印尼3个地区热泉中可培养细菌的分子鉴定与系统发育分析

Molecular Identification and Phylogenetic Analysis of Cultivable Bacteria From Three Hot Springs in Indonesia

采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyu wedang地区的热泉水样、泥样以及沉积物中根据菌落的形态学特征共分离获得69株细菌,通过菌株的16S rDNA的限制性片段长度多态性(PCRRFLP)分析、序列比对和生理生化分析,对其进行鉴定并构建系统发育树。结果表明,分离获得的69株细菌菌株分别属于8种基因型(Ⅰ～Ⅷ),其中1类(基因型I)属于短芽孢杆菌属(Brevibacillus),约占20.28%;1类(基因型Ⅷ)属于地芽孢杆菌属(Geobacillus),约占14.49%;其余6类(基因型Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,Ⅶ)均属于芽孢杆菌属(Bacillus),约占总菌株数量的65.22%,为优势菌群。分离获得的8类细菌均为革兰氏阳性兼性好氧菌,接触酶、氧化酶反应均呈阳性,吲哚反应都呈阴性。产酶实验表明,分离到的菌株在中性pH值,37～70℃范围内能够分泌多种耐热的水解酶类,4种基因型(Ⅰ,Ⅱ,Ⅲ,Ⅳ)的菌株分泌蛋白酶,5种基因型(Ⅳ,Ⅴ,Ⅵ,Ⅶ,Ⅷ)的菌株分泌水解脂肪酶,1种基因型(Ⅳ)的菌株分泌淀粉酶,并且发现其中1种基因型(Ⅳ)的芽孢杆菌可同时产生3种水解酶类。利用限制性内切酶AluI对16S rDNA进行的RFLP技术分析结果表明,利用该技术能够对嗜热细菌的16SrDNA多样性进行有效的分析,经凝胶电泳图谱将菌种鉴定到属的水平,揭示了印尼热泉生态系统中蕴藏着丰富的嗜热菌菌种资源。

Based on the biological morphological features of the bacterial communities, totally 69 strains of thermophilic bacteria are isolated from water, soil or sediment samples of three hot springs respectively in Pantai cermin, Kalianda and Banyu wedang of Indonesia. By means of the bacterial 16S rDNA PCR-RFLP analysis, the 16S rDNA sequence comparison among the bacteria, and the physiological and biochemical analysis, the bacteria are identified and their phylogenetic tree is set up. It is shown in the study results that the 69 isolated strains belong to 3 genera including 8 genotypes. Genotype I is Brevibacillus, taking about 20.28% of the total, Genotype Ⅷ is Geobacillus about 14.49%, and the rest （from Ⅱ to Ⅶ） are the dominant genus Bacillus about 65.22%. All the 8 genotypes are gram-positive and belong to facultative aerobic bacteria. They are positive in the catalase or oxidase test, and negtive in the indole test. It is learnt from the enzyme screening experiments that they can secrete a variety of heat-resistant hydrolases under the neutral pH conditions in a temperature range between 37 ℃ and 70 ℃. The 4 genotypes including Ⅰ , Ⅱ , Ⅲ , and Ⅳcan produce proteases, the 5 genotypes （Ⅳ, Ⅴ , Ⅵ, Ⅶand Ⅷ） can produce lipases, and Gen- otype Ⅳ can simultaneously produce 3 kinds of hydrolases i. e. proteases, lipases, and amylases. By means of Endonucleas Alu I, the 16S rDNA diversity of the thermophilic bacteria can be effectively analysed with the RELP technology. The polyacrylamide gel electrophoregram can be used to identify the bacteria to the genus level. Therefore as it is revealed in this study, the thermophile bacterial resources are rich in the hot spring ecosystem of Indonesia.