-pink and ochre-red colours. In general, the pigment was darker and more brown than pink in complex media such as casein- and lecithin-supplemented MA. It was poor in media that did not favour good growth, such as media with the highest or lowest salinity that supported growth (salinity range tests); however, deep pigmentation was also observed in some minimal media supporting poor growth. In any case, the pigmentation of the two strains was markedly similar when they were cultured under the same conditions and in sharp contrast to R. antarcticus, which always had a fainter pigmentation with different intensities of pink, but never ochre/brown.

The main differences in phenotypic traits between T. stenotrophicus and J. cystaugens, according to the original descriptions, are Bchl a production by T. stenotrophicus only and the occasional presence of white inclusion bodies (which could be interpreted as PHB) in J. cystaugens. Both characteristics were investigated in parallel in this study. PHB production was determined by using Nile blue A staining and fluorescence microscopy, with cells grown on BMA plus acetate and on MA for 5 days. Escherichia coli CECT 101 was used as a negative control and R. antarcticus CECT 7023^T as a positive control. Both J. cystaugens and T. stenotrophicus showed the typical bright-orange fluorescence of PHB on BMA plus acetate-grown cells, but very few cells of either strain fluoresced when grown on MA. In contrast, PHB was produced by most cells of R. antarcticus when grown on MA. Therefore, we concluded that T. stenotrophicus is also able to produce PHB, although bright inclusions, a presumptive trait suggesting the presence of this polymer in bacterial cells, are not easily seen on wet mounts.

Bchl a production was determined as described by Takaichi et al. (1991) in acetone extracts of cells of strains CECT 5294^T and LMG 22015^T grown on MA, BMA plus acetate and BMA plus lactate for 7 days in the dark. All six culture extracts showed the typical absorbance peak of Bchl a at an identical position (772 nm), confirming the ability of J. cystaugens LMG 22015^T to produce the photosynthetic pigment.

Identical results were also obtained with the two strains for the following phenotypic traits. They were unable to hydrolyse casein, starch, alginate, agar, lecithin, Tween 80 or DNA. The salinity range for growth was 1·7–7 % (w/v) total salinity. They were unable to grow in STA supplemented with the four salts tested (Na⁺, Mg²⁺, Ca²⁺ and K⁺ cations), provided the inoculum was prepared in 2 % (w/v) NaCl. This indicates that both strains have complex ionic requirements. Strains LMG 22015^T and CECT 5294^T did not grow at 4 or 40 °C, but did grow at 28 and 37 °C. They utilized D-ribose, L-arabinose, D-glucose, D-fructose, D-gluconate, D-glycerate, glycerol, D-mannitol, D-sorbitol, pyruvate, acetate, succinate, fumarate, malate, lactate, L-serine, L-arginine, L-glutamate, L-alanine and L-ornithine as carbon sources for growth on BMA supplemented with 0·01 % yeast extract. D-Trehalose, D-mannose, L-rhamnose, maltose, D-cellobiose, sucrose, lactose, D-melibiose, amygdalin, N-acetyl-D-glucosamine, D-glucuronate, m-inositol, glycine, L-leucine, L-threonine, L-tyrosine, 4-aminobutyrate, L-citrulline, L-histidine and putrescine did not support growth of any of the strains in the same media and under the same conditions. The instability of T. stenotrophicus CECT 5294^T in the use of sole carbon sources is noteworthy, when the results obtained in this study were compared with those included in the original description. This instability, which was observed even when the same methodology was used at the same laboratory, could be due to the influence of factors such as the age of the cells used for the inoculation of BMA plates in different experiments, which was difficult to standardize. In any case, the carbon source profile obtained in this study for strain CECT 5294^T was almost identical to that of strain LMG 22015^T, with only differences in the use of L-aspartate being obtained.

The sequences used for the phylogenetic analysis were retrieved from the EMBL nucleotide sequence database. The analysis showed a similarity of 99·8 % between the 16S rRNA gene sequences of strains CECT 5294^T and LMG 22015^T, a value that clearly indicates that the two strains are related, at least at the genus level and probably also at the species level. To resolve the latter, DNA–DNA hybridization experiments with genomic DNA were performed in triplicate (Ziemke et al., 1998). A mean relatedness value of 72 % was obtained, which is slightly above the suggested boundary for genomic species definition (Wayne et al., 1987).

