Molecular manipulations of Cba. tepidum to investigate sulfur oxidation and energy regulation
Date
2019
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Publisher
University of Delaware
Abstract
Chlorobaculum tepidum is a model organism for anaerobic phototrophic sulfur oxidation and is unusual amongst other microbes that interact with insoluble minerals in that it can both produce and subsequently consume extracellular S(0); however, the enzymes that catalyze S(0) production and consumption are thought to reside in the cytoplasm, periplasm, or membranes of the cell. The mechanism and proteins involved in the formation and transport of biogenic S(0) across the outer membrane during production and consumption are unknown. Differential expression of Cba. tepidum genes during S(0) production and consumption relative to growth on thiosulfate revealed membrane protein candidates that may play a role in biogenic S(0) metabolism. Mutants lacking candidate genes were tested for defects during growth on sulfide, thiosulfate, and sulfur. To date, these mutant strains have displayed no significant growth defects. These results indicate that there may be downstream regulation of expressed genes involved in S(0) metabolism or multiple pathways for the formation and transport of S(0). In addition, the localization of proteins involved in S(0) metabolism is unknown. Interestingly, shotgun proteomics of S(0) globules revealed a subset of proteins expressed during S(0) production. Yellow Fluorescence-Activating and Absorption Shifting Tag (Y-FAST) is a fluorescent label used to visualize the subcellular or extracellular location of proteins fused to Y-FAST. An attempt to develop this technique in Cba. tepidum is presented here. Further development may lead to the successful localization of recombinant proteins from the S(0) globule protein subset and serve as a useful tool to localize other recombinant proteins in Cba. tepidum in future research. Additionally, the work presented here aided in the successful development Y-FAST in a different anaerobe, Clostridium acetobutylicum. Biogenic S(0) metabolism involves the processing and export of S(0) and proteins, which is energetically costly. Therefore making and consuming S(0) likely causes an energy stress. As part of a larger project on energy stress in prokaryotes, global responses to energy metabolism were examined to determine if DNA modification might play a role in energy stress responses.