Sulfobacillus sibiricus sp. nov., a New Moderately Thermophilic Bacterium

In the course of pilot industrial testing of a biohydrometallurgical technology for processing gold-arsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha (Yakutiya)), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe²⁺, S⁰, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55°C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on a medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow in the absence of an inorganic energy source on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G+C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA–DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis), which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9–98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.     

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Brown algae are one of the largest groups of oceanic primary producers for CO₂ removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2^T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing d-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD⁺, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.     

Catalytic Biomineralization of Fluorescent Calcite by the Thermophilic Bacterium Geobacillus thermoglucosidasius

The thermophilic Geobacillus bacterium catalyzed the formation of 100-μm hexagonal crystals at 60°C in a hydrogel containing sodium acetate, calcium chloride, and magnesium sulfate. Under fluorescence microscopy, crystals fluoresced upon excitation at 365 ± 5, 480 ± 20, or 545 ± 15 nm. X-ray diffraction indicated that the crystals were magnesium-calcite in calcite-type calcium carbonate.Biomineralization is defined as the synthesis of inorganic crystalline or amorphous mineral-like materials by living organisms. Most of the crystals formed through biomineralization consist of inorganic minerals, but they may also contain trace amounts of organic compounds, which are thought to regulate the biomineralization process. A Japanese researcher first coined the term biomineralization in the 1940s, and interest in this field of research soon began to grow (28, 29). Biomineralization is a universal process, occurring in both prokaryotes and eukaryotes, including mammals. Biomineralization research is also a diverse and multidisciplinary field, encompassing microscopic analyses of biominerals and tissues (21, 23), mineralogy (25), organic chemistry (30), paleontology (3), and cellular biochemistry (19).Bacteria are capable of forming inorganic crystals either intracellularly (12, 14) or extracellularly (9). Calcite (calcium carbonate) precipitation is a well-known example of extracellular bacterial biomineralization. Certain species of marine bacteria have been shown to precipitate minerals in water supplemented with artificial marine salt media and differing ratios of Mg²⁺ to Ca²⁺ concentrations (20). Several groups have investigated the geobiochemical significance of the presence of calcium carbonate in seafloor sediments in terms of calcite formation by marine bacteria (17). Boquet et al. (4) reported calcite formation by 210 species of soil bacteria cultured on solid growth medium containing calcium acetate, yeast extract, and glucose. Sánchez-Román et al. (22) found that 19 species of moderately halophilic bacteria, grown in nutrient liquid media, catalyzed the precipitation of calcite, magnesium calcite, and struvite in variable proportions depending on the ratio of Mg²⁺ to Ca²⁺ in the media.Researchers and engineers in materials science have only recently begun to focus on biomineralization (2). There is currently great interest in the development of mimic materials based on biomineralization. In our own research, we found that a moderately thermophilic bacterium isolated from thermophilically composted organic waste catalyzes the formation of inorganic crystals extracellularly under oligotrophic conditions. In this paper, we describe the physiological properties of this isolated thermophilic bacterium, the conditions under which it catalyzes the formation of crystals, the elemental composition and photoluminescent properties of the crystals, and the technological implications of our findings.Composted organic waste (0.4 g) was suspended in 20 ml of sterilized water and streaked onto soybean-casein digest (SCD) agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), and