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Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

ABSTRACT: Bacterial morphology is determined primarily by the architecture of the peptidoglycan (PG) cell wall, a mesh-like layer that encases the cell. To identify novel mechanisms that create or maintain cell shape in Escherichia coli, we used flow cytometry to screen a transposon insertion library and identified a wecE mutant that altered cell shape, causing cells to filament and swell. WecE is a sugar aminotransferase involved in the biosynthesis of enterobacterial common antigen (ECA), a non-essential outer membrane glycolipid of the Enterobacteriaceae. Loss of wecE interrupts biosynthesis of ECA and causes the accumulation of the undecaprenyl pyrophosphate-linked intermediate ECA-lipid II. The wecE shape defects were reversed by: (i) preventing initiation of ECA biosynthesis, (ii) increasing the synthesis of the lipid carrier undecaprenyl phosphate (Und-P), (iii) diverting Und-P to PG synthesis or (iv) promoting Und-P recycling. The results argue that the buildup of ECA-lipid II sequesters part of the pool of Und-P, which, in turn, adversely affects PG synthesis. The data strongly suggest there is competition for a common pool of Und-P, whose proper distribution to alternate metabolic pathways is required to maintain normal cell shape in E.?coli.

SUBMITTER: Jorgenson MA

PROVIDER: S-EPMC4845916 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

Jorgenson Matthew A MA Kannan Suresh S Laubacher Mary E ME Young Kevin D KD

Molecular microbiology 20151222 1

Bacterial morphology is determined primarily by the architecture of the peptidoglycan (PG) cell wall, a mesh-like layer that encases the cell. To identify novel mechanisms that create or maintain cell shape in Escherichia coli, we used flow cytometry to screen a transposon insertion library and identified a wecE mutant that altered cell shape, causing cells to filament and swell. WecE is a sugar aminotransferase involved in the biosynthesis of enterobacterial common antigen (ECA), a non-essentia ...[more]

PMID: 26593043

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Project description: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.ImportanceBacteria 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.

Project description:The peptidoglycan exoskeleton shapes bacteria and protects them against osmotic forces, making its synthesis the target of many current antibiotics. Peptidoglycan precursors are attached to a lipid carrier and flipped from the cytoplasm into the periplasm to be incorporated into the cell wall. In Escherichia coli, this carrier is undecaprenyl phosphate (Und-P), which is synthesized as a diphosphate by the enzyme undecaprenyl pyrophosphate synthase (UppS). E. coli MG1655 exhibits wild-type morphology at all temperatures, but one of our laboratory strains (CS109) was highly aberrant when grown at 42°C. This strain contained mutations affecting the Und-P synthetic pathway genes uppS, ispH, and idi Normal morphology was restored by overexpressing uppS or by replacing the mutant (uppS31) with the wild-type allele. Importantly, moving uppS31 into MG1655 was lethal even at 30°C, indicating that the altered enzyme was highly deleterious, but growth was restored by adding the CS109 versions of ispH and idi Purified UppSW31R was enzymatically defective at all temperatures, suggesting that it could not supply enough Und-P during rapid growth unless suppressor mutations were present. We conclude that cell wall synthesis is profoundly sensitive to changes in the pool of polyisoprenoids and that isoprenoid homeostasis exerts a particularly strong evolutionary pressure.IMPORTANCE Bacterial morphology is determined primarily by the overall structure of the semirigid macromolecule peptidoglycan. Not only does peptidoglycan contribute to cell shape, but it also protects cells against lysis caused by excess osmotic pressure. Because it is critical for bacterial survival, it is no surprise that many antibiotics target peptidoglycan biosynthesis. However, important gaps remain in our understanding about how this process is affected by peptidoglycan precursor availability. Here, we report that a mutation altering the enzyme that synthesizes Und-P prevents cells from growing at high temperatures and that compensatory mutations in enzymes functioning upstream of uppS can reverse this phenotype. The results highlight the importance of Und-P metabolism for maintaining normal cell wall synthesis and shape.

Dataset Information

Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

Publications

Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate.

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