Response to Nutrient Stress in the Industrial Model Bacterium <i>Cupriavidus necator</i> : A Thermal Proteome Profiling (TPP) Investigation

The facultative chemolithoautotroph Cupriavidus necator is capable of heterotrophic growth on diverse carbon sources, or of autotrophic growth using CO₂ fixation with H₂ as an energy source. Under stress conditions, it produces biodegradable polyesters (polyhydroxyalkanoates, PHAs) as a storage material occupying a high proportion of the total biomass. This metabolic versatility means that C. necator is under intense study for sustainable biotechnology processes; however, a relative lack of understanding of the overall regulatory architecture has limited its application. The major mechanisms by which proteins can respond to shifting cellular demands are protein expression change and/or allosteric regulation. Here, we use two powerful proteomics methods to investigate these responses in C. necator cells grown on balanced or low nitrogen (PHA-inducing) media. Using quantitative proteomics and protein stability analysis (which can report on conformation change), we find that proteins across different pathways respond through one or both of these regulatory modes, including coordinated adaptation to nutrient stress by the PHA pathway, the Calvin cycle and ribosomal proteins. Overall, the study offers a valuable overview of global protein changes evoked by nutritional stress, and shows how the combined use of both proteomics approaches can identify key responsive proteins that would otherwise be undetected. SUMMARY: We report a comprehensive proteomics analysis of the important industrial bacterium Cupriavidus necator, using two state-of-the-art approaches: expression proteomics and thermal proteome profiling. With intense interest worldwide in finding substitutes for petrochemical based plastics, organisms such as C. necator are under active investigation, since they produce a storage bioplastic material (PHA) and have a versatile metabolism including growth on carbon dioxide. To our knowledge, this is the first thermal proteome analysis of a lithoautotrophic organism. We compared global protein expression the under conditions that induce PHA production, and we analysed the thermal proteome under the same conditions. Each experiment yielded novel, interesting results pertinent to individual proteins or pathways; moreover, by combining both approaches, proteins regulated by expression change and/or conformation change were highlighted.