the agar plate was incubated at 60°C for 48 h. A single visible colony was picked and recultured by streaking on a fresh SCD agar plate incubated at 60°C. Another single colony was isolated from this plate and cultured in the same manner to obtain a pure culture of the thermophilic bacterium. Gram staining was carried out according to conventional methodology. Morphology and photoluminescence properties were observed using a fluorescence microscope (Axioskop 2 plus; Zeiss, Oberkochen, Germany). Growth under anaerobic conditions was evaluated by culturing the bacterium in an anaerobic chamber.The organism we isolated from composted organic waste was a Gram-positive, rod-shaped (0.5 μm wide and 2 to 5 μm long), facultative aerobic bacterium. Oval-shaped endospores were readily discerned microscopically by their intracellular terminal sites. The bacterium was categorized as moderately thermophilic, as it grew maximally in SCD broth (pH 7.0) at 60°C but did not grow above 70°C or below 45°C (5, 32). The inability of the bacterium to grow at 37°C indicated that the organism was an obligate thermophile (16). The maximal pH for growth in SCD broth culture at 60°C was 6.0 to 7.0, but a pH of 5.0 strongly inhibited growth. There was no growth under acidic conditions (pH 3.0 to 4.0).Total DNA was extracted from 1.5 ml of cultured thermophilic bacteria (approximately 0.04 g of cells) using a DNA extraction kit (InstaGene Matrix; Bio-Rad, CA) according to the manufacturer''s instructions. Sense and antisense primers were designed (5′-GAGTTTGATCCTGGCTCAG-3′ and 5′-GGCTACCTTGTTACGA-3′, respectively) and used in PCR with total DNA serving as the template. The reaction mixture contained 5 μg of total DNA, 400 pmol of each oligonucleotide primer, and 0.1 U of Taq DNA polymerase in a volume of 50 μl. Thirty thermal cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min were carried out. PCR products were purified by agarose gel electrophoresis, and the resulting DNA fragment (approximately 1,500 bp) was ligated into a pCRII vector (Invitrogen, CA). The PCR-amplified fragment was sequenced using the dideoxy-chain termination method.The small subunit rRNA gene of the isolated bacterium was amplified from bulk genomic DNA by PCR, and it constituted 1,483 bp of the 1,500 bp that were originally amplified by PCR. Searches for similar sequences were carried out using the BLAST program. According to the GenBank/DDBJ/EMBL database, the sequence of this gene showed 100% homology with Geobacillus thermoglucosidasius ACTT43742 ({"type":"entrez-nucleotide","attrs":{"text":"AB021197","term_id":"7209544","term_text":"AB021197"}}AB021197 in GenBank). The isolated bacterium was thus identified as Geobacillus thermoglucosidasius and designated strain NY05.For crystal formation on a gel surface, bacteria were first inoculated onto an SCD agar plate and incubated at 60°C for 18 h. A total of 1 mg (wet weight) of bacterial cells were inoculated with a loop onto a crystal-promoting hydrogel (25 mM sodium acetate, 7.0 mM calcium chloride, 2.0 mM magnesium sulfate, and 1.5% [wt/vol] agar) and incubated at 60°C for 24 h (hydrogel method).When cells were cultured using the hydrogel method, crystals of about 20 μm in size appeared in and around bacterial colonies during the course of a 3- to 4-h incubation. As shown in Fig. ​Fig.11 B, 100- to 200-μm hexagon-like crystals appeared when incubation was extended to 24 h. The importance of metabolic activity for crystal formation was evidenced by the absence of crystallization in control experiments, i.e., those performed without bacteria, or with cultures that had been autoclaved. These results demonstrate that bacteria are not simply heterogeneous nuclei for crystallization but actively mediate the process.Open in a separate windowFIG. 1.