A high-resolution reference map for cytoplasmic and membrane-associated proteins ofCorynebacterium glutamicum

Abstract

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.

Toward the Complete Membrane Proteome

“…Such transport proteins are among the roughly 660 annotated integral membrane proteins and represent about 22% of all 3099 proteins forming the proteome of C. glutamicum (14,15). In contrast, previous analysis of the membrane proteome of C. glutamicum by 2D electrophoresis failed to identify membrane integral proteins, and only cytosolic and water-soluble membrane-associated proteins were detected (16,17). Because IEF is unsuitable for the separation of hydrophobic integral membrane proteins (6,18), we have recently developed a method that replaces the IEF step of 2D electrophoresis by ion exchange chromatography.…”

Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

“…1, lanes 1 and 2). The band was excised and digested with trypsin, and the peptide masses determined by MALDI-TOF mass spectrometry were compared to the masses in a tryptic digest database of 3,746 predicted C. glutamicum proteins as described previously (28). Thus, we verified that supernatants of C. glutamicum WT(pEKEx2-ushA) contained UshA.…”

“…To identify proteins separated by SDS-PAGE, the bands of interest were excised and digested with trypsin, and the peptide masses determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry were compared to the masses in a tryptic digest database of 3,746 predicted C. glutamicum proteins as described previously (28). To determine the length of UshA from supernatants, the bands of interest were excised and electroeleuted using a DiaEx midi kit (Serva, Heidelberg, Germany) according to the manufacturer's recommendations, and the protein mass was determined using MALDI-TOF mass spectrometry as described previously (28).…”

Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

“…It is very likely that FdhD from C. glutamicum ATCC 13032 also acts as a sulfur transferase between a desulfurase and FdhF and therefore is essential for the formation of an active FDH. Several homologues of IscS from E. coli can also be found in the genomes mentioned above and have also been described in C. glutamicum ATCC 13032 (cg1214, cg1388 and cg1761) (see Schaffer et al, 2001;Marienhagen et al, 2005;Teramoto et al, 2010).…”