The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria

Genetic Dissection of the Divergent Activities of the Multifunctional Membrane Sensor BglF▿ †

BglF catalyzes β-glucoside phosphotransfer across the cytoplasmic membrane in Escherichia coli. In addition, BglF acts as a sugar sensor that controls expression of β-glucoside utilization genes by reversibly phosphorylating the transcriptional antiterminator BglG. Thus, BglF can exist in two opposed states: a nonstimulated state that inactivates BglG by phosphorylation and a sugar-stimulated state that activates BglG by dephosphorylation and phosphorylates the incoming sugar. Sugar phosphorylation and BglG (de)phosphorylation are both catalyzed by the same residue, Cys24. To investigate the coordination and the structural requirements of the opposing activities of BglF, we conducted a genetic screen that led to the isolation of mutations that shift the balance toward BglG phosphorylation. We show that some of the mutants that are impaired in dephosphorylation of BglG retained the ability to catalyze the concurrent activity of sugar phosphotransfer. These mutations map to two regions in the BglF membrane domain that, based on their predicted topology, were suggested to be implicated in activity. Using in vivo cross-linking, we show that a glycine in the membrane domain, whose substitution impaired the ability of BglF to dephosphorylate BglG, is spatially close to the active-site cysteine located in a hydrophilic domain. This residue is part of a newly identified motif conserved among β-glucoside permeases associated with RNA-binding transcriptional antiterminators. The phenotype of the BglF mutants could be suppressed by BglG mutants that were isolated by a second genetic screen. In summary, we identified distinct sites in BglF that are involved in regulating phosphate flow via the common active-site residue in response to environmental cues