Role of peptidoglycan amidases in the development and morphology of the division septum in Escherichia coli

Abstract

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.

FIG. 1.

Cell division is required for development of SP rings in amidase mutants of E. coli. Sacculi were prepared and observed by TEM. (A) PG rings in E. coli RP77 (ΔamiABC). Arrowheads point to examples of typical PG rings (large-diameter rings at sites with little or no constriction) and SP rings (small-diameter rings located at sites with obvious cell constriction). (B) E. coli CS109 forced to grow as filaments in the presence of the PBP3-inhibitor aztreonam. No PG rings are visible, though the cells continued to elongate. (C) E. coli RP77 incubated in the presence of aztreonam for 40 min before preparation of sacculi. Although preexisting circular and partial PG rings remain, in the elongated portions of these cells no new rings formed (e.g., arrow in left-hand chain). Also, the continued development of early-stage PG rings at the time of antibiotic addition was aborted (e.g., arrows in right-hand chain). (D) E. coli RP77 forced to grow as filaments by inhibiting FtsZ by expressing the sulA gene from plasmid pFAD38. SulA production was induced with 0.2% arabinose for 1 h before preparation of sacculi. No new PG rings of any sort are visible within the elongated cells.

FIG. 2.

PG ring detection by staining with NHS-Texas Red-X. (A) The outer surface of sacculi from E. coli RP77 was labeled uniformly with anti-murein antibody and AlexaFluor 488-labeled goat anti-rabbit antibody. (B) RP77 sacculi labeled with NHS-Texas Red-X. Heavily labeled PG ring bands appear along the length of the sacculi. Arrows indicate the three rings magnified in panel C. (C) Area from panel B at a higher magnification, showing a large (center) PG ring bracketed on either side by more advanced-stage (smaller) SP rings.

FIG. 3.

PG rings behave as though composed of inert PG. E. coli RP77 (ΔamiABC) was grown at 37°C in LB medium containing d-cysteine and then shifted to grow an additional 30 min in d-cysteine-free medium without (A to C and G) or with (D to F and H) the antibiotic aztreonam (1 μg/ml). Sacculi were stained with NHS-Oregon Green and also with anti-biotin mouse primary antibody followed by AlexaFluor 594-labeled goat anti-mouse secondary antibody. (A) Oregon Green-stained sacculi. PG rings are visible as brightly fluorescent bands along the length of the sacculus chain. (B) Location of newly incorporated PG. Older (d-cysteine-containing) PG stains red, while newly synthesized PG (d-cysteine-free material incorporated during the chase period) is unstained (dark areas). (C) Merged image of panels A and B. PG rings present in newly synthesized PG are green, while PG rings in preexisting PG are yellow. (D) Sacculi forced to grow as filaments in the presence of aztreonam and then stained with Oregon Green. PG ring material is more highly fluorescent. (E) Location of newly incorporated PG in sacculi forced to grow as filaments in the presence of aztreonam. Older (d-cysteine-containing) PG stains red, while newly synthesized PG (d-cysteine-free material incorporated during the chase period) is unstained (dark areas). (F) Merger of panels A and B. PG ring material (yellow) is almost exclusively localized in areas of preexisting PG. (G) Fluorescence intensity profiles of sacculi in panels A and B. The two profiles display the distribution of preexisting (red) and newly incorporated (green) PG along the length of the chain of sacculi. Black arrows indicate the location of PG rings that actively incorporated new PG, defined as ring positions that exhibit a low-intensity red trace coupled with a high-intensity green trace. Red arrows indicate the location of PG rings composed of inert PG, defined as ring positions that exhibit high intensity peaks in both the red and green traces. (H) Fluorescence intensity profiles of sacculi in panels D and E. Red arrows indicate the location of PG rings composed of inert PG (ring positions with high-intensity peaks in both the red and green traces). No PG rings incorporated new PG, as denoted by the absence of PG rings with a low-intensity red trace coupled with a high-intensity green trace.

FIG. 4.

Poles and incomplete septa of amidase mutants attract IcsA-GFP. E. coli RP161 (ΔamiAC) was grown at 30°C in LB-ampicillin medium, production of IcsA-GFP variants was induced by adding 0.2% arabinose for 40 min, and the resulting chains of cells were visualized by fluorescence microscopy. (A) IcsA_507-620-GFP, a fusion protein that attaches specifically to cell poles, localizes to complete and incomplete septa in cell chains of the double amidase mutant. (B) Diffuse cytoplasmic localization of IcsA_Δ507-729-GFP, a variant protein from which polar localization signals have been removed.

FIG. 5.

Cytoplasmic compartmentalization between cells in chains of E. coli lacking AmiABC. A cytoplasmic form of GFP was expressed from plasmid pGFPuv in E. coli RP77 (ΔamiABC), and the cells were labeled with FM4-64 to stain the inner membrane. Cells were prepared for confocal microscopy, and potential connections between cells were tested by the technique of FLIP. The size of the area to be bleached was set equal to the width of a single cell, and the areas to be bleached (indicated by arrows) were exposed to a beam of 488-nm light from an argon laser. A chain was selected, and individual cells (A, B, and C) were bleached in sequence. Cells were photographed immediately before, immediately after, and 10 s after bleaching (columns from left to right). Fully compartmentalized cells were those whose neighbors retained their fluorescence (A and C), while cells that were not compartmentalized were identified as those where fluorescence disappeared from one or more neighboring cells (B).

FIG. 6.

FtsZ rings do not persist at older division sites in amidase mutants. E. coli RP75 (ΔamiAB) containing plasmid pDSW230 was grown in LB medium at 37°C to an OD₆₀₀ of 0.2, and IPTG (5 μM final concentration) was added to induce expression of ftsZ-gfp. After 1 h cells were collected for microscopy and examined by phase-contrast and fluorescence microscopy. Representative chains of cells are presented as pairs of photographs, the first showing the location of FtsZ-GFP (fluorescence only) and the second showing the merged phase and fluorescence photos. Arrows indicate septation sites that have no associated FtsZ-GFP ring.

FIG. 7.

Abnormal septa in amidase mutants. Cultures of E. coli RP77 (ΔamiABC) (marked with an asterisk) and E. coli RP108 (ΔamiABC ΔPBP5) (unmarked) were stained to visualize septal cross-walls. (A and B) Paired representative chains stained with NHS-Oregon Green 488-X to label the outer membrane (A) or with FM4-64 to label the inner membrane (B). (C) Chains of cells producing GFP-FtsI from plasmid pDSW234. Arrows indicate some of the abnormal septa that are visibly misshapen or tilted relative to the longitudinal axes of the chains.

FIG. 8.

Schematic models of murein hydrolase activity during septation in E. coli. (A) Proposed sequence of hydrolase functions during septal invagination. (B) Invagination of the outer membrane during division in the presence (1) or absence (2) of murein hydrolases. Details are presented in the text.