A novel microbial interaction: obligate commensalism between a new gram-negative thermophile and a thermophilic Bacillus strain

Obligately commensal interaction between a new gram-negative thermophile and a thermophilic Bacillus strain was investigated. From compost samples, a mixed culture showing tyrosine phenol-lyase activity was enriched at 60°C. The mixed culture consisted of a thermophilic gram-negative strain, SC-1, and a gram-positive spore-forming strain, SK-1. In mixed cultures, strain SC-1 started to grow only when strain SK-1 entered the stationary phase. Although strain SC-1 showed tyrosine phenol lyase activity, we could not isolate a colony with any nutrient medium. For the isolation and cultivation of strain SC-1, we added culture supernatant and cell extract of the mixed culture to the basal medium. The supernatant and cell extract of the mixed culture contained heat-stable and heat-labile factors, respectively, that are essential to the growth of strain SC-1. During pure cultures of strain SK-1, the heat-stable growth factors were released during the growth phase and the heat-labile growth factors were produced intracellularly at the early stationary phase. Strain SC-1 was gram-negative and microaerophilic, and grows optimally at 60°C. Based on these results, we propose a novel commensal interaction between a new gram-negative thermophile, strain SC-1, and Bacillus sp. strain SK-1. Received: November 18, 1999 / Accepted: December 2, 1999     

Geobacillus thermoleovorans subsp. stromboliensis subsp. nov., isolated from the geothermal volcanic environment

A novel thermophilic, aerobic, endospore-forming bacterium, designated strain PizzoT, was isolated from geothermal volcanic environment. Samples were collected from the Pizzo sopra la Fossa site at Stromboli Island (Eolian Islands, south of Italy) at the high altitude of 918 m. Cells of strain PizzoT were rod-shaped and stained Gram-positive. Growth was observed between 50 and 75 degrees C (optimum 70 degrees C) and at pH 5.0-8.0 (optimum pH 7.0). NaCl (0.4%, w/v) supported growth and among the hydrocarbons tested none induced growth. The G+C content of the DNA was 54.1 mol% and the sequence analysis of the 16S rRNA gene showed that the new isolate was phylogenetically closely related to the members of the Bacillus rRNA Group 5. DNA-DNA hybridization studies revealed a borderline similarity between the new isolate and Geobacillus thermoleovorans DSM 5366T (69.8%) and Geobacillus kaustophilus DSM 7263T (63.4%). On the basis of phylogenetic analysis and physiological traits of the isolate, it should be described as a new member of the Geobacillus thermoleovorans species and it is proposed that strain PizzoT can be classified as Geobacillus thermoleovorans subsp. stromboliensis, subsp. nov. (ATCC BAA-979T; DSM 15393T).     

Methanol utilization by a novel thermophilic homoacetogenic bacterium,Moorella mulderi sp. nov., isolated from a bioreactor

A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as the energy source. Cells were gram-positive straight rods, 0.4-0.6 microm x 2-8 microm, growing as single cells or in pairs. The temperature range for growth was 40-70 degrees C with an optimum at 65 degrees C. Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7. The salinity range for growth was 0-45 g NaCl l(-1 )with an optimum at 10 g l(-1). The isolate was able to grow on methanol, H(2)-CO(2 )(80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin. The bacterium reduced thiosulfate to sulfide. The G+C content of the DNA was 53 mol%. Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%). On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp. nov. (=DSM 14980, =ATCC BAA-608).     

一株新型产淀粉酶中度嗜热菌的分离鉴定及酶学性质研究

用稀释分离法从广西象州温泉筛选分离到一株可水解淀粉的中度嗜热细菌GXS1,该茵株革兰氏染色为阳性,端生芽孢,细胞呈杆状,最适生长温度为60℃～65℃,最适生长pH为6.0～7.0.结合生理生化特征和16S rDNA序列分析,初步鉴定该茵株为Geobacillus sp.GXS1.对该茵所产高温淀粉酶的性质研究表明:酶的...     

