Stackebrandt E, Liesack W, Goebel B M
Department of Microbiology, University of Queensland, St. Lucia, Australia.
FASEB J. 1993 Jan;7(1):232-6. doi: 10.1096/fasebj.7.1.8422969.
In order to investigate the genetic diversity of streptomycetes in an acid forested soil sample from Mt. Coot-tha, Brisbane, Australia, cells were mechanically lysed within the soil matrix and genomic DNA was isolated and purified. 16S ribosomal (r)DNA was amplified by the polymerase chain reaction (PCR) method using one primer conserved for members of the domain Bacteria and a second designed specifically for streptomycetes and related taxa. PCR amplification products were cloned into phage vector M13 mp19 and the diversity of 16S rDNA genes was determined by sequence analysis and oligonucleotide probing of the resultant clone library. Comparison of partial 16S rDNA sequences with published sequences revealed that few sequences originated from streptomycetes. The majority of sequences belonged to members of the alpha subclass of Proteobacteria. Other clones were related to planctomycetes, actinomycetes, or represented novel lines of descent. Bacteria that are customarily isolated from soil of pH 4-7 such as thiobacilli, bacilli, spore- and nonsporeforming actinomycetes, and pseudomonads are represented in the clone library in small numbers or were not detected at all. Parameters influencing the recovery, amplification, quantification, and interpretation of genetic information from natural sites are discussed.
为了研究澳大利亚布里斯班库塔山酸性森林土壤样本中链霉菌的遗传多样性，在土壤基质中对细胞进行机械裂解，然后分离并纯化基因组DNA。使用一种对细菌域成员保守的引物和另一种专门为链霉菌及相关分类群设计的引物，通过聚合酶链反应（PCR）方法扩增16S核糖体（r）DNA。将PCR扩增产物克隆到噬菌体载体M13 mp19中，并通过对所得克隆文库进行序列分析和寡核苷酸探测来确定16S rDNA基因的多样性。将部分16S rDNA序列与已发表序列进行比较，结果显示源自链霉菌的序列很少。大多数序列属于变形菌门α亚类的成员。其他克隆与浮霉菌门、放线菌有关，或代表新的谱系。通常从pH值为4 - 7的土壤中分离出的细菌，如硫杆菌、芽孢杆菌、产孢和不产孢放线菌以及假单胞菌，在克隆文库中数量较少或根本未被检测到。文中还讨论了影响从自然环境中获取、扩增、定量和解读遗传信息的参数。
