苍白芽孢杆菌和安徽鞘氨醇杆菌的分离鉴定及多相分类学研究
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摘要
从安徽森林土壤中分离到两株细胞和菌落形态以及保守性16S rRNA基因序列与它们各自属内相邻菌株具有较大差别的新菌株,分别命名为CW 7T和CW 186T。通过革兰氏染色反应,确定菌株CW 7T和CW 186T分别为革兰氏阳性细菌和革兰氏阴性细菌,利用两类菌株各自不同的分类学特征,通过对菌株CW 7T和CW 186T的形态学、生理生化试验、细胞化学成分和16S rRNA基因序列系统进化等多相分类学指标分析,最终确定CW 7T的分类学地位为芽孢杆菌科,芽孢杆菌属内的一个新种,将其命名为苍白芽孢杆菌；而CW 186T的分类学地位为鞘氨醇杆菌科,鞘氨醇杆菌属内的一个新种,将其命名为安徽鞘氨醇杆菌。
CW 7T革兰氏染色反应呈阳性,菌体呈杆状,周身鞭毛；培养2-3天后,菌落颜色为浅白或淡粉红色,圆形且平坦,边缘光滑,并且相互粘连,单菌落较难分开。菌体具运动性,细胞长有周身菌毛,产生孢子,好氧,化能异养型；能较好的生长于TYB复合培养基,在LB培养基中生长缓慢,很难或不能生长于营养琼脂和麦康凯琼脂等单一培养基。
菌株生长的温度范围是15-42℃,最适生长温度是30-37℃。CW 7T能较好的利用七叶树素、葡萄糖酸盐、2-酮基-葡萄糖酸盐和D-木糖为唯一碳源生长；并能够利用葡萄糖酸盐、2-酮基-葡萄糖酸盐和D-木糖发酵产酸。蛋白酶反应阳性,过氧化氢酶弱阳性；其他如氧化酶、p-半乳糖苷酶、精氨酸双水解酶、脂肪酶、α-甲基-D-葡萄糖苷酶、鸟氨酸脱羧酶、赖氨酸脱羧酶和脲酶均为阴性。不能还原硝酸盐和亚硝酸盐；不能水解酪氨酸、吐温80和淀粉；不产H2S和吲哚,VP反应为阳性。能生长于0-2% NaCl盐浓度范围内,无Na+等盐离子依赖性。
CW 7T的主要脂肪酸是ai-C_(15:0), i-C_(15:0)和ai-C17：0。呼吸链异戊二烯醌的种类为七碳烷基侧链的甲基奈醌(MK-7)；细胞壁主要肽聚糖类型为2,6-二氨基庚二酸(meso-diaminopimelic acid, meso-DAP); G+C摩尔百分含量为42.3%。16S rDNA序列同源性分析表明,CW 7T与芽孢杆菌属内其他成员的同源性小于96.5%,并在系统进化树中形成一个独立的分支。
通过以上形态学、生理生化、脂肪酸、细胞壁组分和16S rRNA基因序列同源性等多相分析结果,最终确定CW 7T的分类地位为：细菌域(Bacteria),厚壁菌门(Firmicutes),芽孢杆菌纲(Bacilli),芽孢杆菌目(Bacillales),芽孢杆菌科(Bacillaceae),芽孢杆菌属(Bacillus)的一个新种,并将其命名为苍白芽孢杆菌(Bacillus pallida)。
CW 186T革兰氏染色反应呈阴性,菌体呈椭圆形,具周生纤毛,不具有端生或周生鞭毛,但在适宜的环境下能够滑行,运动能力较差,生长快速,30℃培养2-3天后观察菌落颜色为橙黄色,圆形并向上微微凸起,边缘光滑,单菌落较容易分开。无内生孢子,好氧,化能异养型。不仅能较好的生长于TYB、LB等复合培养基,也能生长于麦康凯琼脂。
该菌株生长的温度范围为4-35℃,最适生长温度为25-30℃。CW 186T能较好的利用N-乙酰葡萄糖胺、七叶树素、D-阿拉伯糖、L-阿拉伯糖、熊果苷、纤维二糖、蔗糖、D-果糖、L-果糖、D-半乳糖、葡萄糖、菊糖、D-乳糖、苦杏仁苷、麦芽糖、D-甘露糖、蜜二糖、α-甲基-D-葡萄糖苷和α-甲基-D-甘露糖苷。过氧化氢酶、氧化酶、α-甲基-D-葡萄糖苷酶和p-半乳糖苷酶呈阳性；其他如精氨酸双水解酶、鸟氨酸脱羧酶、赖氨酸脱羧酶、色氨酸脱羧酶和脲酶均为阴性。不能还原硝酸盐和亚硝酸盐；能水解淀粉和DNA,但不能水解酪氨酸、吐温80和尿素；不产H2S和吲哚,VP反应为阳性。能生长于0-3% NaCl盐浓度范围内,无Na+等盐离子依赖性。
CW 186T主要脂肪酸为iso-C_(15:0)、iso-C_(17:0) 3-OH和Summed feature 3(iso-C_(15.0) 2-OH and/or C_(16:1ω7c))(含量分别为32.2%,9.8%,33.7%),呼吸链异戊二烯醌种类为七碳烷基侧链的甲基奈醌(MK-7)。G+C摩尔百分含量为36.3%。16S rDNA序列同源性分析表明,CW 186T与其所属属内相邻已发表菌种16S rDNA序列同源性差别都小于94%,在系统进化树中形成一个独立的分支。通过以上多相分类学分析结果,最终确定菌株CW 186T的分类地位为鞘氨醇杆菌属中的一个新种,我们将菌株CW 186T命名为安徽鞘氨醇杆菌(Sphingobacterium anhuiense)。
Two novel bacteria strains designated CW 7T and CW 186T were isolated from forest soil, Anhui Province, China. Strains CW 7T and CW 186T showed obviously distincted morphological, chemotaxonomic and phylogenetic characteristics from their closest neighbors. The polyphasic taxonomic study including phenotypic and chemotaxonomic and phylogenetic analysis displayed that strain CW 7T represents a novel species within the genus Bacillus and strain CW 186T represents a novel species within the genus Sphingobacterium, for which the name Bacillus pallida sp. nov. and Sphingobacterium anhuiense sp. nov. were proposed.
