TY - JOUR T1 - In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at the peptide level JF - bioRxiv DO - 10.1101/094961 SP - 094961 AU - Luitzen de Jong AU - Edward A. de Koning AU - Winfried Roseboom AU - Hansuk Buncherd AU - Martin J. Wanner AU - Irena Dapic AU - Petra J. Jansen AU - Jan H. van Maarseveen AU - Garry L. Corthals AU - Peter J. Lewis AU - Leendert W. Hamoen AU - Chris G. de Koster Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/02/07/094961.abstract N2 - Identification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacterium Bacillus subtilis as an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species, Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth. ER -