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International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1872–1875 Rheinheimera chironomi sp. nov., isolated from achironomid (Diptera; Chironomidae) egg mass Malka Halpern,1 Yigal Senderovich1 and Sagi Snir2 1Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim, 2Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel A Gram-negative, rod-shaped bacterial strain, designated K19414T, was isolated from achironomid (Diptera; Chironomidae) egg mass which was sampled from Kishon River in northernIsrael. Phylogenetic analysis based on the 16S rRNA gene sequence positioned the novelstrain among the genus Rheinheimera, with closest similarity to Rheinheimera pacifica KMM1406T. The levels of similarity to type strains of Rheinheimera species were lower than 96.5 %.
Isolate K19414T is aerobic, motile by means of a single polar flagellum, catalase-negative andoxidase-positive; growth was observed at salinities of 0–2 % NaCl and the temperature for growthranged from 4 to 40 6C. The major cellular fatty acids are 16 : 0 (14.8 %) and 16 : 1v7cand/or 15 : 0 iso 2-OH (25.76 %). The DNA G+C content is 49.9 mol%. On the basis of itsphenotypic properties and phylogenetic distinctiveness, strain K19414T (5LMG 23818T 5DSM18694T) was classified in the genus Rheinheimera as the type strain of a novel species, forwhich the name Rheinheimera chironomi sp. nov. is proposed.
The genus Rheinheimera was created by Brettar et al. (2002) study was recently characterized as the type strain of while screening blue-coloured bacterial isolates from differ- Oceanobacillus chironomi (Raats & Halpern, 2007).
ent depth stations in the central Baltic Sea. At present, the Strain K19414T is a motile Gram-negative rod, psychro- genus comprises three species: Rheinheimera baltica (Brettar tolerant and strictly aerobic. Its exact taxonomic position et al., 2002), Rheinheimera pacifica (Romanenko et al., was determined by means of a polyphasic approach that 2003) and Rheinheimera perlucida (Brettar et al., 2006). All included phenotypic properties and phylogenetic analysis three species were isolated from marine environments. In based on the 16S rRNA gene sequence.
this study, a new member of the genus Rheinheimera thatwas isolated from a freshwater insect egg mass is proposed 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 photo- Strain K19414T was isolated from a chironomid (non- graphed under a JEM-1200EX electron microscope (JEOL).
biting midge) egg mass while the diversity of the culturable Electron microscopy showed that the cells were rods with bacteria in chironomid egg masses was under study(Halpern et al., 2007). Chironomid egg masses were a polar flagellum (0.3–0.761.0–2.4 mm) (Supplementary sampled in September 2004, as described previously (Halpern et al., 2007). Egg masses were washed thoroughly The 16S rRNA gene was analysed to determine its phylo- with sterile saline water and then their homogenates were genetic position. Universal bacterial primers 8f and 1512r diluted and cultured directly on bacteriological media (Felske et al., 1997) were used to amplify internal fragments (Halpern et al., 2007). Strain K19414T was isolated from an of the 16S rRNA gene. The amplified PCR product was egg mass that was sampled from Kishon River in northern purified with the Wizard PCR product purification kit Israel, cultured on LB agar and incubated at 30 uC for 48 h (Promega). Purified PCR products were sequenced with in the dark. Another isolate from the above-described primers 8f, 534r, 968f and 1512r as described in detail byRaats & Halpern (2007). This resulted in a sequence of Abbreviations: ML, maximum likelihood; MP, maximum parsimony; NJ, For identification of closest relatives, the 16S rRNA gene of The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain K19414T is DQ298025.
strain K19414T was compared with those of previouslyreported strains available in EMBL (http://www.ebi.ac.uk/).
An electron micrograph of cells of strain K19414T and details of its fattyacid profile in comparison with those of related type strains are available Using the BLAST program (version 2.0), the closest se- as supplementary material with the online version of this paper.
quences obtained were those of R. baltica OS140 (96.9 %), R. baltica OSBAC5 (96.7 %), R. pacifica KMM 1406T profiles of fatty acid methyl esters. The major fatty acid (96.5 %), R. perlucida BA131T (96.0 %), R. baltica OSBAC1T components (exceeding 10 %) of strain K19414T are 16 : 0 (95.8 %), Alishewanella fetalis CCUG 30811T (95.8 %), (14.80 %) and summed feature 3 (16 : 1v7c and/or 15 : 0 iso Brenneria salicis DSM 30166T (91.5 %) and Colwellia piezo- 2-OH; 25.76 %) (Supplementary Table S1).
