Towards a phosphoproteome map of Corynebacterium glutamicum

In this study, the phosphoproteome of Corynebacterium glutamicum , an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram‐positive bacteria, was investigated by two different detection methods: first, by in vivo radio‐labeling using [ 33 P]‐phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid‐specific monoclonal antibodies. After two‐dimensional gel electrophoresis (2‐DE), around 60 [ 33 P]‐labeled protein spots were visualized and around 90 antibody‐decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5‐enolpyruvylshikimate 3‐phosphate synthase, aconitase, acyl‐CoA carboxylase, acyl‐CoA synthetase, ATP (synthase α‐ and β‐chain), carbamoyl‐phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat‐inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol‐acid reductoisomerase, lactate dehydrogenase, leucine‐tRNA ligase, lipoamide dehydrogenase, methionine synthase, O ‐acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (β‐subunit), succinyl‐CoA:CoA transferase, transketolase and UDP‐ N ‐acetylmuramoyl‐ L ‐alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.