A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity

Abstract

Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment.

Co-option and Detoxification of a Phage Lysin for Housekeeping Function

“…S4). Despite previous reports of inactivity [18] and our own data predicting inactivation of its catalytic cleft due to its D20L mutation, we found that purified SpmX muramidase exhibited hydrolytic activity. We used remazol brilliant blue (RBB) assays, which are standard for measuring lysozyme activity [33], to compare the activity of SpmX muramidase from C. crescentus (SpmX-Mur-Cc) to that of the P22 lysozyme (P22Lyso) and a P22Lyso mutant with a similarly inactivated catalytic cleft (P22Lyso-D20L) ( Fig.…”

“…Previously, it was suggested that the muramidase domain was co-opted entirely for the purpose of protein-protein and self-oligomerizing interactions in SpmX's role as a developmental regulator and scaffold in C. crescentus [18]. This conclusion was based on the lack of detectable activity from the purified domain and the inability of the catalytic E11R (E19R in SpmX numbering) mutant to self-oligomerize.…”

“…As part of SpmX, this domain is critical for SpmX's role in both developmental regulation and stalk biogenesis. In particular, the muramidase domain is necessary for proper localization of SpmX in both C. crescentus [15,18] and the Asticcacaulis genus [17]. Various studies have shown that SpmX localizes with polar scaffold PopZ in C. crescentus [19,20] and this interaction occurs entirely through the muramidase domain [18].…”

“…In particular, the muramidase domain is necessary for proper localization of SpmX in both C. crescentus [15,18] and the Asticcacaulis genus [17]. Various studies have shown that SpmX localizes with polar scaffold PopZ in C. crescentus [19,20] and this interaction occurs entirely through the muramidase domain [18]. A previous study was unable to measure enzymatic activity from purified C. crescentus SpmX muramidase domain and consequently concluded that the domain was repurposed for protein interactions and oligomeric assembly [18].…”

“…Various studies have shown that SpmX localizes with polar scaffold PopZ in C. crescentus [19,20] and this interaction occurs entirely through the muramidase domain [18]. A previous study was unable to measure enzymatic activity from purified C. crescentus SpmX muramidase domain and consequently concluded that the domain was repurposed for protein interactions and oligomeric assembly [18]. However, given the remarkable sequence similarity of the SpmX muramidase domain to functional phage lysozymes, including the canonical catalytic glutamate, it seems unlikely that its enzymatic activity is entirely lost.…”