Accumulation of periplasmic enterobactin impairs the growth and morphology of Escherichia coli tolC mutants.

TolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram-negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wild-type cells in minimal media. These phenotypes have no physiological explanation as yet. Here, we find that tolC mutants have highly aberrant shapes when grown in M9-glucose medium but that adding iron restores wild-type morphology. When starved for iron, E. coli tolC mutants synthesize but cannot secrete the siderophore enterobactin, which collects in the periplasm. tolC mutants unable to synthesize enterobactin display no growth or morphological defects, and adding exogenous enterobactin recreates these aberrations, implicating this compound as the causative agent. Cells unable to import enterobactin across the outer membrane grow normally, whereas cells that import enterobactin only to the periplasm become morphologically aberrant. Thus, tolC mutants grown in low iron conditions accumulate periplasmic enterobactin, which impairs bacterial morphology, possibly by sequestering iron and inhibiting an iron-dependent reaction involved in cell division or peptidoglycan synthesis. The results also highlight the need to supply sufficient iron when studying TolC-directed export or efflux, to eliminate extraneous physiological effects.