Phosphoproteomic Study Of Clostridium Acetobutylicum

Clostridium acetobutylicum is a gram positive anaerobic bacterium, which belongs to the clostridium family. It can produce acetic acid and butyric acid in early and middle exponential growth phase (acidogenesis), and can generate acetone, butanol and alcohol in late exponential growth phase and stationary phase (solventogenesis). Thanks to the development of biological energy technology, the mechanism of solvent production and industrial application are emerging more and more important in the field of studying C. acetobutylicum. Protein phosphorylation, playing a pivotal role in cellular signal pathway and metabolism, is one of the most well studied post translational modifications so far. Phosphoproteomic study is promoted by the improvement of enriched phosphorylated peptides through affinity chromatography and good identification of the modified peptides via accurate mass spectrometry. A bunch of experimental evidence in recent years indicates the existence of protein phosphorylation in prokaryotes. There is, however, barely any report in protein phosphorylation in C. acetobutylicum, not to mention phosphoproteome.In this study, we initiated to profile the phosphoproteome of wild type Clostridium acetobutylicum ATCC824 at the stages of acidogenesis and solventogenesis, using affinity enrichment and mass spectrometry. Also we attempted to explore the possible roles of protein phosphorylation involved in metabolic pathways in such bacterium.Acidogenesis and solventogenesis were carefully evaluated by the medium pH values and the solvent products. We harvested the bacterial cells and extracted whole proteins from the two individual phases followed by proteins separation with two-dimensional electrophoresis (2DE). The 2DE images exhibited some iso-spots with similar molecular weights and diverse pⅠ values, suggesting that wide modifications of proteins occurred in C. acetobutylicum. In order to fully analyze the phosphoproteome of the organism, we employed TiO2 columns to enrich phosphorylated peptides, and optimized the enrichment efficiency with different conditions. Based upon the peptide detection upon an ion-trap mass spectrometer, a total of 82 phosphorylated peptides were identified, which belong to 61 unique proteins containing 16 proteins with 2 or more phosphorylated peptides. Further analysis revealed that 52 phosphorylated peptides from 44 unique proteins were detected in acidogenesis, while 70 phosphorylated peptides from 51 unique proteins were detected in solventogenesis. The two stages shared 34 proteins with 40 phosphorylated peptides. About 11% of the identified proteins were functionally categorized to the pathways of solvent production pathway, implicating the crucial role of the protein phosphorylation in solvent production.On the list of phosphorylated proteins in C. acetobutylicum, we realized the importance of acetate kinase, which is a key enzyme in acid production but never be reported involvement of phosphorylation. We deeply investigated the functional changes responding to phosphorylation of acetate kinase. For in vivo study, we generated the monoclonal antibodies against the phosphorylated peptide and wild acetate kinase, and found significantly higher phosphorylation of acetate kinase in acidogenesis than that in solventogenesis. The catalytic activity of acetate kinase in the cell lysate in acidogenesis was almost 100% percent higher compared to that of solventogenesis. The influence of phosphorylation on protein activity was further scrutinized in the in vitro study. We generated wild and mutated recombinant acetate kinase, in which the threonine residue at 239 were converted as non-phosphorylated (T239A) or permanently phosphorylated (T239E) enzyme. Comparison of the acetate kinase activity demonstrated that the permanently phosphorylated enzyme displayed more than twice higher catalytic activity than that of wild type and non-phosphorylated proteins, and provided strong evidence that the phosphorylation at threonine 239 in C. acetobutylicum acetate kinase could result in enzyme activation, possibly involved in regulation of solvent production pathways.The current study is the first attempt to globally evaluate the protein phosphorylation in C. acetobutylicum at different stages of solvent generation with the state-of-the-art means of proteomics. Through functional analysis of the phosphorylated acetate kinase, we raise a hypothesis that enzyme phosphorylation is likely to participate in regulation of acid/solvent production in C. acetobutylicum.