phila Y223GT (89.0 %). The sequences were aligned by the For determination of the DNA G+C content, genomic multiple alignment package CLUSTAL W. Phylogenetic trees DNA of strain K19414T was prepared according to a were generated by neighbour joining (NJ), maximum modification of the procedure of Wilson (1987). The DNA parsimony (MP) and maximum likelihood (ML). NJ and G+C content was determined using HPLC analysis of MP trees were obtained using the MEGA software package(Kumar et al., 2004), while ML was computed by hydrolysed DNA according to Mesbah et al. (1989). The (Felsenstein, 1993). For NJ, distance correction was per- analysis was performed by the BCCM/LMG Bacteria formed using the Kimura two-parameter model (K2ST) Collection Identification Service (Laboratory of Micro- using rate variation across sites. The bootstrap values biology, Ghent University, Belgium). The G+C content of obtained were from 1000 iterations. The NJ tree was drawn by the MEGA software (Fig. 1). In contrast to the highest The morphological, physiological and biochemical traits of sequence similarity to R. baltica OS140 as found by BLAST, strain K19414T are summarized in the species description all phylogenetic inference methods located the novel strain and in Table 1. API ZYM tests of strain K19414T revealed a as a sibling to R. pacifica KMM 1406T.
broad set of 12 enzyme activities (Table 1). Phylogenetic For phenotypic characterization, LB agar was used as the relationships between strain K19414T and some related basal medium with a final concentration of 0.5 % NaCl.
taxa are shown in Fig. 1. Comparative 16S rRNA gene Salt tolerance was determined in LB agar containing varying sequence analysis showed that the new isolate was phylo- concentrations of NaCl at 37 uC. Growth at various tem- genetically most closely related to Rheinheimera species, peratures (4, 6, 8, 10, 15, 18, 25, 30, 32, 35, 37, 39, 40, 41 with 95.8–96.5 % sequence similarity to the type strains.
and 43 uC) was measured on LB agar. Growth under anaer- The new isolate shared its main characteristics with obic conditions was determined after incubation in an Rheinheimera species, such as being aerobic and oxidase- anaerobic chamber on LB agar supplemented with glucose positive, motile rods by means of polar flagella, assimilat- or nitrate. Strain K19414T had optimal growth between 0.5 ing N-acetylglucosamine and hydrolysing gelatin. Its and 1 % NaCl and at 30–37 uC (Table 1).
DNA G+C content was close to those of the otherdescribed Rheinheimera species (48.9–49.9 mol%) (Table 1).
Biochemical tests were performed by means of API Unsaturated fatty acids formed a major fraction of the 20E, API 20NE and API ZYM identification systems total fatty acids in strain K19414T, as in the other described (bioMe´rieux) at 37 uC. Carbon assimilation was analysed strains of the genus Rheinheimera (Supplementary Table S1).
using Biolog GN microwell plates according to the manu- Together with the findings mentioned above, strain K19414T facturer’s instructions (release 3.50, version DE; Biolog).
showed significant characteristics that allowed clear differ- The plates were incubated for 48 h at 37 uC. Wells that entiation from all three described Rheinheimera species. It changed to purple were marked as positive for metabolic lacked catalase activity, grew at relatively low NaCl con- activity. Sensitivity of the strain to antibiotics was tested centrations (0–2 %), grew at 40 uC, which is the highest by means of LB agar and Sensi-discs (BBL). Tests were growth temperature described for species of this genus, and was positive for malate assimilation and for valine arylami- For the cellular fatty acid analysis, cells were cultured on a dase, cystine arylamidase and a-mannosidase (Table 1). On tryptic soy agar (Difco) at 28 uC for 24 h and then the fatty the basis of phenotypic characterization and phylogenetic acids were extracted. The fatty acid profile was analysed by analysis (Stackebrandt & Goebel, 1994), we propose that means of the MIDI/Hewlett Packard Microbial Identifi- strain K19414T should be classified as the type strain of a cation system (Analytical Services Inc.), which uses GC novel species, Rheinheimera chironomi sp. nov.
Fig. 1. Unrooted NJ tree (MEGA3, based onalignment from CLUSTAL W) of strain K19414Tand its closest relatives from the genusRheinheimera and related taxa based on 16SrRNA gene sequences. Bootstrap values areindicated along branches. Branches found byML with P values ,0.01 are marked by anasterisk. Branches marked by a plus sign are inconsensus among all reconstruction methods.
Bar, 0.01 accumulated changes per nucleotideposition.
Table 1. Differential properties of strain K19414T and Rheinheimera type strains Strains: 1, strain K19414T; 2, R. perlucida BA131T (data from Brettar et al., 2006); 3, R. baltica OSBAC1T(Brettar et al., 2002); 4, R. pacifica KMM 1406T (Romanenko et al., 2003). +, Positive; W, weakly positive;2, negative; ND, no data available.
*R. baltica OS550 grew in the presence of 6 % NaCl (Brettar et al., 2002).
International Journal of Systematic and Evolutionary Microbiology 57 Description of Rheinheimera chironomi sp. nov.
monomethyl ester, L-alanine, L-alanyl glycine, glycyl L-aspartic acid and glycyl Rheinheimera chironomi [chi.ro9no.mi. N.L. gen. n. chiro- strates; all other Biolog GN2 substrates are not utilized. The nomi of Chironomus, named after the non-biting midge G+C content of the type strain is 49.9 mol%.
insect from the genus Chironomus (Diptera; Chirono-midae) from which the type strain was isolated].