In addition, we also observed a high concordance in both the mol% G+C content and cellular fatty acid compositions reported in the original descriptions (Adachi et al., 2004; Macián et al., 2005a). The G+C content of T. stenotrophicus CECT 5294^T was 59·1 mol%, and those of J. cystaugens LMG 22015^T and LMG 22016 were 59·1 and 59·2 %, respectively. All three strains contained 67–78 % of 18 : 17c and minor amounts of 18 : 17c 11-methyl (6·9–7·3 %), 18 : 19c (1·2–3·6 %), 18 : 0 (2·6–2·8 %), 10 : 0 3-OH (3·1–3·2 %) and unknown ECL 11·798 (2·9–3·6 %). T. stenotrophicus CECT 5294^T was reported to have a small amount (1·47 %) of an undetermined 19-carbon fatty acid, which could be 19 : 16c or 19 : 0 cyclo. In the case of J. cystaugens, both strains completely lacked 19 : 0 cyclo, which was the characteristic second most abundant fatty acid of Jannaschia helgolandensis (20–25 %) and was also present in Jannaschia rubra in smaller amounts (5·7 %) (Wagner-Döbler et al., 2003; Macián et al., 2005b). Moreover, during the course of this work the polar lipid compositions of T. stenotrophicus CECT 5294^T and J. cystaugens LMG 22015^T were determined at the DSMZ. Polar lipid analyses were carried out by the Identification Service of the DSMZ and Dr B. J. Tindall (Braunschweig, Germany). The results reinforce the high similarity between the two strains, since their polar lipid compositions were essentially identical: phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids and one unidentified aminolipid. T. stenotrophicus CECT 5294^T also had minor amounts of a third unidentified phospholipid. The genus Jannaschia was described as containing phosphatidylethanolamine, which was absent in T. stenotrophicus CECT 5294^T and J. cystaugens LMG 22015^T. However, the distantly related Bchl a-producing species R. antarcticus (Labrenz et al., 2005) has a polar lipid composition that is virtually identical to those of T. stenotrophicus and J. cystaugens.

Transfer of Jannaschia cystaugens Adachi et al. 2004 to Thalassobacter stenotrophicus Macián et al. 2005
All the results reported here support the idea that T. stenotrophicus and J. cystaugens should be considered as representing a single species, as they were clearly separate in terms of genomic similarity from their closest phylogenetic neighbours and readily recognizable by their phenotypic characteristics, in agreement with the criteria of Wayne et al. (1987). The names Jannaschia cystaugens Adachi et al. 2004 and Thalassobacter stenotrophicus Macián et al. 2005a can be considered as heterotypic synonyms. Following the general interpretation of Rules 15 and 17 of the Bacteriological Code, we conclude that the species should be given the name Thalassobacter stenotrophicus, with strain CECT 5294^T (=DSM 16310^T) as the type strain.

Emended description of the genus Thalassobacter Macián et al. 2005
The description is identical to that given by Macián et al. (2005a) with the following amendments. Positive for PHB accumulation. Major polar lipids are phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine. Also contain two unidentified phospholipids and an unidentified aminolipid, but not phosphatidylethanolamine.

Differentiation between T. stenotrophicus and R. antarcticus
The original description of T. stenotrophicus included a comparison of the new genus with its related genera. At the time of writing of that manuscript, the description of the new Bchl a-containing genus and species R. antarcticus (Labrenz et al., 2005) was not available. Therefore, we included the type strain, CECT 7023^T, in this study and we present here a comparison with T. stenotrophicus. The phenotypic traits as well as analysis of the 16S rRNA gene sequence (GenBank accession no. AJ605747) and G+C mol% content confirmed that, despite its ability to synthesize Bchl a under aerobic conditions and its very similar polar lipid composition, Roseisalinus is distantly related to Thalassobacter, showing only 92–93 % 16S rRNA gene sequence similarity. Moreover, when a phylogenetic analysis of members of the whole order ‘Rhodobacterales’ was performed, the Roseisalinus sequence was seen to be more closely related to those of other genera such as Oceanicola, Ketogulonicigenium or Loktanella, whereas Thalassobacter is a neighbour of the genus Jannaschia (Fig. 1). On phenotypic grounds, the two genera could be clearly distinguished by growth at 10 % total salinity, ionic requirements (Roseisalinus being able to grow on STA supplemented with Na⁺, Mg²⁺, Ca²⁺ and K⁺ cations, even when the inoculum was prepared in a 2 %, w/v, NaCl solution), rosette formation (observed only in Roseisalinus), cellular fatty acid composition, G+C content of the DNA and carbon sources that support growth (Table 1).

View larger version (58K): [in this window] [in a new window]   Fig. 1. Unrooted phylogenetic consensus tree based on trees obtained using various datasets of 16S RNA gene sequences and alternative treeing methods (Ludwig et al., 1998). Multifurcations indicate branches for which their relative evolutionary order cannot be ascertained.

	ACKNOWLEDGEMENTS

 
We are grateful to A. Camacho, Universitat de València, for Bchl a spectrophotometry. This work was supported by project AGL-2002-04075-C02-C02 of the Spanish Ministerio de Ciencia y Tecnología. D. R. A. has a contract with the Universitat de València under the Ramón y Cajal program (Ministerio de Ciencia y Tecnología).

	REFERENCES

TOP ABSTRACT MAIN TEXT REFERENCES

 
Adachi, M., Kanno, T., Okamoto, R., Shinozaki, A., Fujikawa-Adachi, K. & Nishijima, T. (2004). Jannaschia cystaugens sp. nov., an Alexandrium (Dinophyceae) cyst formation-promoting bacterium from Hiroshima Bay, Japan. Int J Syst Evol Microbiol 54, 1687–1692.[Abstract/Free Full Text]

Baumann, P. & Baumann, L. (1981). The marine Gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In The Prokaryotes, vol. II, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. G. Schlegel. Berlin: Springer.

