Graduation Year
2021
Document Type
Thesis
Degree
M.S.
Degree Name
Master of Science (M.S.)
Degree Granting Department
Biology (Integrative Biology)
Major Professor
Kathleen Scott, Ph.D.
Committee Member
Mya Breitbart , Ph.D.
Committee Member
Valerie Harwood, Ph.D.
Keywords
autotroph, carbon dioxide concentrating mechanism, carbon fixation
Abstract
Autotrophic organisms are responsible for introducing carbon into food webs. These organisms take up dissolved inorganic carbon (DIC) and use it as their major carbon source, converting it to biomass using a variety of carbon fixing pathways (e.g., the Calvin-Benson-Bassham cycle). In nature, DIC concentrations can be low or temporally heterogeneous, so autotrophic organisms adapt with CO2 concentrating mechanisms (CCMs) to facilitate the acquisition and fixation of this dissolved gas. CCMs consist of membrane-spanning DIC transporters, which elevate intracellular DIC concentrations, which in turn are harvested by carboxysomes, proteinaceous microcompartments that contain the carboxylase Ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) and carbonic anhydrase. CCMs have been well-studied in Cyanobacteria, but much remains to be learned about their presence and activity in other bacterial phyla. Indeed, a novel multisubunit DIC transporter (MDT) was recently discovered, and its activity has been preliminarily described in three organisms. It is broadly distributed throughout a diversity of phyla and consists of either one, two or three subunits. Given the breadth of MDT distribution among organisms with diverse physiologies, and its extensive differences in structure, it was important to determine whether DIC uptake was universal among these transporters. To study these transporters, a carbonic anhydrase-deficient strain of E. coli Lemo21(DE3)yadF-cynT- that is unable to grow under low-CO2 conditions was created, and can be rescued via heterologous expression of DIC transporters. We chose seven transporters from the MDT family from four phyla (Actinobacteria, Aquificae, Proteobacteria and Campylobacterota), representative of organisms from a variety of habitats (acidic to basic, cold to hot), with all three subunit configurations found among MDT. Expression of all seven transporters rescued the ability of E. coli Lemo21(DE3)yadF-cynT- to grow under low CO2 conditions. Six of the seven constructs showed increased intracellular DIC. In MDT with multiple subunits, all subunits were necessary to transport DIC. CO2 appeared to be the source of DIC used by the MDT. In addition, collapsing the proton gradient using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) prevented the accumulation of intracellular DIC, suggesting this family of transporters is using cellular proton potential to drive CO2 transport. The MDT is the first CO2 transporter described for bacteria and is confirmed to function as a DIC transporter throughout its broad taxonomic distribution.
Scholar Commons Citation
Schmid, Sarah E., "Novel CO2 transporters from autotrophic bacteria from extreme environments" (2021). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/9607
