Abstract
dc:description.abstractLiving systems extract energy from the environment by coupling charge-transfer reactions between available electron donors and acceptors with the generation of electrochemical gradients across cellular membranes. The variety of strategies that organisms have evolved to harness the many environmental sources of energy is astounding and still not fully understood. Cyanobacteria, uniquely in the prokaryotic kingdom, can harvest solar energy to couple water oxidation with the reduction of carbon dioxide. This results in photosynthetic production of sugars, which provide energy to many ecosystems on Earth, and the release of molecular oxygen in the atmosphere, which is thought to have driven the Great Oxidation Event (GOE) more than 2 billion years ago, and the subsequent emergence of aerobic life forms. Redox-active molecules form the basis for these bioenergetic processes inside living cells, which, entwined with their environment, must constantly modulate intracellular electron fluxes in response to metabolic needs and external conditions through interactions with the extracellular space. Supporting this, an emerging amount of evidence demonstrates that cyanobacteria, like most other microorganisms, can detect, adapt to, and even generate extracellular electrochemical stimuli. Nevertheless, the genetic pathways (hereafter referred to as “electrogenetic”) responsible for cyanobacteria’s redox interactions with their environment are mostly unknown. This limits our understanding of the evolutionary history, biochemical mechanisms and physiological functions of electrochemical signal transduction in oxygen-evolving photosynthetic bacteria, hindering the development of optimised, solar-powered biotechnologies. Aiming to address this, this dissertation reports results from bioinformatic, microbiological and bioelectrochemical studies on some previously-implicated and newly-discovered electrogenetic factors in cyanobacteria. An introductory chapter reviews available literature on the development and state-of-the art of bio- electrochemical research in cyanobacteria, alongside biochemical considerations used to define electrogenetic pathways. To highlight the significance of this research for the development of a sustainable, biological-based economy, the discussion also provides examples of biotechnological applications leveraging cyanobacterial’s bioelectrical properties. The second chapter describes the materials and methods employed throughout this study. The third presents the results of a bioinformatic analysis aimed at delineating the phylogenomic landscape of cyanobacteria with a focus on electrogenetic pathways. A reference cyanobacterial phylogeny, enriched with phenotypic and ecological species traits, was established. The distributions of putative electrogenetic factors from cyanobacteria or better-characterised electroactive bacteria were mapped onto it. This revealed universally conserved redox-sensitive transcription factors (e.g. OxyR, PerR) and membrane translocation systems (e.g. ABC) alongside more sporadically conserved regulators (e.g. PrqR, SoxR) and transporters (e.g. MATE), suggesting a complex evolutionary history of ancient mechanisms and environment-specific adaptations. Moreover, the widespread conservation of genetic components for reducing (e.g. type II NADH dehydrogenase) and exporting electron shuttles (e.g. flavins) suggests that cyanobacteria use indirect mechanisms for extracellular electron transfer. Finally, the unexpected conservation of genes encoding putative reactive oxygen species (ROS)-generating NADPH oxidases (NOXs), including eukaryotic-like isoforms, suggests polyphyletic evolution of cyanobacterial *nox* genes and a possible eukaryote-to-prokaryote horizontal gene transfer event. These results provide valuable information for the identification of electrogenetic components in cyanobacteria and lay the groundwork for the experimental investigations presented in the following chapters. The fourth chapter reports experimental investigations into the mechanisms and functions of the PrqRA system found in the model cyanobacterium *Synechocystis* sp. PCC 6803, which was previously implicated as a potential redox-sensitive component. Transcript analysis confirmed the hypothesis of bicistronic transcription of the *prqR* and *prqA* genes. Use of plasmid-encoded reporters for gene expression revealed that a hairpin structure in the intergenic region was responsible for transcriptional attenuation of the second cistron (*prqA*). Additionally, it was observed that the gyrase inhibitor novobiocin, but not the redox-active molecule methyl viologen, could inhibit PrqR autorepressor activity, alongside inducing intracellular oxidative stress. *Synechocystis* strains transformed with P*prq*R reporter constructs were applied to the surface of electrodes, and the effects of applying potentials were investigated. The results indicated that PrqR can function as a biomolecular voltage detector and demonstrated the feasibility of using electrical signals for control of cyanobacterial gene expression. Finally, functional characterisation of the efflux transporter PrqA showed a role in conferring tolerance to novobiocin, rhodamine and in the efflux of phenazines, which are attractive redox inducers for electrogenetic systems. A medium-resolution cryo-EM structure of PrqA was generated. The fifth chapter reports adaptive laboratory evolution experiments on *Synechocystis*, aimed at evolving strains resistant to the redox-mediator and photosynthesis inhibitor methyl viologen (MV). Starting from two distinct parent substrains, eight MV-resistant strains emerged, with up to 30-fold more resistance than wild types. Genome sequencing identified mutations that were likely to be responsible. Surprisingly, a number of these mutations were also found at a low frequency in wild types (which are highly polyploid). This indicates that there may be genetic flexibility that permits balancing selection mechanisms. In the presence of MV, MV-resistant strains could still perform oxygenic photosynthesis, but they did so less efficiently than wild types when MV was absent, suggesting trade-offs in cellular fitness associated with the evolution of MV resistance. The mode of resistance was demonstrated to be lower intracellular MV accumulation, as shown by electrochemical experiments. The sixth chapter addresses the surprising presence of a putative ROS-generating (NOX) in cyanobacteria, which - considering their pre-existing struggle for oxidative stress mitigation - raises questions about the functional roles of NOX. A gene encoding a putative prokaryotic NOX isoform from *Chroococcidiopsis thermalis* PCC 7203 was experimentally characterised. Validating prior bioinformatic insights, the findings indicated that *nox* forms a bicistronic unit with an adjacent gene coding for a membrane-tethered superoxide dismutase (SodA). The *C. thermalis nox* was introduced into *nox*-deficient *Synechocystis*. Strains harbouring the monocistronic *nox*-expressing plasmid exhibited enhanced extracellular superoxide production and a notable growth impairment compared to empty vector controls. However, *Synechocystis* expressing *nox*-*sod* operonically resulted in rescued growth phenotypes, whilst maintaining enhanced extracellular ROS levels. Taken together, these findings confirmed the functional activity of cyanobacterial *nox*. The operon arrangement may function akin to a ROS-dependent toxin-antitoxin system, facilitating the extracellular reduction of ferric ions, and potentially explaining the sporadic evolution of NOXs in cyanobacteria. Finally, a concluding chapter provides a general discussion and conclusions of the findings, relating them to the introduction and highlighting their significance and limitations.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Scarampi Del Cairo Di Prunetto, Alberto
- Advisor dc:contributor.advisor
-
- Howe, Christopher
Subjects
dc:subject × 9Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.113017
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/375286