(A and B) Polarized (A) and light (B) microscopic observation of crystals formed using the hydrogel method. (C to E) Fluorescence microscopic image of crystals excited at 480 ± 20 nm, (C) 365 ± 5 nm (D), and 545 ± 15 nm (E).To isolate crystals formed using the hydrogel method, the part of gel on which crystals formed was excised using a sterilized spatula and solubilized in buffer QX1 (Qiagen, CA) in a test tube at room temperature. Crystals were recovered at the bottom of the test tube and washed repeatedly with sterilized distilled water and ethanol and then dried at 60°C. Crystals were observed using an Eclipse-ME600 polarized microscope (Nikon, Tokyo, Japan) and an Axioskop 2 plus fluorescence microscope. For fluorescence microscopy, the following filter blocks were used: exciter, 360 to 370 nm, 460 to 500 nm, and 530 to 560 nm; dichroic mirror, 380 nm, 505 nm, and 570 nm; emitter, >399, 510 to 560 nm, and 573 to 684 nm.The exceptionally strong polarizing property of crystals when examined under polarized microscopy confirmed that the material was composed of crystallized inorganic mineral (Fig. ​(Fig.1A).1A). The remarkable fluorescent property of the crystals was demonstrated by fluorescence microscopy. As shown in Fig. 1C to E, the crystals fluoresced green, blue, and red when excited at 480 ± 20 nm, 365 ± 5 nm, and 545 ± 15 nm, respectively.Geobacillus catalyzed the formation of crystals on gel media containing 25 mM sodium acetate, 7.0 mM calcium chloride, and 2.0 mM magnesium sulfate. Gels lacking magnesium sulfate did not prevent bacteria from catalyzing crystal formation, but the removal of sodium acetate or calcium chloride from media did preclude crystal formation. Thus, calcium and carboxyl carbon were both essential for crystal formation, while magnesium sulfate was inessential. Crystals formed when incubation temperatures were in the range of 60 to 70°C, but when the temperature was kept in the range of 40 to 50°C, fewer crystals formed. Crystallization was precluded by incubation above 80°C and below 30°C. The optimal temperature for crystal formation was therefore chosen to be 60°C. Crystal formation was observed only under aerobic conditions, suggesting that atmospheric oxygen and/or biological oxidative molecules are essential for the catalytic processes leading to crystal formation. The effect of sodium acetate concentration on crystal formation was also investigated using the hydrogel method. The concentrations of calcium chloride and magnesium sulfate were maintained at 7.0 mM and 2.0 mM, respectively, while the concentration of sodium acetate was varied. Sodium acetate in the range of 12.5 mM to 50 mM induced satisfactory crystal formation. However, at a concentration of 75 mM, sodium acetate decreased the efficiency of crystal formation, and no crystals were formed on gels with 100 mM sodium acetate. The effect of different sources of carbon on the ability of Geobacillus to catalyze crystal formation was investigated using the hydrogel method. The presence of sodium formate or acetate at 25 mM induced calcite formation, but 25 mM sodium propionate, sodium citrate, methyl alcohol, and ethyl alcohol did not promote crystal formation. These results indicated that Geobacillus-catalyzed crystal formation requires a carbon source derived from C₁ and C₂ carboxylates. The optimal concentration of calcium ion for crystal formation was also determined using the hydrogel method, in which the concentrations of sodium acetate and magnesium sulfate were 25 mM and 2.0 mM, respectively. More than 200 crystals per milligram (wet weight) of cells were obtained using 40 mM calcium chloride.The effect of heavy metal ions on crystal formation was investigated by doping the hydrogel with heavy metal ions (CuCl₂, NiCl₂, CdCl₂) at concentrations ranging from 0.01 to 0.5 mM. In general, results obtained for hydrogel doped with Cu²⁺ showed that a high concentration (0.5 mM) of heavy metal ions completely inhibited crystal formation. Doping hydrogel with 0.1 mM Cd²⁺ strongly inhibited crystal formation. On the other hand, low concentrations (0.01 mM) of metal ions did not impede crystal formation. The presence of Ni²⁺ at a concentration of 0.01 mM enhanced crystal formation.Crystals were examined for fluorescence properties using an SPEX Fluorolog LF3-22-Tau3 fluorescence spectrophotometer (Horiba, Tokyo, Japan). Crystals were studied at room temperature, and matrix scanning was employed for the measurement of fluorescence spectra. Wavelength scans were performed at 250 to 400 nm at step intervals of 5 nm. The emission spectrum was measured for each excitation wavelength between 310 and 700 nm. Crystals were excited in the range of 260 to 400 nm and emitted energy in the range of 350 to 600 nm. The maximum excitation and emission wavelengths were 369.6 nm and 446.9 nm, respectively (Fig. ​(Fig.22 A). Figure ​Figure2B2B shows an example of a fluorescence spectrum for crystals excited at a wavelength of 350 nm, which were emitted at a wavelength of 375 to 550 nm with the maximum emission at 439 nm.Open in a separate windowFIG. 