【报告题目】：Genome scale analysis of gene function in the hydrogenotrophic methanogenic archaeon Methanococcus maripaludis
【报告人】： William B. Whitman教授
Department of Microbiology，University of Georgia, USA
Methanogenic archaea are obligate anaerobic prokaryotes and widely distributed in O2-free environments where electron acceptors other than CO2 have been depleted. Methanogenesis is a highly specialized anaerobic respiration with a distinctive biochemistry composed of unusual coenzymes and catalysts whose roles are poorly understood. In anaerobic environments, it plays a key role, catalyzing the terminal step of carbon mineralization and maintaining an extremely low partial pressure of H2. Methane, the final product of this process, is also a significant greenhouse gas with about 80% of the atmospheric methane produced by these archaea .
Our understanding of the methanogenic archaea is far from complete. For instance, the methanogen Methanococcus maripaludis S2 possesses 1,728 protein coding genes, only a few of which have been characterized and an even smaller portion has been studied in detail. Close to 800 genes remain annotated as hypothetical proteins awaiting proper identification (1). Much of this uncertainty is shared with other archaea, where many of the fundamental life processes have not been investigated in the same detail as in bacteria and eukaryotes. To address these issues, the essentiality of methanococcal genes was evaluated by a saturation mutagenesis technique. Whole-genome libraries of Tn5 transposon mutants were constructed, and about 89,000 individual mutations were mapped following enrichment of the transposon-chromosomal DNA junctions and Illumina sequencing (Tn-seq). Because mutations in genes that are likely to be essential or strongly advantageous for growth are lethal or rapidly lost from the library, they may be identified by their low frequency in the libraries. Although definitive assignments of essentiality still require detailed analyses of each gene, the methodology generates hypotheses about the nature of specific genes and a great deal of insight into specific questions regarding methanogens as well as more general questions about the genetics, biochemistry and physiology of archaea.
By this methodology, about 30% of the genome appears to be possibly essential or strongly advantageous for growth. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as essential were unique to archaeal or methanococcal lineages, such as the genes encoding the DNA polymerase PolD. In contrast, the gene encoding the DNA polymerase B was not essential for growth, a conclusion which was confirmed by constructing an independent deletion mutant. Thus, PolD and not PolB is likely to play a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possible essential and are likely to play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.