This project sought to understand how a microbe synthesizes and degrades an extracellular, inorganic, insoluble material: elemental sulfur or S(0). We investigated Chlorobaculum tepidum (Cba. tepidum), which makes and consumes S(0) as part of its energy metabolism using sulfur-derived electrons and light to fix carbon dioxide (CO₂) into biomass for growth. S(0) is an important intermediate in sulfur cycling in both natural and engineered settings (e.g. hot springs, stratified lakes and estuaries, wastewater, petroleum), and so S(0)-related reactions could dictate biogeochemical reaction rates and ecological impacts of industrial processes.  Ultimately, understanding S(0) metabolism may enable the use of Cba. tepidum in biotechnology to harness sulfur compounds to make biofuels or other products from CO₂. Many microbes make and use S(0), and although we had some idea of the genes and proteins involved in S oxidation inside the cell, we had very little understanding of how cells interacted with the solid mineral outside the cell. By studying the model Cba. tepidum, we found that the cell’s ability to make and use S(0) is likely connected to the distinctive composition of its S(0), as well as physiological, genetic, and biochemical adaptations.

Cba. tepidum cells produce and consume micron-sized globules of S(0) approximately the same size as the cells, or larger, and do so largely remotely. While a subset of cells attach to S(0), most of the cell-mineral interactions appear to occur via soluble compounds, likely polysulfides, which can serve as both precursors and degradation intermediates of S(0). The attached cells are key, though, because they seem to initiate a cascade of degradation reactions. In the course of these experiments, we also found that Cba. tepidum can move, which had never been reported. Cells moved non-randomly, which may explain how they are able to find and attach to S(0).  We identified motility genes and will test whether or not their function is critical for S(0) metabolism.

Cba. tepidum is only able to consume its own S(0), but not commercially-available S(0), so we were interested in understanding the differences in S(0) composition and mineralogy. We found that the S(0) formed by Cba. tepidum differs from geologic and industrially-produced S(0), which consists of large crystals of sulfur in the α-S₈ phase. Instead, the microbially-produced S(0) contains nanometer-scale α-S₈ crystals that aggregate into globules. These tiny minerals with high surface area are more reactive, which makes them more bioavailable. In addition, the biogenic S(0) is covered in organics, including proteins. Because the surface is the first part of a mineral that cells and solutes see, it is likely the key to cell-mineral interactions. Our characterization of the coating’s composition and proteome gives insight into how the S(0) is made, and why its surface properties differ from commercial synthetic S(0).

The fact that S(0) synthesis and degradation occur in cultures at different times suggests that Cba. tepidum must regulate gene expression to switch between these two opposed.  Therefore, we sought to understand more about transcription and sulfur-dependent gene regulation in Cba. tepidum.  We defined basal promoter elements across the Cba. tepidum genome and identified motifs associated with genes whose expression was regulated during growth on different sulfur compounds.  We also were able to show that the gene CT1277 encodes a negative regulator of genes that are more highly expressed on sulfide.  Our data suggest that the presence of sulfide is the major signal that regulates genes for S(0) and thiosulfate regulation.  These basic findings on how gene expression is controlled and the tools developed to uncover them set the stage for engineering Cba. tepidum for sulfur-based autotrophic biotechnology.

The findings were disseminated in multiple peer-reviewed publications, presentations at local/national/international scientific meetings, and invited seminars.  Beyond communicating with peers, the project supported general science outreach to the public by team members participating in organized STEM enrichment programs both at the University of Delaware (school tours of the Delaware Biotechnology Institute, Coast Day) and on site (Serviam Girls Academy, Havre de Grace High School).

This project supported the career development of the Principal (PI) and Co-Principal Investigators (Co-PI).  The Co-PI was promoted from Assistant Professor to Associate Professor with tenure and the PI was promoted from Associate Professor to Professor during this project.  The project supported significant professional development of trainees as well.  A total of eight trainees, three postdoctoral researchers, three graduate students, and two undergraduates were associated with the project, seven of them female.  Of the five trainees that graduated (two B.S. and one Ph.D.) or took new positions over the course of the project, all are employed in STEM positions at MIDI Inc., Janssen Pharmaceuticals, Siemens Diagnostics, the Noble Research Institute, and Niagara University.

Last Modified: 06/19/2017
Modified by: Thomas E Hanson