CW 7T Cells are Gram-positive, motile by means of peritrichous flagella, spore-forming and rod-shaped (0.7-1.0×1.8-3.5 mm). Colonies are circular, flat and white or light pink after 2 days cultivation at 37℃on TYB medium. Growth occurs at 15-42℃(optimum 30-37℃) and pH 6.0-8.5 (optimum pH 7.0-8.0). Grows in the absence of NaCl but does not grow in the presence of 3% NaCl. Positive for hydrolysis of casein and gelatin, but negative for hydrolysis of Tween 80, chitin, starch, DNA, pectin and tyrosine. Weak catalase activity is present. Oxidase,β-galactosidase, arginine dihydrolase, lipase, methyl a-D-glucosidase, ornithine decarboxylase, lysine decarboxylase and urease activities are absent. Nitrate and nitrite are not reduced. H2S (triple-sugar iron test) is not produced. Voges-Proskauer test is positive. Indole is not produced. Utilizes aesculin, gluconate, 2-ketogluconate and D-xylose, but not N-acetylglucosamine, D-adonitol, D-arabinose, L-arabinose, D-arabitol, L-arabitol, arbutin, cellobiose, dulcitol, erythritol, D-fructose, D-fucose, L-fucose, D-galactose, D-gentiobiose, D-glucose, glycerol, glycogen, inositol, inulin,5-ketogluconate, D-lactose, amygdalin, D-lyxose, maltose, mannitol, D-mannose, melezitose, melibiose, methyl a-Dglucoside, methylα-D-mannoside, methyl P-D-xyloside, raffinose, L-rhamnose, D-ribose, D-salicin, sorbitol, Lsorbitose, starch, sucrose, D-tagatose, trehalose, turanose, xylitol or L-xylose (API CHB tests). Predominant isoprenoid quinone is MK-7. Major cellular fatty acids are iso-C_(15:0), anteiso-C_(15:0)and anteiso-C_(17:0). Cell-wall peptidoglycan contains meso-diaminopimelic acid. The DNA G+C content of the type strain is 42.3 mol%(HPLC). Phylogenetic analysis indicated that strain CW 7T belonged to a monophyletic cluster within the genus Bacillus and showed 16S rRNA gene sequence similarities of less than 96.5% to recognized species of the genus Bacillus. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain CW 7T represents a novel species of the genus Bacillus, for which the name Bacillus pallidus sp. nov. is proposed. The type strain is CW 7T.
