Ligand specificities of <i>Synechococcus</i> substrate-binding proteins: Structural and biophysical characterisation

<p>Paradigms of metabolic strategies employed by marine <em>Synechococcus</em>, generally regarded as an obligate photoautotroph, have been challenged in recent years. The presence of genes annotated as substrate-binding proteins (SBPs) responsible for mediating the uptake of both organic and inorganic nutrients has led to a mixed-mode metabolic strategy (mixotrophy) being proposed for these bacteria, where photoautotrophic growth is supplemented by scavenging organic nutrients from the environment. Despite the profound implications this metabolic strategy has for our understanding of marine ecosystems and nutrient cycling in the oceans, mixotrophy in <em>Synechococcus </em>remains contentious due to a lack of direct evidence to support these functional annotations, and the limited sequence identity underpinning them. This thesis describes the use of bioinformatics, structural, and biophysical approaches to determine the function of novel SBPs from diverse marine <em>Synechococcus </em>spp. To understand their ecological significance.  </p> <p>Structural and functional characterisation of a conserved SBP likely involved in binding modified sugars (MsBP) demonstrated a specific and pronounced affinity for zinc (K<em>D </em>= 1.3 + 0.03 μM). This represents a significant deviation between the predicted and apparent ligand chemistry for this SBP. Two crystal structures were obtained for this protein (6WPM; 6WPN) showing it conformed closely to other sugarbinding proteins. Further <em>in vitro </em>characterisation highlighted altered behaviour in the presence of zinc, leading to the inference that Zn exerts functional control over binding of modified sugar substrates.  </p> <p>Analysis of a predicted glycine betaine-binding protein (ProX) indicated it was only present in selected picocyanobacteria strains. In solution, ProX exists as a dimer and displays high affinity (K<em>D </em>= 0.6 ± 0.12 μM) to glycine betaine, but not any other osmoprotectants screened. ProX displayed altered kinetic behaviour under a range of salt conditions, highlighting a role for additional regulation of function, both through quaternary structure, and salt-mediated changes in solution-state behaviour and binding kinetics.</p> <p>In addition to characterised phosphate uptake systems, most picocyanobacteria possess an additional putative ABC-type organic phosphonate transporter, PhnCDE, thought to provide access to an alternative (organic) P pool in low-P environments. Biophysical characterisation of the purified PhnD protein from four distinct marine <em>Synechococcus </em>isolates (CC9311, CC9605, MITS9220, and WH8102) highlights a clear preference (K<em>D </em>range = 0.6 to 4.3 μM) of these proteins for inorganic phosphite, with additional low affinity interactions with both inorganic phosphate (K<em>D </em>range = 7.4 to 88 μM) and simple alkyl phosphonates (K<em>D </em>range = 17 to 47 μM). These findings indicate an affinity series consistent across these four binding proteins, highlighting that marine <em>Synechococcus </em>species likely access phosphite as an alternative phosphorous source rather than organic phosphonates.</p> <p>This thesis highlights that SBP sequence annotations require detailed experimental characterisation to decipher their range of encoded functions. The case studies of individual SBPs highlight the potential for <em>Synechococcus </em>to access alternative nutrient sources and perform cryptic functions which may be underappreciated among this ubiquitous protein family. This research also begins to disentangle the complex nature of phylogenetic conservation and ecological functions, highlighting how <em>Synechococcus, </em>and by extension picocyanobacteria, exploit SBP-mediated nutrient uptake to adapt to different environmental niches. The findings of this thesis lend further evidence to a proposed mixotrophic strategy within these ubiquitous photosynthetic bacteria, which likely contributes to their success across broader marine environments. </p>