The type strain is strain K19414T (5DSM 18694T 5LMG Cells are aerobic, Gram-negative, non-pigmented rods, 23818T), which is of freshwater origin.
1.0–2.4 mm long and 0.3–0.7 mm wide, occurring as singlecells 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 Brettar, I., Christen, R. & Ho¨fle, M. G. (2002). Rheinheimera baltica and able to reduce nitrate to nitrite. Sodium ions are not gen. nov., sp. nov., a blue-coloured bacterium isolated from the required for growth. Growth is observed in 0–2 % (w/v) central Baltic Sea. Int J Syst Evol Microbiol 52, 1851–1857.
NaCl, but not in 3 % NaCl. Grows at 4–40 uC, but not at Brettar, I., Christen, R. & Ho¨fle, M. G. (2006). Rheinheimera perlucida 41 uC. Hydrolyses gelatin and casein but not DNA. Grows sp. nov., a marine bacterium of the Gammaproteobacteria isolated on LB and half-strength marine agar. Does not haemolyse from surface water of the central Baltic Sea. Int J Syst Evol Microbiol56, 2177–2183.
bovine blood and does not grow on MacConkey agar. Themajor measurable fatty acid components (exceeding 5 %) Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version3.5c. Distributed by the author. Department of Genome Sciences, of the type strain are 11 : 0 3-OH (5.53 %), 12 : 0 3-OH University of Washington, Seattle, USA.
(8.54 %) 15 : 0 (6.86 %), 16 : 0 (14.80 %), summed feature 3 Felske, A., Rheims, H., Wolterink, A., Stackebrandt, E. & Akkermans, (16 : 1v7c and/or 15 : 0 iso 2-OH; 25.76 %), 17 : 0 (5.76 %), A. D. L. (1997). Ribosome analysis reveals prominent activity of an 17 : 1v8c (7.56 %) and 18 : 1v7c (6.68 %). The following uncultured member of the class Actinobacteria in grassland soils.
fatty acids are detected in the type strain as minor components: 9 : 0 (0.2 %), 10 : 0 (1.2 %), 11 : 0 (1.35 %), Halpern, M., Landsberg, O., Raats, D. & Rosenberg, E. (2007).
10 : 0 3-OH (0.53 %), unknown ECL 11.799 (2.63 %), 12 : 0 Culturable and VBNC Vibrio cholerae: interactions with chironomid (1.06 %), 13 : 0 (0.62 %), 12 : 0 iso 3-OH (0.41 %), 14 : 0 iso egg masses and their bacterial population. Microb Ecol 53, 285–293.
(0.19 %), 14 : 0 (0.89 %), summed feature 1 (one or more Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software of 13 : 0 3-OH, 15 : 1 iso I and 15 : 1 iso H; 1.9 %), 15 : 0 for molecular evolutionary genetics analysis and sequence alignment.
anteiso (0.28 %), 15 : 1v8c (2.75 %), 15 : 1v6c (1.04 %), summed feature 2 (14 : 0 3-OH and/or 16 : 1 iso I; 0.28 %), Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise 16 : 0 iso (1.45 %), 17 : 0 iso (0.19 %), 17 : 0 anteiso measurement of the G+C content of deoxyribonucleic acid by high- (0.41 %), 17 : 1v6c (0.87 %) and 18 : 0 (0.25 %). Cells are performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
resistant to neomycin, penicillin G and bacitracin but Raats, D. & Halpern, M. (2007). Oceanobacillus chironomi sp. nov., a susceptible to tetracycline, ampicillin, vancomycin, strepto- halotolerant and facultatively alkaliphilic species isolated from a mycin, chloramphenicol, novobiocin and SXT (sulfameth- chironomid egg mass. Int J Syst Evol Microbiol 57, 255–259.
oxazole and trimethoprim). In the API 20NE test system, Romanenko, L. A., Uchino, M., Falsen, E., Zhukova, N. V., Mikhailov, nitrate is reduced to nitrite, glucose, N-acetylglucosamine, V. V. & Uchimura, T. (2003). Rheinheimera pacifica sp. nov., a novel maltose and malate are assimilated and enzyme activities of halotolerant bacterium isolated from deep sea water of the Pacific. IntJ Syst Evol Microbiol 53, 1973–1977.
protease (gelatinase) and b-galactosidase are detected. Inthe API ZYM test system, 12 enzyme activities are detected: Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place forDNA-DNA reassociation and 16S rRNA sequence analysis in the alkaline and acid phosphatases, esterase (C4 and C8), leucine present species definition in bacteriology. Int J Syst Bacteriol 44, arylamidase, valine arylamidase, cystine arylamidase, trypsin, Wilson, K. (1987). Preparation of genomic DNA from bacteria. In acetyl-b-glucosaminidase and a-mannosidase. In the Biolog Current Protocols in Molecular Biology, pp. 2.4.122.4.5. Edited by GN2 test system, Tweens 40 and 80, D-galactose, D-glucose, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith & maltose, sucrose, pyruvic acid methyl ester, succinic acid K. Struhl. New York: Greene Publishing and Wiley-Interscience.

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