The First Purification of Functional Proteins from the Unculturable, Genome-Reduced, Bottlenecked α-Proteobacterium 's Liberibacter solanacearum'.

Liberibacter solanacearum' is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato-a major problem in potato-growing areas, because it affects growth and yield. Developing effective treatments for ' L. solanacearum' has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. ' L. solanacearum' has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying ' L. solanacearum' proteins in by coexpressing them with chaperones. Neither of the representative ' L. solanacearum' enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully coexpressed with GroEL/GroES, while soluble pyruvate kinase was successfully coexpressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size-exclusion chromatography. Additionally, both ' L. solanacearum' enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologs. This is the first study to successfully isolate and functionally characterize proteins from ' L. solanacearum'. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programs to study and manage the pathogenicity of the organism.