In Vivo Cross-Linking Sheds Light on the Salmonella Divisome in Which PBP3 and PBP3SAL Compete for Occupancy.

Abstract

Bacterial cell division is orchestrated by proteins that assemble in dynamic complexes collectively known as the divisome. Essential monofunctional enzymes with glycosyltransferase or transpeptidase (TPase) activities, FtsW and FtsI respectively, engage in the synthesis of septal peptidoglycan (sPG). Enigmatically, Salmonella has two TPases that can promote cell division independently: FtsI (PBP3) and the pathogen-specific paralogue PBP3_SAL. How Salmonella regulates the assembly of the sPG synthase complex with these two TPases, is unknown. Here, we characterized Salmonella division complexes in wild-type cells and isogenic mutants lacking PBP3 or PBP3_SAL. The complexes were cross-linked in vivo and pulled down with antibodies recognizing each enzyme. Proteomics of the immunoprecipitates showed that PBP3 and PBP3_SAL do not extensively cross-link in wild type cells, supporting the presence of independent complexes. More than 40 proteins cross-link in complexes in which these two TPases are present. Those identified with high scores include FtsA, FtsK, FtsQLB, FtsW, PBP1B, SPOR domain-containing proteins (FtsN, DedD, RlpA, DamX), amidase activators (FtsX, EnvC, NlpD) and Tol-Pal proteins. Other cross-linked proteins are the protease Prc, the elongasome TPase PBP2 and, D,D-endo- and D,D-carboxypeptidases. PBP3 and PBP3_SAL localize at midcell and compete for occupying the division complex in response to environmental cues. Thus, a catalytic-dead PBP3_SAL-S300A variant impairs cell division in a high osmolarity and acidic condition in which it is produced at levels exceeding those of PBP3. Salmonella may therefore exploit an 'adjustable' divisome to exchange TPases for ensuring cell division in distinct environments and, in this manner, expand its colonization capacities.