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1 Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan
2 Bio-Nano Electronics Research Centre, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585, Japan
3 Noda Institute for Scientific Research, 399 Noda, Noda, Chiba 278-0037, Japan
Correspondence
Akinobu Echigo
dc0400017{at}toyonet.toyo.ac.jp
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, meso-diaminopimelic acid-type murein. Growth occurred in 5.0–25 % (w/v) NaCl (optimum, 10–15 %, w/v), at pH 5.5–10.0 (optimum, pH 8.5–9.0) and at 20–40 °C. The predominant isoprenoid quinone was menaquinone-7. The major cellular fatty acids were ai-C15 : 0, i-C16 : 0, ai-C17 : 0 and i-C15 : 0. The G+C content of the total DNA of strain BH2T was 35.1±0.4 mol% (±SD; n=5). The phylogenetic distance from species with validly published names was less than 94.1 %. The phylogenetic and phenotypic characteristics indicated that strain BH2T represents a novel genus and species, for which the name Halalkalibacillus halophilus gen. nov., sp. nov. is proposed. The type strain is BH2T (=JCM 14192T=DSM 18494T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain BH2T is AB264529.
An alignment of 16S rRNA gene sequences of members of the family Bacillaceae and a phylogenetic tree reconstructed from the alignment and signature bases of the genera Alkalibacillus, Filobacillus, Tenuibacillus and strain BH2T are available as supplementary material in IJSEM Online.
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; Oren, 2002). Moderate halophiles are defined as those that grow optimally in media containing 3–15 % (w/v) NaCl. They have been isolated from various saline environments such as salt lakes (e.g. the Dead Sea and the Great Salt Lake), salterns, solar salts and subsurface salt formation. It is tacitly believed that the habitats of moderate halophiles are restricted to saline environments and no reports have been published on the isolation of moderately halophilic micro-organisms from non-saline environments. In this study, we propose that a moderately halophilic and alkaliphilic bacterium isolated from a sample from ordinary non-saline garden soil represents a novel genus and species, based on phylogenetic and phenotypic characteristics.
Soil samples were taken from non-saline, ordinary gardens in many cities of Saitama Prefecture, Japan. Most of the gardens were full of trees and flowers. No highly saline environments such as salterns or salt lakes occur in the region. NaCl contents calculated by determining the Cl– content of a soil extract with water using the method of Mohr (Lenore et al., 1998) were less than 0.1 % (w/v). The pH values of the soil extracts were slightly acidic, pH 5.0–6.0.
About 0.5 g of the soil sample was placed on an alkaline agar plate, spread with a spatula and incubated at 37 °C for 3 weeks in a plastic bag to prevent desiccation. The medium used contained the following (l–1): 5.0 g Casamino acids (Difco), 5.0 g yeast extract (Difco), 1.0 g sodium glutamate.H2O, 3.0 g trisodium citrate.2H2O, 2.0 g KCl, 0.2 g MgSO4.7H2O, 36 mg FeCl2.4H2O, 200.0 g (3.4 M) NaCl and 20 g Bacto agar (Difco), pH 7.2. After autoclaving, the pH was adjusted to 9.5 by addition of pre-calculated amounts of sterile Na2CO3 solutions. Colonies were picked, transferred to fresh agar plates, and pure cultures were obtained by plating serial dilutions and repeated transfers on agar plates. Strain BH2T, isolated from non-saline surface soil from a garden in Niiza City, was selected for further characterization after sequencing of PCR-amplified 16S rRNA genes of a few isolates.
Strain BH2T grew in 5.0–25 % (w/v) NaCl (pH 9.5, 37 °C), with optimum growth at 10–15 % (w/v) NaCl. Growth occurred within the pH range 5.5–10.0 [10 % (w/v) NaCl, 37 °C], with optimum growth at pH 8.5–9.0. The temperature range for growth was 20–40 °C [10 % (w/v) NaCl, pH 9.5], with optimum growth at 30–37 °C. Anaerobic growth was not observed using an anaerobic jar (Echigo et al., 2005) after incubation for 7 days at 37 °C.
Colonies of strain BH2T formed on agar (10 % NaCl, pH 9.5, 37 °C) were cream-like and opaque. The cells were rod-shaped and approximately 0.3–0.5x2.0–3.0 µm. The cells were motile with a single polar flagellum, observed according to Kodaka et al. (1982). Endospore formation was examined after spore-staining, according to the method of Wirtz-Conklin (Murray et al., 1999). A spherical terminal endospore with a swollen sporangium was produced (at 10 % NaCl, pH 9.5, 37 °C). Cells stained Gram-positive after acetic acid fixation, as described by Dussault (1955). The KOH test (Gregersen, 1978) and L-alanine aminopeptidase using Bactident Aminopeptidase test strips (Merck) were negative, as reported previously for Gram-positive bacteria (Gregersen, 1978).
