Biochemical, genetic and genomic approaches to studying sulfur metabolism by the hyperthermophilic archaeon Pyrococcus furiosus
Bridger, Stephanie Louise
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The availability of a highly efficient genetic system in the model hyperthermophilic archaeon, Pyrococcus furiosus, has greatly improved our ability to study its physiology and metabolism. This work describes the genome sequencing of the genetically-tractable strain of P. furiosus, COM1, and the characterization of deletion strains of key sulfur responsive proteins, namely NSR1, MBX1, and SIP1. The COM1 genome revealed a surprisingly large number of changes compared to that of the P. furiosus NCBI reference sequence, including chromosomal rearrangements, deletions, and single base changes in both coding and potential regulatory regions. However, in spite of all the genomic changes only a few phenotypic differences could be observed in COM1 compared to its parental strain. COM1 was used to generate deletions of the genes encoding for the cytoplasmic NADPH-dependent coenzyme-A sulfur oxidoreductase (NSR), the membrane-bound oxidoreductase (MBX) and the sulfur-induced protein A (SipA). Characterization of NSR1 revealed a non-essential role for NSR in S0 reduction, unlike MBX, which was shown to play a critical role in S0 reduction and energy conservation. Transcriptional analyses revealed a probable role for NSR in maintaining the thiol-redox balance of the cell and modulating the activity of the redox-dependent S0-response regulator, SurR.