2.(A) Photoluminescence spectrum of a single crystal prepared using the gel method. (B) Emission fluorescence spectrum of a single crystal excited at 350 nm. cps, counts per second.Energy-dispersive X-ray (EDX) elemental analyses of single purified crystals were performed using an EMAX-5770 electron microscope equipped with a microanalysis system (Horiba, Tokyo, Japan). The analyses were carried out at 20 kV of accelerating voltage and 0.26 nA of probe current. As shown in Fig. ​Fig.33 A, crystals contained large amounts of carbon, oxygen, and calcium, with smaller amounts of magnesium, sodium, silica, sulfur, phosphorus, and chlorine. When the number of calcium atoms was converted to 100, the composition with respect to magnesium, sodium, silicon, sulfur, phosphorus, and chlorine was 19.1, 12.2, 5.10, 2.35, 1.22, and 1.83, respectively. We confirmed that the crystals react with hydrochloric acid using the Meingen reaction (4) and that they emit gas (CO₂) upon melting. The crystal is possibly a calcium carbonate species, because its spectrum was similar to that of standard calcium carbonate (Fig. ​(Fig.3B).3B). The inability of Geobacillus to catalyze crystal formation under anaerobic conditions suggests that the oxygen incorporated into the crystals may be derived from atmospheric oxygen.Open in a separate windowFIG. 3.EDX spectrum of crystals. (A) Crystal formed through catalysis by G. thermoglucosidasius. (B) Purified calcium carbonate.The structure of purified crystals was analyzed by the Bragg-Brentano method by using a RINT-2000 powder X-ray diffractometer (Rigaku, Tokyo, Japan) with a graphite monochromator, a scintillation counter scanning rate of 4°/min, and a rotary stage. The X-ray generator was operated at 40 kV and 100 to 200 mA. The divergence and receiving slits for the diffracted beam were set at 0.01° and 0.15 mm, respectively. The scanning regions of the diffraction angle (2 theta) were 20° to 90° at a step interval of 0.02° and a scan rate of 2.0°/min. Spectra were collected by using a diffractometer with a graphite monochromator and Cu Kα radiation (Fig. ​(Fig.4).4). The peak pattern of crystals was consistent with that of CaCO₃ as a reference at up to 60° in 2 theta degrees, but the peak intensity of crystals was not consistent with that of the CaCO₃ reference at over 40° in 2 theta degrees. A comparison of the X-ray diffraction pattern with the powder diffraction file (PDF-2) provided by the International Centre for Diffraction Data suggested that the crystals are magnesian calcite [(Ca, Mg)CO₃] (PDF identification number 00-043-0697). Therefore, the data indicate that the crystals formed by Geobacillus nucleation are calcite-type calcium carbonate, in which some of the calcium in the crystal structure has been replaced by magnesium, sodium, sulfur, chlorine, and phosphorus.Open in a separate windowFIG. 4.Powder X-ray diffraction patterns of the CaCO₃ reference (bottom) and the unidentified crystal formed by G. thermoglucosidasius catalysis (top).We observed that visible calcite crystals formed within 4 h in the area around colonies of G. thermoglucosidasius cultured on crystal formation gel. This observation supports the contention that G. thermoglucosidasius secretes nano-sized nucleus-like factors that are associated with extracellular crystal formation. Addadi et al. (1) reported that acidic matrix macromolecules of polystyrene film, which are involved in regulating calcite growth, often contain aspartic acid-rich domains and covalently bound sulfated polysaccharides. They proposed that sulfates and beta-sheet-structured carboxylates cooperate in oriented calcite crystal nucleation (1). In our study, the rate of crystal formation may have been enhanced due to the gel matrices that regulated the dispersion of calcium ions.G. thermoglucosidasius was unable to catalyze calcite formation under anaerobic conditions, indicating that oxygen is required for the formation of calcite crystals. Accordingly, we proposed that the following two reactions are involved in G. thermoglucosidasius-catalyzed calcite formation when either formate, 2Ca²⁺ + 2HCOO⁻ + O₂ → 2CaCO₃ + 2H⁺, or acetate, 2Ca²⁺ + 2CH₃COO⁻ + 3O₂ → 2CaCO₃ + 2CO₂ + 2H⁺, is involved.The precise role G. thermoglucosidasius-catalyzed calcite formation