Interrupting Biosynthesis of O Antigen or the Lipopolysaccharide Core Produces Morphological Defects in Escherichia coli by Sequestering Undecaprenyl Phosphate

Abstract

Undecaprenyl phosphate (Und-P) is a member of the family of essential polyprenyl phosphate lipid carriers and in the Gram-negative bacterium Escherichia coli is required for synthesizing the peptidoglycan (PG) cell wall, enterobacterial common antigen (ECA), O antigen, and colanic acid. Previously, we found that interruption of ECA biosynthesis indirectly alters PG synthesis by sequestering Und-P via dead-end intermediates, causing morphological defects. To determine if competition for Und-P was a more general phenomenon, we determined if O-antigen intermediates caused similar effects. Indeed, disrupting the synthesis of O antigen or the lipopolysaccharide core oligosaccharide induced cell shape deformities, which were suppressed by preventing the initiation of O-antigen biosynthesis or by manipulating Und-P metabolism. We conclude that accumulation of O-antigen intermediates alters PG synthesis by sequestering Und-P. Importantly, many previous experiments addressed the physiological functions of various oligosaccharides and glycoconjugates, but these studies employed mutants that accumulate deleterious intermediates. Thus, conclusions based on these experiments must be reevaluated to account for possible indirect effects of Und-P sequestration.

Importance: Bacteria use long-chain isoprenoids like undecaprenyl phosphate (Und-P) as lipid carriers to assemble numerous glycan polymers that comprise the cell envelope. In any one bacterium, multiple oligosaccharide biosynthetic pathways compete for a common pool of Und-P, which means that disruptions in one pathway may produce secondary consequences that affect the others. Using the Gram-negative bacterium Escherichia coli as a model, we demonstrate that interruption of the biogenesis of O antigen, a major outer membrane component, indirectly impairs peptidoglycan synthesis by sequestering Und-P into dead-end intermediates. These results strongly argue that the functions of many Und-P-utilizing pathways must be reevaluated, because much of our current understanding is based on experiments that did not control for these unintended secondary effects.

FIG 1

Restoring O-antigen biosynthesis to E. coli K-12. (A) Structure of the O16 antigen from E. coli K-12 (reviewed in reference 43). Abbreviations: Galf, galactofuranose; Glc, glucose; Rha, rhamnose; GlcNAc, N-acetylglucosamine. (B) Detection of O antigen with concanavalin A-AF488 in cells with the indicated genotypes. Cells were labeled with concanavalin A-AF488, washed, and photographed by phase-contrast and fluorescence microscopy (green signal). Bar, 3 μm. Concanavalin A binds α-glucose (underlined in panel A) and α-mannose residues. The strains tested were MAJ1 (wbbL::IS5) and MAJ330 (wbbL⁺).

FIG 2

Disrupting O-antigen biosynthesis induces morphological defects in E. coli. (A) O-antigen biosynthesis pathway. Abbreviations: IM, inner membrane; O-Ag, O antigen; P-Gc, glucose 1-phosphate; Und-P, undecaprenyl phosphate; G, N-acetylglucosamine; Ac, acetyl; Ac-CoA, acetyl coenzyme A; Rh, rhamnose; Gf, galactofuranose. Note that WzxB and WbbH are also known as Wzx(O16) and Wzy(O16), respectively (62). (B) Micrographs of cells with the indicated genotypes. Cells were grown at 37°C in LB for approximately 10 doublings until the culture reached an OD₆₀₀ of 0.5 to 0.6. The cells were then fixed and photographed by phase-contrast microscopy. Additional O-antigen mutants are shown in Fig. S1 in the supplemental material. Micrographs of ΔwzxB cells were from overnight cultures because the strain readily develops suppressor mutations that correct the shape defect, as shown in Fig. S2. WT, wild type. Bar, 3 μm. (C) Flow cytometry data from live cells in panel B. Histograms of the forward scatter area (FSC-A) from 100,000 events (cells) are shown. The mean cell size of the wild type is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of those from two independent experiments. The strains tested were MAJ330 (wild type), MAJ339 (ΔwzxB), MAJ345 (ΔwaaL), MAJ343 (ΔwecA), MAJ369 (ΔwecA ΔwzxB), and MAJ370 (ΔwecA ΔwaaL).

