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1 Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim, Tivon 36006, Israel
2 Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel
Correspondence
Malka Halpern
mhalpern{at}research.haifa.ac.il
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7c and/or 15 : 0 iso 2-OH (25.76 %). The DNA G+C content is 49.9 mol%. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain K19414T (=LMG 23818T =DSM 18694T) was classified in the genus Rheinheimera as the type strain of a novel species, for which the name Rheinheimera chironomi sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain K19414T is DQ298025.
An electron micrograph of cells of strain K19414T and details of its fatty acid profile in comparison with those of related type strains are available as supplementary material with the online version of this paper.
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while screening blue-coloured bacterial isolates from different depth stations in the central Baltic Sea. At present, the genus comprises three species: Rheinheimera baltica (Brettar et al., 2002), Rheinheimera pacifica (Romanenko et al., 2003) and Rheinheimera perlucida (Brettar et al., 2006). All three species were isolated from marine environments. In this study, a new member of the genus Rheinheimera that was isolated from a freshwater insect egg mass is proposed on the basis of polyphasic studies.
Strain K19414T was isolated from a chironomid (non-biting midge) egg mass while the diversity of the culturable bacteria in chironomid egg masses was under study (Halpern et al., 2007). Chironomid egg masses were sampled in September 2004, as described previously (Halpern et al., 2007). Egg masses were washed thoroughly with sterile saline water and then their homogenates were diluted and cultured directly on bacteriological media (Halpern et al., 2007). Strain K19414T was isolated from an egg mass that was sampled from Kishon River in northern Israel, cultured on LB agar and incubated at 30 °C for 48 h in the dark. Another isolate from the above-described study was recently characterized as the type strain of Oceanobacillus chironomi (Raats & Halpern, 2007).
Strain K19414T is a motile Gram-negative rod, psychrotolerant and strictly aerobic. Its exact taxonomic position was determined by means of a polyphasic approach that included phenotypic properties and phylogenetic analysis based on the 16S rRNA gene sequence.
For electron microscopy, bacteria in LB agar medium were suspended in saline. The samples were adhered to a carbon-coated grid and stained with 2 % uranyl acetate and photographed under a JEM-1200EX electron microscope (JEOL). Electron microscopy showed that the cells were rods with a polar flagellum (0.3–0.7x1.0–2.4 µm) (Supplementary Fig. S1 in IJSEM Online).
The 16S rRNA gene was analysed to determine its phylogenetic position. Universal bacterial primers 8f and 1512r (Felske et al., 1997) were used to amplify internal fragments of the 16S rRNA gene. The amplified PCR product was purified with the Wizard PCR product purification kit (Promega). Purified PCR products were sequenced with primers 8f, 534r, 968f and 1512r as described in detail by Raats & Halpern (2007). This resulted in a sequence of 1499 bp.
For identification of closest relatives, the 16S rRNA gene of strain K19414T was compared with those of previously reported strains available in EMBL (http://www.ebi.ac.uk/). Using the BLAST program (version 2.0), the closest sequences obtained were those of R. baltica OS140 (96.9 %), R. baltica OSBAC5 (96.7 %), R. pacifica KMM 1406T (96.5 %), R. perlucida BA131T (96.0 %), R. baltica OSBAC1T (95.8 %), Alishewanella fetalis CCUG 30811T (95.8 %), Brenneria salicis DSM 30166T (91.5 %) and Colwellia piezophila Y223GT (89.0 %). The sequences were aligned by the multiple alignment package CLUSTAL W. Phylogenetic trees were generated by neighbour joining (NJ), maximum parsimony (MP) and maximum likelihood (ML). NJ and MP trees were obtained using the MEGA software package (Kumar et al., 2004), while ML was computed by PHYLIP (Felsenstein, 1993). For NJ, distance correction was performed using the Kimura two-parameter model (K2ST) using rate variation across sites. The bootstrap values obtained were from 1000 iterations. The NJ tree was drawn by the MEGA software (Fig. 1). In contrast to the highest sequence similarity to R. baltica OS140 as found by BLAST, all phylogenetic inference methods located the novel strain as a sibling to R. pacifica KMM 1406T.
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For the cellular fatty acid analysis, cells were cultured on a tryptic soy agar (Difco) at 28 °C for 24 h and then the fatty acids were extracted. The fatty acid profile was analysed by means of the MIDI/Hewlett Packard Microbial Identification system (Analytical Services Inc.), which uses GC profiles of fatty acid methyl esters. The major fatty acid components (exceeding 10 %) of strain K19414T are 16 : 0 (14.80 %) and summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH; 25.76 %) (Supplementary Table S1).
For determination of the DNA G+C content, genomic DNA of strain K19414T was prepared according to a modification of the procedure of Wilson (1987). The DNA G+C content was determined using HPLC analysis of hydrolysed DNA according to Mesbah et al. (1989). The analysis was performed by the BCCM/LMG Bacteria Collection Identification Service (Laboratory of Microbiology, Ghent University, Belgium). The G+C content of strain K19414T was 49.9 mol%.
