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Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim, Tivon 36006, Israel
Correspondence
Malka Halpern
mhalpern{at}research.haifa.ac.il
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, 2002) and at the time of writing comprises three species: O. iheyensis (Lu et al., 2001), whose entire genome sequence has been determined and is now available (Takami et al., 2002), O. oncorhynchi (Yumoto et al., 2005) and O. picturae, which was recently transferred from the genus Virgibacillus (Lee et al., 2006). All of the species belonging to this genus are halophilic or halotolerant and alkaliphilic or alkalitolerant.
Chironomids (Diptera; Chironomidae) are the most abundant insects in freshwater aquatic habitats. Females of the genus Chironomus lay egg masses at the water's edge. Each egg mass contains hundreds of eggs embedded in a thick, gelatinous matrix. Chironomid egg masses harbour Vibrio cholerae and provide a natural reservoir for this bacterium (Broza & Halpern, 2001; Halpern et al., 2003, 2004, 2006). All Vibrio cholerae isolates have the capacity to degrade chironomid egg masses and prevent the eggs from hatching (Halpern et al., 2003, 2004, 2006). Strain T3944DT was isolated in the course of screening of the microbial population of chironomid egg masses (Halpern et al., 2007). The egg masses were sampled in mid-September 2004 from the Tivon waste-stabilization pond near Haifa (northern Israel), using Styrofoam boards as artificial oviposition sites for the adult female chironomids. The egg masses were thoroughly washed and then cultured directly on three bacteriological media, thiosulphate-citrate-bile salts agar (selective for Vibrio cholerae), LB agar and MacConkey agar. Strain T3944DT was one of the colonies that was randomly picked and streaked from the LB agar. More than 102 faecal coliforms ml–1 were counted in the water samples from the Tivon waste-stabilization pond (Halpern et al., 2004), but no faecal coliforms were isolated from the egg mass even though MacConkey agar (selective for Escherichia coli) was used. This confirmed that the egg masses had been rinsed properly and that strain T3944DT and the other isolates had been tightly attached to the egg mass.
Diverse types of bacteria have been isolated from the egg masses and characterized (Halpern et al., 2007). One of these isolates, a Gram-positive, halotolerant, facultatively alkaliphilic, spore-forming, motile bacillus, designated strain T3944DT, is described here. Its exact taxonomic position was determined by using a polyphasic approach that included phenotypic analysis and a phylogenetic investigation based on 16S rRNA gene sequences.
For electron microscopy, bacteria from LB agar were suspended in saline. The samples were attached to a carbon-coated grid, stained with 2 % uranyl acetate and then photographed under a JEM-1200EX electron microscope (JEOL). Electron microscopy showed that the cells were peritrichously flagellated rods (0.8–1.0x1.3–3.0 µm) (Fig. 1).
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. The amplified PCR product (approx. 1.5 kb) was purified with a Wizard PCR product-purification kit (Promega). Purified PCR products were sequenced directly by the dideoxynucleotide chain-termination method, using a DNA sequencer (ABI PRIZM 3100) with BigDye terminator reagents (Applied Biosystems), according to the instructions of the manufacturer (at Technion Medical School, Haifa, Israel). Sequencing was performed using primers 8f (5'-CACGGATCCAGACTTTGATYMTGGCTCAG-3'), 519f (5'-CAGCMGCCGCGGTAATWC-3'), 534r (5'-ATTACCGCGGCTGCTGG-3') and 968f (5'-AACGCGAAGAACCTTAC-3'). This resulted in data relating to approximately 1471 bp. To identify the closest relatives, the newly determined sequence was compared with those available in GenBank (http://www.ebi.ac.uk) by using the WU BLAST program (Washington University Basic Local Alignment Search Tool, version 2.0). Sequence alignment was performed using the CLUSTAL W program, and the phylogenetic tree was generated using the neighbour-joining method in the MEGA3 software (Kumar et al., 2004). Bootstrap values (from 1000 replicates) greater than 50 % are shown at the branch points in the tree presented in Fig. 2.
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).
For the phenotypic characterization, LB agar was used as the basal medium, except for the determination of the pH range, for which peptone-yeast extract medium supplemented with 1 % NaCl was used (according to Lu et al., 2001). Salt tolerance was determined at 37 °C on LB agar containing varying concentrations of NaCl. Growth at various temperatures (4, 6, 10, 11, 12, 15, 25, 30, 32, 35, 37, 39, 40, 44, 46, 47, 48 and 50 °C) was measured on LB agar (pH 8.5) supplemented with 1 % NaCl. Growth under anaerobic conditions was determined after incubation of the novel strain, in an anaerobic chamber, on LB agar containing 1 % NaCl (pH 8.5) and on LB agar supplemented with nitrate. Strain T3944DT showed optimal growth at 1–3 % NaCl, pH 8.5 and 37 °C (Table 1).
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For analysis of the cellular fatty acids, cells were cultured on tryptic soy agar at 28 °C before extraction of the fatty acids. The microbial fatty acid profile was analysed using the MIDI/Hewlett Packard microbial identification system (Analytical Services), which uses GC profiles of fatty acid methyl esters. The measurable fatty acid components of strain T3944DT were as follows: iso-C14 : 0 (5.03 %), C14 : 0 (2.39 %), iso-C15 : 0 (6.08 %), anteiso-C15 : 0 (59.97 %), iso-C16 : 0 (5.92 %), C16 : 0 (7.72 %) and anteiso-C17 : 0 (12.89 %).
