Genetic interaction mapping highlights key roles of the Tol-Pal complex.

ABSTRACT

The conserved Tol-Pal trans-envelope complex is important for outer membrane (OM) stability and cell division in Gram-negative bacteria. It is proposed to mediate OM constriction during cell division via cell wall tethering. Yet, recent studies suggest the complex has additional roles in OM lipid homeostasis and septal wall separation. How Tol-Pal facilitates all these processes is unclear. To gain insights into its function(s), we applied transposon-insertion sequencing, and report here a detailed network of genetic interactions with the tol-pal locus in Escherichia coli. We found one positive and > 20 negative strong interactions based on fitness. Disrupting osmoregulated-periplasmic glucan biosynthesis restores fitness and OM barrier function, but not proper division, in tol-pal mutants. In contrast, deleting genes involved in OM homeostasis and cell wall remodeling causes synthetic growth defects in strains lacking Tol-Pal, especially exacerbating OM barrier and/or division phenotypes. Notably, the ΔtolA mutant having additional defects in OM protein assembly (ΔbamB) exhibited severe division phenotypes, even when single mutants divided normally; this highlights the possibility for OM phenotypes to indirectly impact cell division. Overall, our work underscores the intricate nature of Tol-Pal function, and reinforces its key roles in cell wall-OM tethering, cell wall remodeling, and in particular, OM homeostasis.