The morphological, physiological and biochemical traits of strain K19414T are summarized in the species description and in Table 1
. API ZYM tests of strain K19414T revealed a broad set of 12 enzyme activities (Table 1). Phylogenetic relationships between strain K19414T and some related taxa are shown in Fig. 1. Comparative 16S rRNA gene sequence analysis showed that the new isolate was phylogenetically most closely related to Rheinheimera species, with 95.8–96.5 % sequence similarity to the type strains. The new isolate shared its main characteristics with Rheinheimera species, such as being aerobic and oxidase-positive, motile rods by means of polar flagella, assimilating N-acetylglucosamine and hydrolysing gelatin. Its DNA G+C content was close to those of the other described Rheinheimera species (48.9–49.9 mol%) (Table 1). Unsaturated fatty acids formed a major fraction of the total fatty acids in strain K19414T, as in the other described strains of the genus Rheinheimera (Supplementary Table S1). Together with the findings mentioned above, strain K19414T showed significant characteristics that allowed clear differentiation from all three described Rheinheimera species. It lacked catalase activity, grew at relatively low NaCl concentrations (0–2 %), grew at 40 °C, which is the highest growth temperature described for species of this genus, and was positive for malate assimilation and for valine arylamidase, cystine arylamidase and 
-mannosidase (Table 1). On the basis of phenotypic characterization and phylogenetic analysis (Stackebrandt & Goebel, 1994), we propose that strain K19414T should be classified as the type strain of a novel species, Rheinheimera chironomi sp. nov.
Description of Rheinheimera chironomi sp. nov.
Rheinheimera chironomi [chi.ro'no.mi. N.L. gen. n. chironomi of Chironomus, named after the non-biting midge insect from the genus Chironomus (Diptera; Chironomidae) from which the type strain was isolated].
Cells are aerobic, Gram-negative, non-pigmented rods, 1.0–2.4 µm long and 0.3–0.7 µm wide, occurring as single cells or in pairs, and they are motile by means of a single polar flagellum. Colonies are circular, non-pigmented, smooth and convex. Oxidase-positive, catalase-negative and able to reduce nitrate to nitrite. Sodium ions are not required for growth. Growth is observed in 0–2 % (w/v) NaCl, but not in 3 % NaCl. Grows at 4–40 °C, but not at 41 °C. Hydrolyses gelatin and casein but not DNA. Grows on LB and half-strength marine agar. Does not haemolyse bovine blood and does not grow on MacConkey agar. The major measurable fatty acid components (exceeding 5 %) of the type strain are 11 : 0 3-OH (5.53 %), 12 : 0 3-OH (8.54 %) 15 : 0 (6.86 %), 16 : 0 (14.80 %), summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH; 25.76 %), 17 : 0 (5.76 %), 17 : 18c (7.56 %) and 18 : 17c (6.68 %). The following fatty acids are detected in the type strain as minor components: 9 : 0 (0.2 %), 10 : 0 (1.2 %), 11 : 0 (1.35 %), 10 : 0 3-OH (0.53 %), unknown ECL 11.799 (2.63 %), 12 : 0 (1.06 %), 13 : 0 (0.62 %), 12 : 0 iso 3-OH (0.41 %), 14 : 0 iso (0.19 %), 14 : 0 (0.89 %), summed feature 1 (one or more of 13 : 0 3-OH, 15 : 1 iso I and 15 : 1 iso H; 1.9 %), 15 : 0 anteiso (0.28 %), 15 : 18c (2.75 %), 15 : 16c (1.04 %), summed feature 2 (14 : 0 3-OH and/or 16 : 1 iso I; 0.28 %), 16 : 0 iso (1.45 %), 17 : 0 iso (0.19 %), 17 : 0 anteiso (0.41 %), 17 : 16c (0.87 %) and 18 : 0 (0.25 %). Cells are resistant to neomycin, penicillin G and bacitracin but susceptible to tetracycline, ampicillin, vancomycin, streptomycin, chloramphenicol, novobiocin and SXT (sulfamethoxazole and trimethoprim). In the API 20NE test system, nitrate is reduced to nitrite, glucose, N-acetylglucosamine, maltose and malate are assimilated and enzyme activities of protease (gelatinase) and 
-galactosidase are detected. In the API ZYM test system, 12 enzyme activities are detected: alkaline and acid phosphatases, esterase (C4 and C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -chymotrypsin, naphthol-AS-BI-phosphohydrolase, N-acetyl--glucosaminidase and -mannosidase. In the Biolog GN2 test system, Tweens 40 and 80, D-galactose, D-glucose, maltose, sucrose, pyruvic acid methyl ester, succinic acid monomethyl ester, L-alanine, L-alanyl glycine, glycyl L-aspartic acid and glycyl L-glutamic acid are utilized as substrates; all other Biolog GN2 substrates are not utilized. The G+C content of the type strain is 49.9 mol%.
The type strain is strain K19414T (=DSM 18694T =LMG 23818T), which is of freshwater origin.
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REFERENCES |
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Brettar, I., Christen, R. & Höfle, M. G. (2006). Rheinheimera perlucida sp. nov., a marine bacterium of the Gammaproteobacteria isolated from surface water of the central Baltic Sea. Int J Syst Evol Microbiol 56, 2177–2183.
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
Raats, D. & Halpern, M. (2007). Oceanobacillus chironomi sp. nov., a halotolerant and facultatively alkaliphilic species isolated from a chironomid egg mass. Int J Syst Evol Microbiol 57, 255–259.
Romanenko, L. A., Uchino, M., Falsen, E., Zhukova, N. V., Mikhailov, V. V. & Uchimura, T. (2003). Rheinheimera pacifica sp. nov., a novel halotolerant bacterium isolated from deep sea water of the Pacific. Int J Syst Evol Microbiol 53, 1973–1977.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Wilson, K. (1987). Preparation of genomic DNA from bacteria. In Current Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith & K. Struhl. New York: Greene Publishing and Wiley-Interscience.
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