CW 186T Cells are Gram-negative, non-motile, nonspore-forming and rod-shaped(0.4-0.8μm in width and 1.8-2.5μm in length). Colonies are circular, convex and bright yellow-coloured after 24 hours cultivation at 30℃on TYB agar. Growth occurs at 4-35℃(optimum 25-30℃) and pH 6.0-8.0 (optimum pH 6.5-7.5); growth occurs with 0-3 NaCl, but does not occur with 4% NaCl in modified TYB broth. Starch and DNA are hydrolyzed. Tween 80, casein and urea are not hydrolyzed. Catalase, oxidase, a-methyl-D-glucosidase and beta-galactosidase activities are present; arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, tryptophan decarboxylase and urease activities are absent; H2S is not produced; Voges-Proskauer tests are positive; citrate is not utilized; indole is not produced. N-acetyl-glucosamine, aesculin, D-arabinose, L-arabinose, arbutin, D-cellobiose, D-fructose, D-fucose, L-fucose, D-galactose, D-glucose, inulin, D-lactose, laetrile, D-maltose, D-mannose, melibiose,α-methyl-D-glucoside,α-methyl-D-mannoside, D-raffinose, salicoside, starch and D-sucrose are utilized; D-adonite, D-arabitol, L-arabitol, dulcitol, erythritol, D-fucose, D-gentiobiose, glycerol, glycogen, gluconate, inositol,2-keto-gluconate,5-keto-gluconate, D-lyxose, mannitol, D-melezitose, P-methyl-D-xyloside, L-rhamnose, D-ribose, sorbitol, L-sorbitose, D-tagatose, D-turanose, xylitol, D-xylose and L-xylose are not utilized. Predominant isoprenoid quinone is MK-7. Major cellular fatty acids are i-C15:0 (32.1%), i-C17:0 3-OH (9.8%) and Summed Feature 3 (i-C15:0 2-OH and/or C16:1ω7c.33.7%). DNA G+C content is 36.3 mol%(HPLC). Although chemotaxonomy (major fatty acids and quinone) and phylogenetic analysis results unequivocally supported the new isolate to be a member of genus Sphingobacterium; strain CW 186T can be easily differentiated from other related Sphingobacterium species by means of some phenotypic properties such as growth temperature range, starch hydrolysis, acid production from carbohydrates and also by differences in minor fatty acid components. On the basis of the phylogenetic and chemotaxonomic evidence together with the phenotypic characteristics and low 16S rDNA sequence similarities, the newly isolated strain CW 186T was assigned to be a novel species in the genus Sphingobacterium for which the name Sphingobacterium anhuiense sp. nov. was proposed. The type strain is CW 186T.
