Branching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coli

Abstract

Certain mutants in Escherichia coli lacking multiple penicillin-binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild-type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild-type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low-molecular-weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries.

Fig. 1

Outer membrane stability at poles and deformities of E. coli 2443-derived strains. Cells were labelled with Texas Red succinimidyl ester and incubated in the absence of dye and in the presence of aztreonam. Stable regions of outer membrane proteins appear as brightly fluorescent patches at the poles in strain 2443 (left) and at the poles and deformities in the PBP mutant AG70C-12 (right).

Fig. 2. Localization of IcsA–GFP fusion proteins to poles and deformities in E. coli 2443-derived strains. Direct fluorescence (left) and phase (right) micrographs of IcsA_507-620–GFP (A, C and E) and IcsA_Δ507-729–GFP (B, D and F)

A and B. Wild-type strain 2443. C and D. PBP mutant AG70A-6. E and F. PBP mutant AG70C-12.

Fig. 3. Localization of IcsA–GFP and GFP–EpsM in the K-12-derived PBP mutant CS703-1. Direct fluorescence (left) and phase (right) micrographs

A. IcsA_507-620–GFP. B. IcsA_507-620–GFP in cells filamented with aztreonam. C. GFP–EpsM.

Fig. 4. Localization of full-length IcsA to the outer membrane of E. coli 2443-derived strains. Indirect immunofluorescence (left) and phase (right) micrographs of surface-localized IcsA

A. E. coli 2443 ompT. B. PBP mutant AG70C-12 ompT. Arrows, IcsA localized at branches; arrowheads, IcsA localized at split poles.

Fig. 5. Interference of full-length IcsA with IcsA_507-620–GFP localization in PBP mutant AG70C-12 ompT. Direct fluorescence (left) and phase (right) micrographs

A. PBP mutant AG70C-12 ompT expressing IcsA_507-620–GFP alone. B. PBP mutant AG70C-12 ompT expressing both full-length IcsA and IcsA_507-620–GFP.

Fig. 6. Co-localization of full-length IcsA and IcsA_507-620–GFP to poles and sites of deformities of E. coli 2443-derived strains. Phase micro-graphs (Phase), direct fluorescence micrographs (Ics A_507-620–GFP), indirect immunofluorescence micrographs (Full-length IcsA) and overlay of direct fluorescence (green) and indirect immunofluorescence (red) micrographs (Overlay)

A. Wild-type derivative 2443 ompT. B and C. PBP mutant AG70C-12 ompT. Arrows indicate co-localization of cytoplasmic and surface IcsA.