Geobacillus uralicus,a new species of thermophilic bacteria

The K2T 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 degrees C) in the Ural. 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 and of fermentation. Growth occurs within the 40-75 degrees C temperature range (with an optimum of 65 degrees C) and at pH 5-9. The minimum generation time (15 min) was observed at pH 7.5. Ammonium salts and nitrates 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 K2T represents a new species of the genus Geobacillus, Geobacillus uralicus.     

Evaluation of arabinofuranosidase and xylanase activities of Geobacillus spp. isolated from some hot springs in Turkey

Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about 60 degrees C on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by > or = 97%, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima (80 degrees C), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at 75 degrees C, and only isolate AC 15 has the lowest pH of 5.5.     

Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon-degrading, heavy metal resistant bacterium from hot compost

A thermophilic, spore-forming bacterial strain L1(T) was isolated from hot compost "Pomigliano Environment" s.p.a., Pomigliano, Naples, Italy. The strain was identified by using a polyphasic taxonomic approach. L1(T) resulted in an aerobic, gram-positive, rod-shaped, thermophilic with an optimum growth temperature of 68 degrees C chemorganotrophic bacterium which grew on hydrocarbons as unique carbon and energy sources and was resistant to heavy metals. The G+C DNA content was 43.5 mol%. Phylogenetic analysis of 16S rRNA gene sequence and Random Amplified Polymorphic DNA-PCR (RAPD-PCR) analysis of L1(T) and related strains showed that it forms within Geobacillus toebii, a separate cluster in the Geobacillus genus. The composition of cellular fatty acids analyses by Gas-Mass Spectroscopy differed from that typical for the genus Geobacillus in that it is lacking in iso-C15 fatty acid, while iso-C16 and iso-C17 were predominant. Isolates grew on a rich complex medium at temperatures between 55-75 degrees C and presented a doubling time (t(d)) of 2 h and 6 h using complex media and hydrocarbon media, respectively. Among hydrocarbons tested, n-decane (2%) was the more effective to support the growth (1 g/L of wet cells). The microorganism showed resistance to heavy metal tested during the growth. Furthermore, intracellular alpha-galactosidase and alpha-glucosidase enzymatic activities were detectable in the L1(T) strain. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA-DNA hybridization, we propose assigning a novel subspecies of Geobacillus toebii, to be named Geobacillus toebii subsp. decanicus subsp. nov., with the type strain L1(T) (=DSM 17041=ATCC BAA 1004).     

Purification and characterization of an extremely thermostable cyclomaltodextrin glucanotransferase from a newly isolated hyperthermophilic archaeon, a Thermococcus sp

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 micrometers in diameter. The new isolate grew at temperatures between 60 and 95 degrees C (optimum, 85 degrees C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120 degrees C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100 degrees C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110 degrees C. The enzyme formed mainly alpha-cyclodextrin with small amounts of beta- and gamma-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Geobacillus thermodenitrificans subsp. calidus, subsp. nov., a thermophilic and α-glucosidase producing bacterium isolated from Kizilcahamam, Turkey