Biebl, H., Allgaier, M., Tindall, B. J., Koblizek, M., Lünsdorf, H., Pukall, R. & Wagner-Döbler, I. (2005). Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates. Int J Syst Evol Microbiol 55, 1089–1096.[Abstract/Free Full Text]

Garrity, G. M., Bell, J. A. & Lilburn, T. G. (2004). Taxonomic outline of the Prokaryotes. In Bergey's Manual of Systematic Bacteriology, 2nd edn, release 5.0. New York: Springer. doi: 10.1007/bergeysoutline200405

Labrenz, M., Collins, M. D., Lawson, P. A., Tindall, B. J., Schumann, P. & Hirsch, P. (1999). Roseovarius tolerans gen. nov., sp. nov., a budding bacterium with variable bacteriochlorophyll a production from hypersaline Ekho Lake. Int J Syst Bacteriol 49, 137–147.[Abstract/Free Full Text]

Labrenz, M., Tindall, B. J., Lawson, P. A., Collins, M. D., Schumann, P. & Hirsch, P. (2000). Staleya guttiformis gen. nov., sp. nov. and Sulfitobacter brevis sp. nov., -3-Proteobacteria from hypersaline, heliothermal and meromictic antarctic Ekho Lake. Int J Syst Evol Microbiol 50, 303–313.[Abstract]

Labrenz, M., Lawson, P. A., Tindall, B. J., Collins, M. D. & Hirsch, P. (2005). Roseisalinus antarcticus gen. nov., sp. nov., a novel aerobic bacteriochlorophyll a-producing -proteobacterium isolated from hypersaline Ekho Lake, Antarctica. Int J Syst Evol Microbiol 55, 41–47.[Abstract/Free Full Text]

Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K.-H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554–568.[CrossRef][Medline]

Ludwig, W., Strunk, O., Westram, R. & 29 other authors (2004). ARB: a software environment for sequence data. Nucleic Acids Res 32, 1363–1371.[Abstract/Free Full Text]

Macián, M. C., Arahal, D. R., Garay, E., Ludwig, W., Schleifer, K. H. & Pujalte, M. J. (2005a). Thalassobacter stenotrophicus gen. nov., sp. nov., a novel marine -proteobacterium isolated from Mediterranean sea water. Int J Syst Evol Microbiol 55, 105–110.[Abstract/Free Full Text]

Macián, M. C., Arahal, D. R., Garay, E., Ludwig, W., Schleifer, K. H. & Pujalte, M. J. (2005b). Jannaschia rubra sp. nov., a red-pigmented bacterium isolated from sea water. Int J Syst Evol Microbiol 55, 649–653.[Abstract/Free Full Text]

Shiba, T. (1991). Roseobacter litoralis gen. nov., sp. nov., and Roseobacter denitrificans sp. nov., aerobic pink-pigmented bacteria which contain bacteriochlorophyll a. Syst Appl Microbiol 14, 140–145.

Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607–654. Edited by P. Gerhardt, R. G. E. Murray, W. A. Wood & N. R. Krieg. Washington, DC: American Society for Microbiology.

Suzuki, T., Muroga, Y., Takahama, M., Shiba, T. & Nishimura, Y. (1999a). Rubrimonas cliftonensis gen. nov., sp. nov., an aerobic bacteriochlorophyll-containing bacterium isolated from a saline lake. Int J Syst Bacteriol 49, 201–205.[Abstract/Free Full Text]

Suzuki, T., Muroga, Y., Takahama, M. & Nishimura, Y. (1999b). Roseivivax halodurans gen. nov., sp. nov. and Roseivivax halotolerans sp. nov., aerobic bacteriochlorophyll-containing bacteria isolated from a saline lake. Int J Syst Bacteriol 49, 629–634.[Abstract/Free Full Text]

Takaichi, S., Furihata, K., Ishidu, J. & Shimada, K. (1991). Carotenoid sulphates from the aerobic photosynthetic bacterium, Erythrobacter longus. Phytochemistry 30, 3411–3415.[CrossRef]

Wagner-Döbler, I., Rheims, H., Felske, A., Pukall, R. & Tindall, B. J. (2003). Jannaschia helgolandensis gen. nov., sp. nov., a novel abundant member of the marine Roseobacter clade from the North Sea. Int J Syst Evol Microbiol 53, 731–738.[Abstract/Free Full Text]

Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[Free Full Text]

Ziemke, F., Höfle, M. G., Lalucat, J. & Rosselló-Mora, R. (1998). Reclassification of Shewanella putrefaciens Owen's genomic group II as Shewanella baltica sp. nov. Int J Syst Bacteriol 48, 179–186.[Abstract/Free Full Text]

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