FIG 3

Synthetically misshapen mutant combinations in the O-antigen and ECA pathways. (A) Biosynthetic pathway illustrating how the ECA and O-antigen synthesis pathways compete for Und-PP–GlcNAc. Abbreviations: GlcNAc, N-acetylglucosamine; ManNAcA, N-acetylmannosaminuronic acid; Und-P, undecaprenyl phosphate; Und-PP, undecaprenyl pyrophosphate; ECA, enterobacterial common antigen. (B) Micrographs of cells with the indicated genotypes. Cells were grown and imaged as described in the legend to Fig. 2. Bar, 3 μm. (C) Flow cytometry data from live cells in panel B. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size for wild-type cells (red graph) is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of those from two independent experiments. The strains tested were MAJ330 (wild type), MAJ356 (ΔwecB), MAJ346 (ΔwbbJ), MAJ344 (ΔwbbK), MAJ398 (ΔwecB ΔwbbJ), and MAJ397 (ΔwecB ΔwbbK).

FIG 4

Suppression of ΔwaaL shape defects. (A) Micrographs of ΔwaaL cells containing derivatives of pDSW361 that express the indicated genes. Cells were grown at 37°C in LB containing 100 μM IPTG (0 μM IPTG for pwecG) until the culture reached an OD₆₀₀ of 0.5 to 0.6. The cells were then fixed and photographed by phase-contrast microscopy. Bar, 3 μm. Further characterization of wecG overexpression is found in Fig. S3 in the supplemental material. (B) Flow cytometry data from live cells in panel A. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size for ΔwaaL cells expressing waaL in trans (red graph) is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of those from two independent experiments. The strains tested were MAJ434 (pwaaL), MAJ437 (puppS), MAJ436 (pwecG), MAJ435 (pmurA), and MAJ433 (vector). The effect of expression of the aforementioned derivatives of pDSW361 on wild-type cells is shown in Fig. S4.

FIG 5

Disrupting LPS core biosynthesis induces morphological defects in E. coli. (A) Maturation of Und-PP-linked O-antigen intermediates and their ligation to the lipid A core. Mutations that truncate the core oligosaccharide (e.g., ΔwaaC) prevent attachment of the O antigen (right). Abbreviations: OM, outer membrane; A, lipid A. The definitions of the other abbreviations are listed in the legend to Fig. 2. (B) General structure of the E. coli K-12 core oligosaccharide (shaded in gray) (reviewed in reference 77). WaaC and WaaF transfer Hep-I and Hep-II, respectively, onto the growing O-antigen chain. (C) Micrographs of cells with the indicated genotypes. Cells were grown and imaged as described in the legend to Fig. 2. The cells were fixed and photographed by phase-contrast microscopy. Bar, 3 μm. Similar results were obtained for mutants of waaF, as shown in Fig. S5 in the supplemental material. (D) Flow cytometry data from live cells in panel C. Histograms of the FSC-A from 100,000 events (cells) are shown. The mean cell size of the wild-type strain (red graph) is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of those from two independent experiments. The strains tested were MAJ330 (wild type), MAJ374 (ΔwaaC), MAJ343 (ΔwecA), and MAJ384 (ΔwecA ΔwaaC).

FIG 6

Suppression of ΔwaaC shape defects. (A) Micrographs of ΔwaaC cells containing derivatives of pDSW361 that express the indicated genes. Cells were grown and imaged as described in the legend to Fig. 4, except that IPTG was added to 25 μM for the pwecG strain. Overexpression of murA does not fully suppress the shape defects of ΔwaaC cells (e.g., pmurA cells, inset). Bar, 3 μm. (B) Flow cytometry data from live cells in panel A. Histograms of the forward scatter area from 100,000 events (cells) are shown. The mean cell size area for ΔwaaC cells expressing waaC in trans (red graph) is represented by the dashed line and is expressed in arbitrary units (AU). Data are representative of those from two independent experiments. The strains tested were MAJ439 (pwaaC), MAJ442 (puppS), MAJ441 (pwecG), MAJ440 (pmurA), and MAJ438 (vector).

FIG 7

Model of competition for Und-P in E. coli. The PG, ECA, and O-antigen biosynthesis pathways compete for pools of Und-P (red) and UDP-GlcNAc (blue). Interruption of the biosynthesis of ECA, O antigen, or the lipid A core sequesters part of the pool of Und-P and therefore restricts its availability for PG synthesis. Note that the colanic acid biosynthesis pathway, which also utilizes Und-P, is omitted from this illustration because this compound is not appreciably produced under normal growth conditions, though it is highly expressed during stress. Abbreviations: PG, peptidoglycan; UDP-M5, UDP–N-acetylmuramic acid–l-alanine–d-glutamate–meso-diaminopimelic acid–d-alanine–d-alanine; Und-P, undecaprenyl phosphate; GlcNAc, N-acetylglucosamine; Und-PP, undecaprenyl pyrophosphate; ECA, enterobacterial common antigen; LPS, lipopolysaccharide.