An α-glucosidase producing, thermophilic, facultatively anaerobic, and endospore-forming, motile, rod-shaped bacterial strain F84b(T) was isolated from a high temperature well-pipeline sediment sample in Kizilcahamam, Turkey. The growth occurred at temperatures, pH and salinities ranging from 45 to 69oC (optimum 60oC), 7.0 to 8.5 (optimum 8.0) and 0 to 5% (w/v) (optimum 3.5%), respectively. Strain F84b(T) was able to grow on a wide range of carbon sources. Starch and tyrosine utilization, amylase, catalase and oxidase activities, nitrate reduction, and gas production from nitrate were all positive. The G+C content of the genomic DNA was 49.6 mol%. The menaquinone content was MK-7. The dominant cellular fatty acids were iso-C17:0, iso-C15:0, and C16:0. In phylogenetic analysis of 16S rRNA gene sequence, strain F84b(T) showed high sequence similarity to Geobacillus thermodenitrificans (99.8%) and to Geobacillus subterraneus (99.3%) with DNA hybridization values of 74.3% and 29.1%, respectively. In addition, the Rep-PCR and the intergenic 16S-23S rRNA gene fingerprinting profiles differentiated strain F84b(T) from the Geobacillus species studied. The results obtained from the physiological and biochemical characters, the menaquinone contents, the borderline DNA-DNA hybridization homology, and the genomic fingerprinting patterns had allowed phenotypic, chemotaxonomic and genotypic differentiation of strain F84b(T) from G. thermodenitrificans. Therefore, strain F84b(T) is assigned to be a new subspecies of G. thermodenitrificans, for which the name Geobacillus thermodenitrificans subsp. calidus, subsp. nov. is proposed (The type strain F84b(T) = DSM 22629(T) = NCIMB 14582(T)).     

嗜热菌的耐热L—乳酸脱氢酶的研究

About 200 strains of extreme thermophilic bacteria were isolated from hot springs in Guandong province. A strain, HG25, was found to produce thermostable intracellular L-lactate dehydrogenase (EC. 1.1.1.27). It has the characteristic of Thermus sp. The cells were gram-negative, non-sporulating, nonmotile, aerobic rods containing yellow pigment. The optimum temperature for growth was between 65 degrees C to 75 degrees C, the maximum 85 degrees C, and minimum 40 degrees C. The generation time at the optimum was about 80 min. Starch was not hydrolyzed. Acid was not produced from glucose. The G+C content in DNA was 62-65 mol% (Tm). As the properties of strain HG25 is similar to those of Thermus aquaticus and T. thermophilus HB 8 belonging to the genus Thermus. The thermostable L-lactate dehydrogenase was partially purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. For pyruvate reduction, the optimum temperature of the enzyme was 60 degrees C and pH 8.0. After incubation in 0.1 mol/L phosphate buffer pH 7.4 at 70 degrees C for 10 min, the enzyme retained about 85% of its original activity. The half-live time (t1/2) at 85 degrees C was 10 min.     

Thermaerobacter nagasakiensis sp. nov., a novel aerobic and extremely thermophilic marine bacterium

A novel, extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent at depth of 22 m in Tachibana Bay, Nagasaki Prefecture, Japan. Cells were gram-negative, non-spore-forming, motile rods. Growth was observed between 52 and 78 degrees C (optimum 70 degrees C), pH 5 and 8 (optimum pH 7) and 0-4.5% NaCl (optimum 1.0%). The isolate was a strictly aerobic heterotroph utilizing yeast extract and trypticase peptone. The G+C content of the genomic DNA is 69 mol%. Analysis of 16S rDNA sequences indicated that strain Ts1a is closely related to Thermaerobacter marianensis. The differences in physiology and DNA-DNA similarity between strain Ts1a and T. marianensis showed that strain Ts1a represents a new species of Thermaerobacter. The type strain of T. nagasakiensis is strain Ts1a (=JCM11223, DSM 14512).     

Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1

A novel lipase-producing thermophilic strain TW1, assigned to Geobacillus sp. TW1 based on 16S rRNA sequence, was isolated from a hot spring in China. Based on this strain, a lipase gene encoding 417 amino acids was cloned. Subsequently, the lipase gene was expressed in Escherichia coli and purified as a fusion protein with glutathione S-transferase. The results showed that the recombinant lipase had an activity optimum at 40 degrees C and pH at 7.0-8.0. It was active up to 90 degrees C at pH 7.5, and stable over a wide pH ranging from 6.0 to 9.0. The recombinant lipase was stable in 1 mM enzyme inhibitors (EDTA, 2-ME, SDS, PMSF or DTT), as well as in 0.1% detergents (Tween 20, Chaps or Triton X-100). Its catalytic function was enhanced in the presence of Ca(2+), Mg(2+), Zn(2+), Fe(2+) or Fe(3+), but inhibited by Cu(2+), Mn(2+), and Li(+). By comparison with the crude lipase, the recombinant lipase had similar properties and was characteristic of thermostable enzymes. Our study presented a rapid overexpression and purification of the lipase gene from thermophile, aimed at improving the enzyme yield for industrial applications.     

