Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes
Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes
复制标题
DOI：
10.1128/aem.66.3.930-936.2000
复制
发表时间：
2000-03
影响因子：
4.4
通讯作者：
S. Peters;Stefanie Koschinsky;F. Schwieger;C. Tebbe
中科院分区：
文献类型：
--
作者：
S. Peters;Stefanie Koschinsky;F. Schwieger;C. Tebbe
摘要采用PCR扩增小亚基rRNA基因（SSU rDNA）的非培养依赖性遗传分析技术，研究了有机农业基质堆肥过程中微生物群落的多样性和演替。用直接从堆肥样品中提取的DNA和靶向（i）真细菌16 S rRNA基因的V4-V5区、（ii）放线菌16 S rRNA基因的V3区或（iii）真菌18 S rRNA基因的V8-V9区的引物进行PCR扩增。同源PCR产物通过核酸外切酶消化转化为单链DNA分子，随后通过其单链构象多态性（SSCP）进行电泳分离。通过该技术获得的遗传图谱显示了所有引物的微生物种群的继承和增加的多样性。通过PCR再扩增和克隆，共分离到19个单一产物。这些分子分离物的DNA测序显示与已知的具有低或高G+C DNA含量的革兰氏阳性细菌以及γ-变形菌门的SSU rDNA的相似性在92.3%至100%的范围内。扩增的18 S rRNA基因序列与克柔念珠菌和热带念珠菌各自的基因区域相关。特定的分子分离物可以归因于不同的堆肥阶段。从堆肥过程结束时采集的样品中分离出的培养细菌的多样性很低。共290个分离物仅与6个不同的物种有关。在SSCP群落图谱中也可以检测到其中的两到三个物种。我们的研究表明，社区SSCP配置文件可以是非常有用的监测细菌多样性和社区演替的生物技术相关的过程。
ABSTRACT A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.