Environmental determination and dynamic regulation of the carbon concentrating mechanism in Fremyella diplosiphon

"Cyanobacteria are single-celled photoautotrophic organisms that are major contributors to global carbon fixation. Since the accumulation of significant amounts of oxygen (O2) in the atmosphere, they have contended with decreased amounts of available inorganic carbon (Ci) with which to supply photosynthesis. This has led to the evolution of a carbon concentrating mechanism (CCM) that involves increasing the Ci uptake into the cell and utilizing a specialized bacterial microcompartment (BMC) called the carboxysome to trap carbon dioxide (CO2) around the carbon-fixing enzyme, rubisco. Since the CCM is an integral part of cyanobacterial photosynthesis, it is expected to be regulated by many environmental factors that affect photosynthesis. Here, I present findings from studies on Fremyella diplosiphon, a model cyanobacterium that exhibits complementary chromatic acclimation (CCA), wherein photosynthetic pigmentation and efficiency are tuned in response to environmental light cues. A photoreceptor, RcaE, that controls CCA was shown to be important for determining the abundance and stoichiometry of CCM components. The size and abundance of carboxysomes were found to correlate with the ratio of carboxysomal cargo:shell in a Delta-rcaE mutant strain, suggesting a role for RcaE in regulating carboxysome morphology. Additionally, F. diplosiphon is one of many cyanobacteria that express an activase-like cyanobacterial (ALC) protein, a homologue to rubisco activase that is an essential protein in plants for rubisco activity. The ALC protein is not coded for in the genomes of many model cyanobacteria but was predicted to be targeted to the carboxysome when present, thus representing an important factor in the nuanced regulation of the CCM. Through contributions to a study highlighting the carboxysomal localization and enzymatic activity of the ALC protein, I provide evidence that ALC is involved in the cellular response to Ci availability. Additionally, computational modeling of the interaction between ALC and rubisco contrasted two potential binding sites and suggested that the interaction could depend on species of origin and post-transcriptional modification. Given the myriad of factors impacting carboxysome regulation, I then analyzed the carbon fixation capabilities of F. diplosiphon strains under variations in light quality, light quantity, and Ci availability. Assessment of carbon fixation behavior utilizing a novel application of carbon response curves to cyanobacteria was consistent with expectations and provided additional insight into which components of the CCM respond to environmental cues. The Delta-rcaE mutant exhibited a noteworthy green-light-dependent limitation in carbon fixation, and further analyses suggested that this depended on rubisco levels and the expression of Ci-uptake genes. These findings, alongside the behavior of other cultivation conditions and F. diplosiphon strains, were used to distinguish at a preliminary level the components of carbon response curves in cyanobacteria as a method to assess carbon fixation behavior and the functional impacts of CCM regulation."--Pages ii-iii.