Production of acid from carbohydrates was tested using media in which the amounts of Casamino acids and yeast extract were reduced to 1.0 g l–1, supplemented with 10.0 g l–1 of the test carbohydrate. The cultures were incubated at 37 °C under aerobic conditions for 3 days. Growth was determined visually and the pH was measured with a pH electrode. Strain BH2T did not produce acid from D-galactose, maltose, sucrose, D-trehalose, D-mannitol, D-fructose, D-glucose or D-xylose. Tests for catalase and oxidase activities and hydrolysis of starch, gelatin, casein, DNA, hippurate, aesculin, pullulan and Tween 80 were performed according to the standard or modified procedures of Smibert & Krieg (1994), Oren et al. (1997) and Schlesner et al. (2001). Strain BH2T was catalase- and oxidase-positive and did not hydrolyse starch, casein, gelatin, DNA, hippurate, aesculin, pullulan or Tween 80. Reduction of nitrate was not detected using the sulfanilic acid and 
-naphthylamine reagent (Smibert & Krieg, 1981) and formation of gas from nitrate was not detected using Durham tubes under anaerobic conditions.
The sensitivity to antimicrobial agents was tested with discs impregnated with the particular antimicrobial agents placed on pre-inoculated plates. Inhibitory zones around the discs were recorded after incubation for 3 days at 37 °C. Strain BH2T was sensitive to ampicillin (50 µg disc–1), bacitracin (25 µg disc–1), chloramphenicol (25 µg disc–1) and novobiocin (25 µg disc–1), whereas it was resistant to kanamycin (50 µg disc–1), streptomycin (100 µg disc–1), tetracycline (50 µg disc–1) and anisomycin (50 µg disc–1).
HPLC analysis of isoprenoid quinones and GC/MS analysis of fatty acid methyl esters were performed according to the modified procedures of Tamaoka (1986) and Komagata & Suzuki (1987). The predominant isoprenoid quinone of strain BH2T was menaquinone-7 (MK-7). The cellular fatty acid profile of strain BH2T was characterized by saturated branched fatty acids such as ai-C15 : 0, i-C16 : 0, ai-C17 : 0 and i-C15 : 0.
Preparation of peptidoglycan and determination of its structure were performed according to the modified procedures of Schleifer & Kandler (1972), Schleifer (1985) and Schlesner et al. (2001). Purified peptidoglycan was hydrolysed in 4 M HCl at 100 °C for 16 h (total hydrolysate) or 45 min (partial hydrolysate). Diamino acids were identified from the total hydrolysate by using one-dimensional TLC in methanol/pyridine/4 M HCl/water (80 : 10 : 4 : 26, by vol.) solvent system. Amino acids and peptides were identified from the partial hydrolysate by using two-dimensional TLC. The first direction was developed in isopropyl alcohol/acetic acid/water (75 : 10 : 15, by vol.) and the second in -dipicolin/25 % ammonium hydrate/water (70 : 2 : 28, by vol). The resulting fingerprints were compared with those from known peptidoglycan structures. Strain BH2T possessed A1, meso-diaminopimelic (m-Dpm)-type murein.
Total DNA was extracted by using the method of Saito & Miura (1963). The 16S rRNA genes were amplified by PCR with the following forward and reverse primers: 5'-AGAGTTTGATCCTGGCTCAG-3' (positions 8–27 according to Escherichia coli numbering) and 5'-GGCTACCTTGTTACGACTT-3' (positions 1510–1492). The amplified DNA was cloned using a TA Cloning kit (Invitrogen) and sequenced using an ABI PRISM BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) with the following primers: 5'-GGAAACAGCTATGACCATG-3' (vector side), 5'-GACTACCAGGGTATCTAATC-3' (positions 805–786), 5'-AGGGTTGCGCTCGTTG-3' (positions 1115–1100) and 5'-GTAAAACGACGGCCAGT-3' (vector side), using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). There was little (2 or 3 bases) heterogeneity among the sequences (1492 bp) of several clones. The sequence of strain BH2T was shown to be most closely related to three species of the genus Alkalibacillus (Alkalibacillus haloalkaliphilus, Alkalibacillus filiformis and Alkalibacillus salilacus; 94.1–93.6 % sequence similarity), Thalassobacillus devorans (94.0 %), Filobacillus milosensis (94.0 %) and Tenuibacillus multivorans (92.9 %) by FASTA analysis. The sequences of related strains retrieved from the DNA Database of Japan (DDBJ) (Miyazaki et al., 2003; Pearson & Lipman, 1988; Lipman & Pearson, 1985) were aligned using the CLUSTAL W multiple sequence alignment program (Thompson et al., 1994). A phylogenetic tree was reconstructed by using the neighbour-joining method (Saitou & Nei, 1987) and evaluated by bootstrap sampling (Felsenstein, 1985). Fig. 1 shows that strain BH2T is a member of the family Bacillaceae and is most closely related to the three species of the genus Alkalibacillus, F. milosensis and Tenuibacillus multivorans. The G+C content of the total DNA of strain BH2T, determined using the HPLC method of Tamaoka & Komagata (1984), was 35.1±0.4 mol% (±SD; n=5).
