Graduation Year
2025
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Integrative Biology
Major Professor
Kathleen K. Scott, Ph.D.
Committee Member
Valerie J. Harwood, Ph.D.
Committee Member
John J. Parkinson, Ph.D.
Committee Member
George G. Philippidis, Ph.D.
Keywords
bicarbonate transporters, carbon concentrating mechanisms, carbon fixation, carbonic anhydrase, carboxysomes, SulP transporters
Abstract
Dissolved inorganic carbon (DIC: CO2, HCO3⁻, CO32⁻) plays a central role in the metabolism of all living organisms. DIC is necessary for steps in essential biosynthetic processes, such as the synthesis of amino acids and nucleobases; in autotrophic bacteria, organic molecules are synthesized entirely from DIC. To ensure a steady supply of CO2 and HCO3 - needed for biosynthesis and/or autotrophy, organisms use bicarbonate transporters to increase the cytoplasmic concentration of this anion, and carbonic anhydrase (CA) to speed the interconversion of CO2 and HCO3 -, both in the cytoplasm and at the cell surface. This dissertation investigates DIC metabolism by autotrophic and heterotrophic members of Bacteria. Autotrophic organisms using the Calvin-Benson-Bassham cycle must minimize the wasteful oxygenase reaction of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). They have evolved carbon dioxide concentrating mechanisms (CCMs). These systems include DIC transporters to elevate intracellular bicarbonate concentrations and carboxysomes, which are proteinaceous microcompartments which encapsulate RubisCO and CA to increase local CO2 concentrations and enhance the carboxylation efficiency of RubisCO. Chapters 2 and 3 of this dissertation focus on genus Thiomicrospira, especially T. pelophila, an autotrophic member of class Gammaproteobacteria. In Chapter 2, we demonstrated that ιCA, a newly discovered form of CA, is active within carboxysomes from members of genus Thiomicrospira and furthermore, we demonstrated for T. pelophila that ιCA is essential for growth under DIC limitation, both in native cells and when heterologously expressed in E. coli. This is the first time that ιCA has been described within a CCM context. In Chapter 3, we showed that T. pelophila encodes an unusually large number of putative DIC transporters. Four of these transporters were able to mediate bicarbonate uptake when expressed in E. coli. Interestingly, only one transporter was dramatically transcriptionally upregulated under DIC limitation, while transcripts from carboxysome genes did not differ as dramatically. This suggests that CCMs in T. pelophila might function differently than CCMs of other autotrophs, in which genes encoding multiple DIC transporters and carboxysome components are all strongly upregulated under low DIC conditions. Chapter 4 broadens the scope of DIC metabolism to all microorganisms, with phylogenetic and functional analysis of the sulfate permease (SulP) family of transporters to determine how widespread bicarbonate transport is among this family. Phylogenic analysis suggests that SulP transporters can be divided into 5 different clades (A-E). In our first round of screening a library of 210 sulP genes, we found that many members of the C-clade are capable of bicarbonate uptake, while bicarbonate transport in other clades is less common. In some cases, results from this first screen of the library were not reliably replicable. These findings advance our understanding of microbial DIC uptake and fixation, and have implications for microbial ecology, global carbon cycling, and biotechnological applications.
Scholar Commons Citation
Wieschollek, Jana, "Dissolved Inorganic Carbon Metabolism in Bacteria" (2025). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/11104