Anoxybacillus mongoliensis sp. nov., a novel thermophilic proteinase producing bacterium isolated from alkaline hot spring, central Mongolia

A Gram reaction positive, spore-forming, facultative anaerobic bacterium belonging to the Phylum Firmicutes, was isolated from alkaline hot (80 degrees C, pH 9.8 spring Tsenher, central Mongolia. The cells were rod shaped, feebly motile, peritrichously flagellated. Strain T4 was moderately thermophilic with optimum growth at 60 degrees C. Maximum temperature for growth was between 70 and 75 degrees C; minimum temperature for growth was between 35 and 30 degrees C. Alkalitolerant, optimum pH for growth was 8.0; minimum pH for growth was between 5.0 and 5.5 and maximum was between 10.5 and 10.8. The growth was observed at NaCl concentrations of 0-5% (w/v) with the optimum at 0.2-0.5%. No growth was observed at 6% NaCl (w/v). Aerobically, the strain utilized proteinaceous substrates, organic acids and a range of carbohydrates including glucose, ribose, sucrose and xylose as well. Anaerobically, only glucose and sucrose were utilized. Strain T4T produced thermostable alkaline subtilisin-like serine proteinase. The G + C content was 44.2 mol. % (td). On the basis of 16S rRNA gene sequence similarity strain T4(T) was shown to be closely related to the members of the genus Anoxybacillus (family Bacillaceae, class "Bacilli"). DNA-DNA hybridization data revealed that strain T4T had only 38% relatedness to A. flavithermus and 28% relatedness to A. pushchinoensis. Based on its morphology, physiology, phylogenetic relationship and its low DNA-DNA relatedness values with validly published species of Anoxybacillus, it is proposed that strain T4T represents a novel species Anoxybacillus mongoliensis sp. nov., with the type strain T4(T) (=DSM 19169 = VKM 2407).     

嗜热菌来源的生淀粉酶分离纯化及其酶学性质

从嗜热菌库中分离到两株能水解生淀粉的菌株173和174,通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明,所分离两株菌属于Geobacillus属的细菌.液体摇瓶发酵菌株173、174,其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL.通过生淀粉吸附-熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化,得到纯化的RSDE173和RSDE174,纯化倍数分别为50和29,活力回收率分别为34%和41%.有关RSDE173和RSDE174酶学性质研究显示.对熟淀粉水解的最适作用温度均为70℃,而对生淀粉水解则分别在50℃～60℃和40℃～60℃下表现出高水解活力;对不同底物的最适作用pH值均为5.0～5.5;它们对大多数试验离子的敏感性较低,但个别离子如Co2 、Cu'2 对RSDE173或u'2 对RSDE174的酶活力有一定的抑制作用.纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同.RSDE173底物专一性顺序为红薯淀粉>小麦淀粉>玉米淀粉>木薯淀粉>糯米淀粉;而RSDE174的糯米淀粉>小麦淀粉>红薯淀粉>玉米淀粉>木薯淀粉.RSDE173对生红薯淀粉有很好的降解,其水解糊化淀粉与生红薯淀粉的比值为1.48;而RSDE174优先降解生糯米淀粉,其相应比值为1.69.     

