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Dr. Patricia C. Dos Santos

Dos Santos Research Group
Research Group 2011

 

Research Information

Iron and sulfur atoms can form chemical structures of biological importance named [Fe-S] clusters. The importance of Fe-S clusters is intimately associated with the essential role of Fe-S proteins in a wide range of life sustaining processes including respiration, carbon metabolism, photosynthesis and nitrogen fixation. These chemical structures are organized in fundamental units of [2Fe-2S] and [4Fe-4S] clusters, or multimers, or fragments of these units. Although [Fe-S] clusters can be formed in vitro from their toxic individual components, almost all forms of life have developed specialized machineries dedicated to the assembly of these metal-centers. Our research group is interested in understanding the biochemical formation of [Fe-S] clusters in Gram-positive bacteria.

Bioinformatic analysis shows that Gram-positive organisms, including those members of pathogenic relevance, likely use a novel strategy to assemble [Fe-S] clusters that is different from the one used by Gram-negative bacteria and humans. Therefore, the study of this biochemical process is relevant for at least three reasons: (i) it provides a novel system for the biosynthesis, structure and function of metallocenters and their associated proteins, (ii) it establishes a relationship between evolution and differentiation/specialization of [Fe-S] cluster formation when compared to other studied systems, and (iii) it directs future work toward the development of new drug targets against Gram-positive pathogens.

The main objective of this research is to identify unique features associated with the assembly and regulation of [Fe-S] cluster formation in Gram-positive bacteria using Bacillus subtilis as the model organism. Our initial efforts are geared towards understanding the chemical mechanism of [Fe-S] cluster formation in B. subtilis, through application of bioinformatics, molecular genetics, biochemical, and biophysical approaches.

The work in our laboratory involves, initially, the identification of the genes in this biochemical pathway using genomic analyses. It is followed by the use of molecular biology and biochemical techniques to accomplish cloning, expression, purification, and characterization of all the components in this pathway. Towards this aim, we are interested in identifying the mechanisms of [Fe-S] cluster formation using in vitro experiments to monitor the kinetics of [Fe-S] cluster assembly and [Fe-S] cluster transfer from the biosynthetic apparatus to various acceptor proteins.

Complementary to the in vitro protein-assisted [Fe-S] cluster formation, this project also aims to identify the in vivo mechanistic differences among various [Fe-S] cluster biosynthetic machineries. Our genetic approach involves the construction of B. subtilis strains that have controlled expression of the genes required for assembly of [Fe-S] clusters and the analysis of Fe-S proteins in cells depleted for the [Fe-S] cluster biosynthetic machinery under different physiological conditions. A complete understanding of the key differences of [Fe-S] cluster formation at a molecular level will contribute directly to future studies undertaken to develop strategies that inhibit the key unique steps in this pathway among B. subtilis and other infectious microbes.

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