plays in the environment has not been elucidated. Reports demonstrating that bacteria adsorbed onto inorganic minerals are resistant to various chemical and physical stressors suggest a possible role for bacterially catalyzed calcite formation (7, 13, 26). We found endospore-like substances adsorbed on the surface of purified calcite. Purified calcite formed by G. thermoglucosidasius nucleation and exposed to extreme conditions (i.e., washing with 70% ethyl alcohol, treatment with protease and DNase, UV irradiation at 254 nm, boiling for 15 min, freezing at −80°C for 48 h) was incubated on the surface of SCD nutrient agar at 60°C for 14 to 18 h, resulting in the formation of a bacterial colony around the calcite. Though the calcite crystals are inorganic substances and were completely dehydrated, G. thermoglucosidasius cells and/or endospores could have been adsorbed to the surface and protected from the extreme conditions in a cryptobiotic state (6, 10). We confirmed that the colony was growing concentrically around the calcite that had been exposed to the extreme environment. These results indicated that calcite-bound cells were more resistant to physicochemical stressors.The calcite formation was initiated under oligotrophic conditions in which formate or acetate was present, as were trace amounts of calcium. If G. thermoglucosidasius cells were exposed to conditions unsuitable for their growth in the natural environment, they would be expected to catalyze the formation of calcite and adsorb onto it, living in a cryptobiotic state until conditions improved. It is assumed that G. thermoglucosidasius cells adsorb onto calcite as a means of surviving unfavorable conditions but remain there and continue to grow under favorable oligotrophic conditions. Calcite formation may therefore be one of the means these organisms use to survive in extreme environments.G. thermoglucosidasius-catalyzed calcite crystals may have a number of applications that can be exploited. Metal oxide-type phosphors, which are conventionally used in fluorescent lamps and cathode ray tubes, are generally prepared by doping high-purity and precious rare earth with an activator (31). Different CaCO₃ modifications doped with Eu³⁺, Tb³⁺, or Ce³⁺ have been synthesized as phosphor hosts, and their luminescent properties have been determined (8). The maximum excitation and emission wavelengths for Eu³⁺-doped calcite-type red phosphor are 393 nm and 611 nm, respectively (18). Vaterite-type CaCO₃ doped with Ce³⁺ and Tb³⁺ emits green light, and its maximum excitation and emission wavelengths are 275 nm and 545 nm, respectively (11). However, the drastic loss of fluorescence intensity these phosphors display at room temperature is disadvantageous. In addition, these phosphors have a very narrow emission wavelength interval of 50 nm. The calcite crystals formed by G. thermoglucosidasius nucleation, on the other hand, are excited by a wavelength interval of 260 to 400 nm, and their emission wavelengths are from 350 to 600 nm. The wide emission wavelength interval is a novel fluorescence property of G. thermoglucosidasius-catalyzed calcite crystals. Unlike conventional phosphors, calcite crystals formed by G. thermoglucosidasius nucleation can be prepared without rare earth, and they accommodate magnesium, sodium, sulfur, and phosphorus atoms in the carbonate host lattice. We may expect new industrial applications for G. thermoglucosidasius-catalyzed calcite phosphor, owing to its fluorescence stability without loss of intensity.G. thermoglucosidasius calcite phosphor could have potential advantages as a filler in rubber and plastics, fluorescent particles in stationery ink, and a fluorescent marker for biochemistry applications (15) and also with uses in other biodevices (24). Our study may provide novel inorganic materials with a range of photoluminescent properties that can be fabricated from calcite doped with various heavy-metal ions (e.g., Cu²⁺, Ni²⁺, Cd²⁺). In materials engineering, environmentally friendly systems with minimal energy consumption and resource depletion are required for producing materials and composites. Biological processes serve as impressive archetypes of sustainable materials technologies. Because of its potential benefits in this regard, the study of biominerals has gained recognition as an important area of biomimetic materials science (27).     

