Genetic relationships and the effects of an antimicrobial agent on the hyperthermophile Pyrococcus furiosus
Hamilton-Brehm, Scott David
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The goals of this research were to develop whole genome microarray technology for the hyperthermophilic archaeon, Pyrococcus furiosus, and to use this approach to a) evaluate genetic relationships between P. furiosus and P. woesei, and b) determine the mechanism of action of the an antimicrobial agent, Roussin’s Black Salt (RBS) on P. furiosus. Microarrays have been demonstrated to be one of the most powerful molecular biology techniques and allow the rapid analysis of whole genomic transcript responses of organisms to metabolic stresses. The processing and biochemical techniques involved with microarray have undergone many revisions and improvements as innovative products are made available. Genomic comparisons between P. furiosus and P. woesei were made possible by advances in microarray design. It was shown that of the 2,192 annotated open reading frames (ORFs) in the P. furiosus genome, at least 104 of them were absent from the genome of P. woesei. These "missing" ORFs were arranged in distinct groups within the P. furiosus genome and flanked by insertion sequences which are believed to be involved with lateral gene transfer (LGT). The likely mechanism of LGT was derived from sequence analysis of the two genomes, and it is concluded that P. furiosus is a sister or parent strain of P. woesei. RBS is a broad spectrum bactericide that has proven very effective in controlling pathogenic anaerobes such as Clostridium both in the vegetative and spore state. RBS has the formula [Fe4S3(NO)7] and it has been generally assumed that the release of nitric oxide (NO) is the cause of the cytotoxic effects although the mechanism is unknown. The effects of NO release were to be assessed by DNA microarray analyses. However, it was demonstrated using growth studies, membrane analyses, and scanning electron microscopy that NO does not play a role; rather, the mechanism of toxicity involves membrane disruption. Moreover, insoluble elemental sulfur, which is reduced by P. furiosus to hydrogen sulfide, prevents membrane disruption by RBS. RBS therefore appears to be a novel type of inorganic surfactant, and its mechanism is independent of NO and involves membrane specific disruption.