Frias-Lopez Jorge, Shi Yanmei, Tyson Gene W, Coleman Maureen L, Schuster Stephan C, Chisholm Sallie W, Delong Edward F
Departments of Civil and Environmental Engineering and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3805-10. doi: 10.1073/pnas.0708897105. Epub 2008 Mar 3.
Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species-including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition-and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction ( approximately 50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.
宏基因组学正在拓展我们对自然微生物群落中基因含量、功能意义及遗传变异性的认识。然而，关于环境中基因调控和动态变化的信息仍然有限。我们在此报告对一个自然存在的微生物群落中表达基因的全面分析。我们首先采用RNA扩增技术，从少量的总微生物群落RNA中生成大量的cDNA。RNA扩增程序的保真度通过原绿球藻培养物进行了验证，然后应用于在贫营养的太平洋采集的微生物群落。通过焦磷酸测序分析微生物群落cDNA，并与从同一样本中测定的微生物群落基因组DNA序列进行比较。基于焦磷酸测序的微生物群落基因表达估计与使用定量PCR对单个基因表达的独立评估结果相当。与开阔海洋微生物物种关键代谢途径相关的基因——包括参与光合作用、碳固定和氮获取的基因——以及许多编码假定蛋白质的基因在cDNA文库中高度富集。原绿球藻基因组可变区域中的基因是表达量最高的基因之一，这表明这些基因编码特定基因型细胞过程中的核心蛋白质。尽管检测到的许多转录本与先前在海洋宏基因组学调查中检测到的基因高度相似，但仍有很大一部分（约50%）是独特的。因此，微生物群落转录组分析不仅揭示了本地基因和分类群特异性的表达模式，还揭示了在先前基于DNA的宏基因组学调查中未检测到的基因类别。