嗜热土芽孢杆菌GSEY01及其高温蛋白酶的初步研究

从云南腾冲热海热泉中分离出一株产高温蛋白酶的菌株GSEY01。该菌株最适生长温度为60℃,16S rRNA基因序列分析表明,该菌株为土芽孢杆菌属(Geobacillus)的耐热菌株。该菌株所产高温蛋白酶可以通过超滤浓缩,硫酸铵分级沉淀和强阴离子交换层析获得纯酶。此高温蛋白酶分子量约为42kD,最适催化温度为80℃,最适催化pH7.5,Mg2+能增强该酶活力,Fe3+,Cd2+和Ni2+对其活性则有抑制作用。PMSF对该酶影响较小,乙二胺四乙酸(EDTA)和十二烷基磺酸钠(SDS)则对其有强烈的抑制作用,此高温蛋白酶和其他土芽孢杆菌所产蛋白酶有较大差异,可以应用于相关的高温催化环境。     

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H₂, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.     

Deinococcus soli sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Rice Field Soil

A Gram-negative, non-motile, short rod-shaped bacterial strain, designated N5^T, was isolated from a rice field soil in South Korea. Phylogenetic analysis based on the 16S rRNA gene sequence of the new isolate showed that strain N5^T belongs to the genus Deinococcus, family Deinococcaceae, showing the highest sequence similarity to Deinococcus grandis KACC 11979^T (98.4 %) and Deinococcus daejeonensis KCTC 13751^T (97.5 %). Strain N5^T exhibits resistance to gamma-radiation similar to that of other members of the genus Deinococcus, with a D₁₀ value in excess of 4 kGy. Chemotaxonomic data showed that the most abundant fatty acids are C_16:1 ω7c (25.25 %), C_15:1 ω6c (19.77 %), C_17:1 ω6c (11.87 %), and C_17:0 (9.41 %), and the major polar lipid is an unknown phosphoglycolipid. The predominant respiratory quinone is menaquinone MK-8. The DNA G+C content is 71.4 mol%. Phenotypic, phylogenetic, and chemotaxonomic data support designation of strain N5^T as a novel species of the genus Deinococcus, for which the name Deinococcus soli sp. nov. is proposed. The type strain is N5^T (=KCTC 33153^T = JCM 19176^T).     

Spirosoma radiotolerans sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Gamma Ray-Irradiated Soil

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A^T, was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A^T belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74^T, 93.4 % with Spirosoma rigui WPCB118^T, 92.8 % with Spirosoma luteum SPM-10^T, 92.7 % with Spirosoma spitsbergense SPM-9^T, and 91.9 % with Spirosoma panaciterrae Gsoil 1519^T. Strain DG5A^T revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C_16:1 ω7c/C_16:1 ω6c (36.90 %), C_16:1 ω5c (29.55 %), and iso-C_15:0 (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A^T was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A^T presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A^T (=KCTC 32455^T = JCM19447^T).     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

一株高乙醇耐受的嗜热细菌Anoxybacillus sp. WP06的性质研究

厌氧芽孢杆菌属(Anoxybacillus)的菌株WP06是一株兼性厌氧的嗜热细菌, 能利用木糖、阿拉伯糖和葡萄糖等产生乙醇。不像绝大多数嗜热细菌, WP06菌株在高温下表现出极高的乙醇耐受力, 60oC时在8%的乙醇胁迫下才出现生长抑制现象, 15%的乙醇胁迫下仍能生长, 是目前已知的乙醇耐受力最高的嗜热细菌。WP06菌株突破了人们对高温下细菌耐受乙醇浓度的极限认识, 是研究高温下乙醇耐受机制的良好出发菌株。     

一株高乙醇耐受的嗜热细菌Anoxybacillus sp. WP06的性质研究

厌氧芽孢杆菌属(Anoxybacillus)的菌株WP06是一株兼性厌氧的嗜热细菌, 能利用木糖、阿拉伯糖和葡萄糖等产生乙醇。不像绝大多数嗜热细菌, WP06菌株在高温下表现出极高的乙醇耐受力, 60oC时在8%的乙醇胁迫下才出现生长抑制现象, 15%的乙醇胁迫下仍能生长, 是目前已知的乙醇耐受力最高的嗜热细菌。WP06菌株突破了人们对高温下细菌耐受乙醇浓度的极限认识, 是研究高温下乙醇耐受机制的良好出发菌株。     

Isolation and Identification of a Pyrogallol Producing Bacterium from Soil

A bacterial strain, which was isolated from soil, and identified as Citrobacter sp., showed an inducible gallic acid decarboxylase activity producing pyrogallol from gallic acid. The strain also decarboxylated protocatechuic acid, pyrocatechuic acid, 3,5-dihydroxybenzoic acid and m-hydroxybenzoic acid as well. The pyrogallol and pyrocatechol produced were isolated from the cultured broths to which gallic acid and protocatechuic acid were added, respectively.     

Salinicola zeshunii sp. nov., a Moderately Halophilic Bacterium Isolated from Soil of a Chicken Farm

The taxonomic status of a moderately halophilic bacterium, strain N4^T, isolated from soil of a chicken farm in China was determined. It was Gram-negative, non-spore-forming, motile, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequence indicated that this strain belonged to the genus Salinicola, as it showed the highest sequence similarities to Salinicola salaries M27^T (98.3 %), Salinicola socius SMB35^T (98.1 %), and Salinicola halophilus CG4.1^T (98.1 %). The major cellular fatty acids were C_16:0 (25.6 %), C_18:1ω7c (35.0 %), and C_19:0 cyclo ω8c (11.9 %), which are properties shared by members of the genus Salinicola. The DNA G+C content of strain N4^T was 69.1 mol %. The level of DNA–DNA relatedness between strain N4^T and the other three type strains of the genus of Salinicola salaries M27^T, Salinicola socius SMB35^T, and Salinicola halophilus CG4.1^T were 34.3, 28.7, and 26.9 %, respectively. Based on the results of phenotypic, chemotaxonomic, DNA–DNA relatedness, and phylogenetic analysis, strain N4^T should be classified as a novel species of the genus Salinicola, for which the name Salinicola zeshunii sp. nov. is proposed, with strain N4^T (=KACC 16602^T = CCTCC AB 2012912^T) as the type strain.     

