In-Culture Cross-Linking of Bacterial Cells Reveals Large-Scale Dynamic Protein–Protein Interactions at the Peptide Level

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 interprotein cross-linked peptides with <1%
false discovery rate by mass spectrometry and genome-wide database
searching. Nearly 60% of the interprotein 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. Data are
available via ProteomeXchange with identifier PXD006287.