CW 7T革兰氏染色反应呈阳性,菌体呈杆状,周身鞭毛；培养2-3天后,菌落颜色为浅白或淡粉红色,圆形且平坦,边缘光滑,并且相互粘连,单菌落较难分开。菌体具运动性,细胞长有周身菌毛,产生孢子,好氧,化能异养型；能较好的生长于TYB复合培养基,在LB培养基中生长缓慢,很难或不能生长于营养琼脂和麦康凯琼脂等单一培养基。
菌株生长的温度范围是15-42℃,最适生长温度是30-37℃。CW 7T能较好的利用七叶树素、葡萄糖酸盐、2-酮基-葡萄糖酸盐和D-木糖为唯一碳源生长；并能够利用葡萄糖酸盐、2-酮基-葡萄糖酸盐和D-木糖发酵产酸。蛋白酶反应阳性,过氧化氢酶弱阳性；其他如氧化酶、p-半乳糖苷酶、精氨酸双水解酶、脂肪酶、α-甲基-D-葡萄糖苷酶、鸟氨酸脱羧酶、赖氨酸脱羧酶和脲酶均为阴性。不能还原硝酸盐和亚硝酸盐；不能水解酪氨酸、吐温80和淀粉；不产H2S和吲哚,VP反应为阳性。能生长于0-2% NaCl盐浓度范围内,无Na+等盐离子依赖性。
CW 7T的主要脂肪酸是ai-C_(15:0), i-C_(15:0)和ai-C17：0。呼吸链异戊二烯醌的种类为七碳烷基侧链的甲基奈醌(MK-7)；细胞壁主要肽聚糖类型为2,6-二氨基庚二酸(meso-diaminopimelic acid, meso-DAP); G+C摩尔百分含量为42.3%。16S rDNA序列同源性分析表明,CW 7T与芽孢杆菌属内其他成员的同源性小于96.5%,并在系统进化树中形成一个独立的分支。
通过以上形态学、生理生化、脂肪酸、细胞壁组分和16S rRNA基因序列同源性等多相分析结果,最终确定CW 7T的分类地位为：细菌域(Bacteria),厚壁菌门(Firmicutes),芽孢杆菌纲(Bacilli),芽孢杆菌目(Bacillales),芽孢杆菌科(Bacillaceae),芽孢杆菌属(Bacillus)的一个新种,并将其命名为苍白芽孢杆菌(Bacillus pallida)。
CW 186T革兰氏染色反应呈阴性,菌体呈椭圆形,具周生纤毛,不具有端生或周生鞭毛,但在适宜的环境下能够滑行,运动能力较差,生长快速,30℃培养2-3天后观察菌落颜色为橙黄色,圆形并向上微微凸起,边缘光滑,单菌落较容易分开。无内生孢子,好氧,化能异养型。不仅能较好的生长于TYB、LB等复合培养基,也能生长于麦康凯琼脂。
该菌株生长的温度范围为4-35℃,最适生长温度为25-30℃。CW 186T能较好的利用N-乙酰葡萄糖胺、七叶树素、D-阿拉伯糖、L-阿拉伯糖、熊果苷、纤维二糖、蔗糖、D-果糖、L-果糖、D-半乳糖、葡萄糖、菊糖、D-乳糖、苦杏仁苷、麦芽糖、D-甘露糖、蜜二糖、α-甲基-D-葡萄糖苷和α-甲基-D-甘露糖苷。过氧化氢酶、氧化酶、α-甲基-D-葡萄糖苷酶和p-半乳糖苷酶呈阳性；其他如精氨酸双水解酶、鸟氨酸脱羧酶、赖氨酸脱羧酶、色氨酸脱羧酶和脲酶均为阴性。不能还原硝酸盐和亚硝酸盐；能水解淀粉和DNA,但不能水解酪氨酸、吐温80和尿素；不产H2S和吲哚,VP反应为阳性。能生长于0-3% NaCl盐浓度范围内,无Na+等盐离子依赖性。
CW 186T主要脂肪酸为iso-C_(15:0)、iso-C_(17:0) 3-OH和Summed feature 3(iso-C_(15.0) 2-OH and/or C_(16:1ω7c))(含量分别为32.2%,9.8%,33.7%),呼吸链异戊二烯醌种类为七碳烷基侧链的甲基奈醌(MK-7)。G+C摩尔百分含量为36.3%。16S rDNA序列同源性分析表明,CW 186T与其所属属内相邻已发表菌种16S rDNA序列同源性差别都小于94%,在系统进化树中形成一个独立的分支。通过以上多相分类学分析结果,最终确定菌株CW 186T的分类地位为鞘氨醇杆菌属中的一个新种,我们将菌株CW 186T命名为安徽鞘氨醇杆菌(Sphingobacterium anhuiense)。
Two novel bacteria strains designated CW 7T and CW 186T were isolated from forest soil, Anhui Province, China. Strains CW 7T and CW 186T showed obviously distincted morphological, chemotaxonomic and phylogenetic characteristics from their closest neighbors. The polyphasic taxonomic study including phenotypic and chemotaxonomic and phylogenetic analysis displayed that strain CW 7T represents a novel species within the genus Bacillus and strain CW 186T represents a novel species within the genus Sphingobacterium, for which the name Bacillus pallida sp. nov. and Sphingobacterium anhuiense sp. nov. were proposed.