Growth of the Thermophilic Bacterium Geobacillus uralicus as a Function of Temperature and pH: An SCM-Based Kinetic Analysis

The synthetic chemostat model (SCM), originally developed to describe nonstationary growth under widely varying concentrations of the limiting substrate, was modified to account for the effects of nontrophic factors such as temperature and pH. The bacterium Geobacillus uralicus, isolated from an ultradeep well (4680 m), was grown at temperatures ranging from 40 to 75°C and at pH varying from 5 to 9. The biomass kinetics was reasonably well described by the SCM, including the phase of growth deceleration observed in the first hours after a change in the cultivation temperature. At an early stage of batch growth in a neutral or alkalescent medium, bacterial cells showed reversible attachment to the glass surface of the fermentation vessel. The temperature dependence of the maximum specific growth rate (_m) was fitted using the equation _m = Aexp(T)/{1 + expB[1 – C/(T + 273)]}, where A, , B, and C are constants. The maximum specific growth rate of 2.7 h^–1 (generation time, 15.4 min) was attained on a complex nutrient medium (peptone and yeast extract) at 66.5°C and pH 7.5. On a synthetic mineral medium with glucose, the specific growth rate declined to 1.2 h^–1, and the optimal temperature for growth decreased to 62.3°C.     

Geobacillus uralicus,a New Species of Thermophilic Bacteria

The K^T ₂ strain of thermophilic spore-forming bacteria was isolated from a biofilm on the surface of a corroded pipeline in an extremely deep well (4680 m, 40–72°C) in the Urals. The cells are rod-shaped, motile, gram-variable. They grow on a complex medium with tryptone and yeast extract and on a synthetic medium with glucose and mineral salts without additional growth factors. The cells use a wide range of organic substances as carbon and energy sources. They exhibit a respiratory metabolism but are also capable of anaerobic growth on a nitrate-containing medium. Growth occurs within the 40–75°C temperature range (with an optimum of 65°C) and at pH 5–9. The minimum generation time (15 min) was observed at pH 7.5. Ammonium salts, nitrates, and arginine are used as nitrogen sources. The G+C content of the DNA is 54.5 mol %. From the morphological, physiological, and biochemical properties and the nucleotide sequence of the 16S rRNA gene, it was concluded that the isolate K^T ₂ represents a new species of the genus Geobacillus, Geobacillus uralicus.     

Pseudomonas creosotenesis sp. n., a Creosote-tolerant Marine Bacterium

In a study of the marine biological environment in which creosoted pilings are located, a previously unreported species of bacteria was isolated. This species was detected on creosoted piling from 11 widely differing locations and was the predominant species of bacteria found on these piling. The new organism was identified as a gram-negative rod belonging to the genus Pseudomonas and has been named Pseudomonas creosotensis. It has been completely described by the standard morphological and biochemical tests.     

Experimental fossilization of the Thermophilic Gram-positive Bacterium Geobacillus SP7A: A Long Duration Preservation Study

Recent experiments to fossilize microorganisms using silica have shown that the fossilization process is far more complex than originally thought; microorganisms not only play an active role in silica precipitation but may also remain alive while silica is precipitating on their cell wall. To better understand the mechanisms that lead to the preservation of fossilized microbes in recent and ancient rocks, we experimentally silicified a Gram-positive bacterium, Geobacillus SP7A, over a period of five years. The microbial response to experimental fossilization was monitored with the use of LIVE/DEAD staining to assess the structural integrity of the cells during fossilization. It documented the crucial role of silicification on the preservation of the cells and of their structural integrity after several years. Electron microscopy observations showed that initial fossilization of Gram-positive bacteria was extremely rapid, thus allowing very good preservation of Geobacillus SP7A cells. A thick layer of silica was deposited on the outer surface of cell walls in the earliest phase of silicification before invading the cytoplasmic space. Eventually, the cell wall was the only recognizable feature. Heavily mineralized cells thus showed morphological similarities with natural microfossils found in the rock record.