Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli

Abstract

Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell. Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway. Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins. Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step. Here we present a method-premature targeting-for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway. We fused FtsQ to the In Escherichia coli, cell division requires the coordinated constriction of the three layers of the Gram-negative cell envelope. Invagination is mediated by the septal ring, an assembly of proteins that localizes to the site of constriction and appears to remain at the site until separation of the two daughter cells. The septal ring is composed of multiple proteins, both essential and nonessential, which localize to the septum and/or are required for optimal cell division. The 10 known essential proteins are strictly required for cell division and localize to the constriction site in a defined hierarchical order ( Fig. 1A; . This hierarchy defines the septal recruitment "pathway," in which a given protein requires all proteins upstream of it to localize to the division site, and is itself required for the localization of those that are farther downstream. The mode of localization to the septum within the pathway, however, is understood for only a few of the proteins, leaving open the questions of how the cell assembles the "divisome" into its final state and ultimately whether the order of assembly is critical to the cell division process.The assembly of the most upstream components of the septal ring is best understood. FtsZ is a widely conserved bacterial homolog of tubulin (Mukherjee et al. 1993;Lowe and Amos 1998) and was the first cell division protein shown to be localized to the bacterial midcell (Bi and Lutkenhaus 1991). FtsZ polymers form a ring or tight spiral at the bacterial midcell, which is thought to provide both a scaffold for assembly of subsequent septal ring components and possibly the force required for constriction (Romberg and Levin 2003). FtsA, a conserved member of the actin superfamily (Bork et al. 1992; van den Ent and Lowe 2000), and ZipA have both been shown to bind directly to the C terminus of FtsZ (Hale and de Boer 1997;Wang et al. 1997;Ma and Margolin 1999;Mosyak et al. 2000;Moy et al. 2000;Yan et al. 2000) and are thought to be required for stabilizing polymerized Z rings at the midcell (Pichoff and Lutkenhaus 2002). The molecular events that underlie recruitment among later proteins remain speculative.Interestingly, the almost strict linearity...

Rapid β-lactam-induced lysis requires successful assembly of the cell division machinery

“…This ordinarily means that old peptidoglycan is not degraded unless the divisome is functioning properly. Consistent with previous studies suggesting a role for FtsN in activation of the divisome and initiation of septation and separation of daughter cells (5,(26)(27)(28), our results suggest that recruitment of FtsN functions to ensure that cell wall degradation does not take place unless new cell wall is being incorporated at the emergent poles. Also consistent with a regulatory rather than, for instance, an essential enzymatic role, mutants that are missing FtsN can be suppressed by mutations in another component of the divisome, FtsA (29).…”

Characterization of CrgA, a New Partner of the Mycobacterium tuberculosis Peptidoglycan Polymerization Complexes

“…The latter two proteins are predicted to be transpeptidases involved in peptidoglycan cross-linking during septum synthesis (12,14). FtsQ is an integral part of the divisome and is believed to play a crucial role in the recruitment of early and late cell division proteins and peptidoglycan polymerases (4,23,51). Our studies showed increased GFP-FtsI MT localization at the septa and enhanced Bocillin-FL staining under conditions of CrgA overproduction.…”

FtsZ and the division of prokaryotic cells and organelles

“…FtsQ can co-purify with its downstream partners FtsL and FtsB 87 . Moreover, when targeted prematurely to the Z ring by fusion to ZapA, FtsQ can recruit FtsL, FtsB and the later protein FtsI (but not FtsN) to the Z ring in the absence of the upstream proteins FtsW or FtsA 88 . The failure of FtsN to be recruited in this system indicates that the proper assembly of all components at the septum, particularly FtsA, might be essential for the localization of FtsN.…”