CW 7T Cells are Gram-positive, motile by means of peritrichous flagella, spore-forming and rod-shaped (0.7-1.0×1.8-3.5 mm). Colonies are circular, flat and white or light pink after 2 days cultivation at 37℃on TYB medium. Growth occurs at 15-42℃(optimum 30-37℃) and pH 6.0-8.5 (optimum pH 7.0-8.0). Grows in the absence of NaCl but does not grow in the presence of 3% NaCl. Positive for hydrolysis of casein and gelatin, but negative for hydrolysis of Tween 80, chitin, starch, DNA, pectin and tyrosine. Weak catalase activity is present. Oxidase,β-galactosidase, arginine dihydrolase, lipase, methyl a-D-glucosidase, ornithine decarboxylase, lysine decarboxylase and urease activities are absent. Nitrate and nitrite are not reduced. H2S (triple-sugar iron test) is not produced. Voges-Proskauer test is positive. Indole is not produced. Utilizes aesculin, gluconate, 2-ketogluconate and D-xylose, but not N-acetylglucosamine, D-adonitol, D-arabinose, L-arabinose, D-arabitol, L-arabitol, arbutin, cellobiose, dulcitol, erythritol, D-fructose, D-fucose, L-fucose, D-galactose, D-gentiobiose, D-glucose, glycerol, glycogen, inositol, inulin,5-ketogluconate, D-lactose, amygdalin, D-lyxose, maltose, mannitol, D-mannose, melezitose, melibiose, methyl a-Dglucoside, methylα-D-mannoside, methyl P-D-xyloside, raffinose, L-rhamnose, D-ribose, D-salicin, sorbitol, Lsorbitose, starch, sucrose, D-tagatose, trehalose, turanose, xylitol or L-xylose (API CHB tests). Predominant isoprenoid quinone is MK-7. Major cellular fatty acids are iso-C_(15:0), anteiso-C_(15:0)and anteiso-C_(17:0). Cell-wall peptidoglycan contains meso-diaminopimelic acid. The DNA G+C content of the type strain is 42.3 mol%(HPLC). Phylogenetic analysis indicated that strain CW 7T belonged to a monophyletic cluster within the genus Bacillus and showed 16S rRNA gene sequence similarities of less than 96.5% to recognized species of the genus Bacillus. The results of the polyphasic taxonomic study, including phenotypic, chemotaxonomic and phylogenetic analyses, showed that strain CW 7T represents a novel species of the genus Bacillus, for which the name Bacillus pallidus sp. nov. is proposed. The type strain is CW 7T.
CW 186T Cells are Gram-negative, non-motile, nonspore-forming and rod-shaped(0.4-0.8μm in width and 1.8-2.5μm in length). Colonies are circular, convex and bright yellow-coloured after 24 hours cultivation at 30℃on TYB agar. Growth occurs at 4-35℃(optimum 25-30℃) and pH 6.0-8.0 (optimum pH 6.5-7.5); growth occurs with 0-3 NaCl, but does not occur with 4% NaCl in modified TYB broth. Starch and DNA are hydrolyzed. Tween 80, casein and urea are not hydrolyzed. Catalase, oxidase, a-methyl-D-glucosidase and beta-galactosidase activities are present; arginine dihydrolase, ornithine decarboxylase, lysine decarboxylase, tryptophan decarboxylase and urease activities are absent; H2S is not produced; Voges-Proskauer tests are positive; citrate is not utilized; indole is not produced. N-acetyl-glucosamine, aesculin, D-arabinose, L-arabinose, arbutin, D-cellobiose, D-fructose, D-fucose, L-fucose, D-galactose, D-glucose, inulin, D-lactose, laetrile, D-maltose, D-mannose, melibiose,α-methyl-D-glucoside,α-methyl-D-mannoside, D-raffinose, salicoside, starch and D-sucrose are utilized; D-adonite, D-arabitol, L-arabitol, dulcitol, erythritol, D-fucose, D-gentiobiose, glycerol, glycogen, gluconate, inositol,2-keto-gluconate,5-keto-gluconate, D-lyxose, mannitol, D-melezitose, P-methyl-D-xyloside, L-rhamnose, D-ribose, sorbitol, L-sorbitose, D-tagatose, D-turanose, xylitol, D-xylose and L-xylose are not utilized. Predominant isoprenoid quinone is MK-7. Major cellular fatty acids are i-C15:0 (32.1%), i-C17:0 3-OH (9.8%) and Summed Feature 3 (i-C15:0 2-OH and/or C16:1ω7c.33.7%). DNA G+C content is 36.3 mol%(HPLC). Although chemotaxonomy (major fatty acids and quinone) and phylogenetic analysis results unequivocally supported the new isolate to be a member of genus Sphingobacterium; strain CW 186T can be easily differentiated from other related Sphingobacterium species by means of some phenotypic properties such as growth temperature range, starch hydrolysis, acid production from carbohydrates and also by differences in minor fatty acid components. On the basis of the phylogenetic and chemotaxonomic evidence together with the phenotypic characteristics and low 16S rDNA sequence similarities, the newly isolated strain CW 186T was assigned to be a novel species in the genus Sphingobacterium for which the name Sphingobacterium anhuiense sp. nov. was proposed. The type strain is CW 186T.
引文
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