For determination of the DNA G+C content, genomic DNA of strain T3944DT was prepared according to a modified version of the procedure of Wilson (1987). The G+C content of the DNA sample was determined in three independent analyses using the HPLC technique (Mesbah et al., 1989) and was performed by the BCCM/LMG Bacteria Collection Identification Service (Laboratory of Microbiology, Ghent University, Ghent, Belgium). The DNA G+C content of strain T3944DT was found to be 38.1 mol%.
Strain T3944DT was isolated from chironomid egg masses, along with Vibrio cholerae and other culturable bacterial populations that inhabit this environment (Halpern et al., 2007). The eggs are embedded in a gelatinous matrix and, although strain T3944DT is able to degrade gelatin (Table 1), unlike all Vibrio cholerae isolates from the same niche (Halpern et al., 2003, 2006), it does not degrade the egg mass.
The genus Oceanobacillus was created by Lu et al. (2001, 2002) and at the time of writing contains three species, which were isolated from quite different environments: (i) O. iheyensis, which is of marine origin; (ii) O. oncorhynchi subsp. oncorhynchi, O. oncorhynchi subsp. incaldanensis and strain T3944DT, which are of freshwater origin; and (iii) O. picturae, which was isolated from a mural painting. The phylogenetic relationships among strain T3944DT and related taxa are shown in Fig. 2. The comparative 16S rRNA gene sequence analysis showed that the novel isolate is phylogenetically most closely related to Oceanobacillus species (94.5–94.9 % sequence similarity). Strain T3944DT shared the main characteristics with Oceanobacillus species, for example, they were positive for catalase and oxidase, they were motile rods (having peritrichous flagella), they produced endospores and they were halotolerant (except for O. oncorhynchi subsp. incaldanensis DSM 16557T, which is halophilic and also does not produce endospores) (Table 1). The DNA G+C content of strain T3944DT (38.1 mol%) is within the range for Oceanobacillus species (35.8–40.1 mol%) and its major cellular fatty acid is anteiso-C15 : 0 (Table 1).
However, strain T3944DT possesses a unique combination of the properties that characterize the members of the genus Oceanobacillus. It is halotolerant and is a facultatively alkaliphilic obligate aerobe (Table 1). It hydrolyses gelatin, is able to reduce nitrite to N2 and has a unique fatty acid profile [including anteiso-C15 : 0 (60.0 %) and anteiso-C17 : 0 (12.9 %)] (Table 1). It grows at 46 °C, which is a higher growth temperature than that of any Oceanobacillus species (Table 1). In the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/blast/), four sequences (GenBank accession numbers DQ346552, DQ346561, DQ346602 and DQ346627) of uncultured and undescribed compost bacteria showed 98.6 % 16S rRNA gene sequence similarity with respect to strain T3944DT. These uncultured strains, which were identified during a clone library study of the bacterial diversity of compost from Korea, probably fall within the lineage of the novel species proposed here (Fig. 2).
On the basis of the phenotypic characterization and the phylogenetic analysis, strain T3944DT should be classified as a novel species, for which the name Oceanobacillus chironomi sp. nov. is proposed.
Description of Oceanobacillus chironomi sp. nov.
Oceanobacillus chironomi [chi.ro'no.mi. N.L. gen. n. chironomi of Chironomus, named after the non-biting midge insect of the genus Chironomus (Chironomidae: Diptera) from which the type strain was isolated].
Cells are Gram-positive, peritrichously flagellated rods (0.8–1.0x1.3–3.0 µm) (Fig. 1) that sometimes form chains. Cells produce ellipsoidal spores terminally or subterminally positioned within swollen sporangia. Colonies are circular and cream–beige in colour. Colony colour darkens from the centre as the culture ages. Obligately aerobic and does not ferment carbohydrates. Facultatively alkaliphilic. Grows at pH 6.5–10, with optimal growth at pH 8.5. Halotolerant and grows with 0–11 % NaCl, with optimal growth at 1–3 % NaCl. Catalase- and oxidase-positive. Growth occurs at 12–46 °C, with optimum growth at 37 °C. Negative for indole production, ONPG hydrolysis and deamination of phenylalanine. Cells are resistant to tetracycline but susceptible to penicillin G, ampicillin, vancomycin, streptomycin, chloramphenicol, bacitracin, novobiocin, gentamicin, neomycin and kanamycin. The following substrates are assimilated in the Biolog test: dextrin, N-acetyl-D-glucosamine, D-fructose, D-galactose, 
-D-glucose, -D-lactose, maltose, maltotriose, D-mannose, D-melibiose, D-ribose, -ketoglutaric acid, -ketovaleric acid, L-alaninamide, 2,3-butanediol, glycerol and adenosine. Cellular fatty acids are iso-C14 : 0 (5.0 %), C14 : 0 (2.4 %), iso-C15 : 0 (6.1 %), anteiso-C15 : 0 (60.0 %), iso-C16 : 0 (6.0 %), C16 : 0 (7.7 %) and anteiso-C17 : 0 (12.9 %).
The DNA G+C content of the type strain is 38.1 mol%. The type strain, T3944DT (=LMG 23627T=DSM 18262T), was isolated from a chironomid egg mass sampled from a waste-stabilization pond in northern Israel.
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