Isolation and characterization of thermophilic,alkaliphilic, cellulose‐degrading bacillus thermoalcaliphilus sp. nov. from termite (odontotermes obesus) mound soil of a semiarid area

A new heterotrophic, thermophilic, alkaliphilic, facultatively anaerobic, cellulose‐degrading bacterium strain STS1 was isolated from mound soils infested with the higher termite Odontotermes obesus in the semiarid ecosystem of Delhi (India). The gram‐positive, spore‐forming, catalase‐positive Bacillus sp. grew on natural and raw celluloses. The taxonomic position of the organism was investigated. The guanine plus cytosine content of the isolate was found to be 48.6 mol% (melting temperature profile). Addition of peptone or yeast extract stimulated growth. The isolate did not grow on silica gel plates or on agar media in which the agar was the sole source of carbon and energy. The high growth temperature of 70°C and the pH of 9.0 are characteristic of this species. The role of this bacterium in the semiarid ecosystem is discussed. Because of its high optimum temperature and high optimum pH for growth, the name Bacillus thermoalcaliphilus is proposed.     

一株产木聚糖酶嗜热菌的鉴定及酶学性质

从云南腾冲热泉水样中分离筛选得到一株产木聚糖酶的菌株。通过细菌形态观察、生理生化特性并结合16S rDNA序列分析,经鉴定,该菌为地芽孢杆菌(Geobacillus sp.),命名为Geobacillus sp.PZH1。对该菌株所产嗜热木聚糖酶及酶学特性进行了初步研究。SDS-PAGE和酶谱分析测得该酶分子量约为69 kD;酶的最适反应pH和最适反应温度分别为7.0和70°C,在pH 5.0-11.0和40°C-100°C范围内均有较高酶活;该酶在pH 5.0-12.0范围内和70°C以内具有较高的稳定性;40°C-100°C范围内,该木聚糖酶没有被检测到纤维素酶活性。     

Identification and over-expression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli

A newly isolated thermophilic strain producing thermostable lipase was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its closeness to Geobacillus thermoleovorans. Thermostable lipase from this bacterium was cloned using consensus degenerate PCR primers. For over-expression in Escherichia coli, the lipase gene was sub-cloned in pET 15b vector with a strong T7 promotor. Lipase activity was approximately 4.5-fold higher than in the wild-type strain. The lipase enzyme was thermostable at 60 degrees C and pH 8, whereas a 30% residual activity was retained when incubated for 1h at 100 degrees C. Optimum lipase expression was obtained in 2 x YT medium after 70min of induction by IPTG.     

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.     

Geobacillus jurassicus sp. nov., a new thermophilic bacterium isolated from a high-temperature petroleum reservoir, and the validation of the Geobacillus species

Four thermophilic, spore-forming bacterial strains, DS1(T), DS2, 46 and 49, were isolated from the high-temperature Dagang oilfield, located in China. The strains were identified by using the polyphasic taxonomy approach. These were aerobic, gram-positive, rod-shaped, moderately thermophilic (with an optimum growth temperature of 60-65 degrees C), chemoorganotrophic bacteria capable of growing on various sugars, carboxylic acids and crude oil. Two strains, DS1(T) and DS2, were capable of growing on individual saturated hydrocarbons. The G + C content of the DNA of strains DS1(T) and DS2 was 54.5 and 53.8 mol%, respectively. The phylogenetic analysis of the 16S rDNA of strains DS1(T) and DS2 showed that they form a separate cluster within the genus Geobacillus. The cellular fatty acids of the isolates were dominated by iso-15:0, iso-16:0 and iso-17:0 acids, which are the typical fatty acids of bacteria from the genus Geobacillus. The DNA-DNA hybridization study and the comparative analysis of the morphological and chemotaxonomic characteristics of strains DS1(T) and DS2 showed that they differ from the previously described Geobacillus species and belong to a new species, which was called Geobacillus jurassicus. DS1(T) (=VKM B2301(T), = DSM 15726(T)) is the type strain of this species. According to both DNA-DNA reassociation studies and 16S rDNA sequence analysis, two other strains, 46 and 49, were assigned to the species G. stearothermophilus. In this paper, we provide evidence that the new combinations G. stearothermophilus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans may be considered to be valid.