Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli

Abstract

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.

FIG. 1

Morphology of PBP mutants before and after filamentation by aztreonam. Bacteria were grown in LB broth at 37°C until reaching an A₅₅₀ of approximately 0.2, at which point 10 μg of aztreonam per ml was added. Samples were prepared for microscopy immediately before and 45 min after addition of the antibiotic. (A) CS109; (B) CS109 plus aztreonam; (C) CS604-2; (D) CS604-2 plus aztreonam; (E) CS701-1; (F) CS701-1 plus aztreonam; (G) CS601-3; (H) CS601-3 plus aztreonam; (I) CS612-1; (J) CS612-1 plus aztreonam.

FIG. 2

Morphology of untreated PBP mutants. Bacteria were grown in LB broth at 37°C until reaching log phase and then photographed. (A) CS502-1; (B) CS508-1; (C) CS403-3; (D) CS506-1; (E) CS509-3.

FIG. 3

Differences in cellular diameter of strains isogenic for dacA. Strains were grown in LB broth at 37°C until reaching an A₅₅₀ of approximately 0.2, at which point samples were prepared for microscopy. “Frequency” indicates the proportion of the population with a particular diameter. n, the number of cells measured for each population.

FIG. 4

Restoration of normal morphology by cloned PBP 5. E. coli CS701-1 pPJ5C was grown at 37°C in LB broth supplemented with 20 μg of chloramphenicol and either 0.005% arabinose or 0.1% glucose until reaching an A₅₅₀ of 0.2, at which point 10 μg of aztreonam per ml was added. Samples were prepared for microscopy 45 min after addition of the antibiotic. (A) PBP 5 expression induced by 0.005% arabinose; (B) inhibition of PBP 5 expression by 0.1% glucose.