A Functional Genomics Study of the Primary Pathways for Carbon Metabolism in a Hyperthermophilic Archaeon

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Well over 200 genomic sequencing efforts have been completed ranging from pathogens and exotic microbes, to yeast, rice and human, yielding a phenomenal array of sequence information, the comprehension of which is only just beginning. Understanding how a living cell works, however, obviously requires much more than simply determining the genome sequence and the number of genes. A fundamental problem is how to find genes and assign functions to these genes. Insights can be obtained by analyzing the sequences of the proteins that the genes encode using the available databases, but by such methods less than half of the genes in a given organism can be assigned a function with any degree of confidence. Hence, the roles of about half of the genes in any given genome is essentially unknown. This suggests that half of life's biochemistry is unknown. A major effort is therefore needed to assign biological roles to these unknown genes through functional genomic approaches.The goal of this research is to identify genes involved in carbon metabolism of Pyrococcus furiosus, an archaeon (archaebacterium) that grows optimally at 100C. The research will largely focus on finding genes involved in carbon metabolism in the 'unknown' half of the microbial genome. The P. furiosus 1.908 Mb genome contains approximately 2,200 putative genes. The fundamental hypothesis to be tested is that many of the more than 1,000 genes of unknown function play key roles in primary carbon metabolism by as yet unknown mechanisms. This will be ascertained using DNA microarray analyses using all 2,200 genes in the P. furiosus genome. Preliminary data have shown that a surprisingly large number of the unknown genes are regulated by the carbon source used to grow the organism. The specific aims are a) to identify previously uncharacterized genes involved in the metabolism of carbohydrates, proteins and C-1 compounds by analyzing expression levels using cells grown under a wide variety of conditions, b) to confirm gene expression data by enzymatic assays and metabolite analyses, and c) to characterize the key enzymes by direct purification using P. furiosus biomass from 600 liter fermentations and by using recombinant proteins that are available through a complementary structural genomics effort with P. furiosus. It is anticipated that the results of this study will provide completely new insights into the metabolism of fixed carbon compounds and the role of what are currently hypothetical genes in both P. furiosus and in other organisms, including human.