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; Jeon et al., 2005; Romano et al., 2005), F. milosensis (Schlesner et al., 2001) and Tenuibacillus multivorans (Ren & Zhou, 2005), as summarized in Table 1. Strain BH2T grew at pH 5.5–10.0 with optimal growth at pH 8.5–9.0, whereas F. milosensis and Tenuibacillus multivorans were neutrophilic with no growth above pH 9.0. The Alkalibacillus species were alkaliphilic with no growth below pH 6.5. The murein type of strain BH2T was m-Dpm, as for the related genera Alkalibacillus and Tenuibacillus. F. milosensis was shown to possess A4
, L-orn-D-Glu-type murein. The major fatty acid of strain BH2T was ai-C15 : 0 (i-C16 : 0 was the second most abundant), whereas those of the three Alkalibacillus species and Tenuibacillus multivorans were i-C15 : 0 (ai-C15 : 0 or ai-C17 : 0) and those of F. milosensis were ai-C15 : 0 (i-C15 : 0). Other differences between strain BH2T and F. milosensis occurred in Gram stain, oxidase reaction, hydrolysis of DNA and hippurate and sensitivity to streptomycin, and other differences between strain BH2T and Tenuibacillus multivorans were in hydrolysis of casein, gelatin and aesculin. An alignment of 16S rRNA gene sequences of members of the genera discussed above as well as Gracilibacillus, Halobacillus, Virgibacillus, etc., and some halophilic strains of the genus Bacillus and a phylogenetic tree reconstructed from the alignment are available as Supplementary Figs S1 and S2, respectively, in IJSEM Online. In addition, a table showing signature bases of the genera Alkalibacillus, Filobacillus and Tenuibacillus and strain BH2T is available as Supplementary Table S1 in IJSEM Online. According to the phylogenetic and phenotypic characteristics, strain BH2T represents a novel species of a new genus, for which the name Halalkalibacillus halophilus gen. nov., sp. nov. is proposed.
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Cells are motile with a single polar flagellum, rod-shaped, 0.3–0.5x2.0–3.0 µm. Spores are spherical, located terminally, with swollen sporangia. Gram-positive. KOH test and L-alanine aminopeptidase are negative. Cell walls contain A1, m-Dpm-type murein. No growth occurs in media without NaCl. Alkaliphilic, mesophilic, aerobic and halophilic. Catalase- and oxidase-positive. Reduction of nitrate and gas formation are not observed. The G+C content of the total DNA is 35.1±0.4 mol% (±SD; n=5). The predominant isoprenoid quinone is MK-7. Major cellular fatty acids are ai-C15 : 0, i-C16 : 0, ai-C17 : 0 and i-C15 : 0. The type species is Halalkalibacillus halophilus.
Description of Halalkalibacillus halophilus sp. nov.
Halalkalibacillus halophilus (ha.lo.phi'lus. Gr. n. hals salt; Gr. adj. philos loving; N.L. masc. adj. halophilus salt loving).
Exhibits the following characteristics in addition to those given in the genus description. Colonies formed on agar are cream-like and opaque. Growth occurs in 5.0–25 % (w/v) NaCl, with optimum growth at 10–15 % (w/v) NaCl. Growth occurs at pH 5.5–10.0, with optimum growth at pH 8.5–9.0. Temperature range for growth is 20–40 °C, with optimum growth at 30–37 °C. Anaerobic growth is not observed. Acid is not produced from D-galactose, maltose, sucrose, D-trehalose, D-mannitol, D-fructose, D-glucose or D-xylose. Starch, gelatin, casein, DNA, hippurate, aesculin, pullulan and Tween 80 are not hydrolysed. Sensitive to ampicillin, bacitracin, novobiocin and chloramphenicol; resistant to tetracycline, streptomycin, kanamycin and anisomycin.
The type strain is strain BH2T (=JCM 14192T=DSM 18494T), which was isolated from non-saline surface soil from a garden in Niiza City, Saitama Prefecture, Japan.
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ACKNOWLEDGEMENTS |
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