1. Volume 23, Issue 2, Pages 267-277 (February 2016)
Cold Stress Makes Escherichia coli Susceptible to Glycopeptide Antibiotics by Altering Outer Membrane Integrity 
Jonathan M. Stokes, Shawn French, Olga G. Ovchinnikova, Catrien Bouwman, Chris Whitfield, Eric D. Brown 
Cell Chemical Biology 
Volume 23, Issue 2, Pages (February 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 267-277DOI: (10. 1016/j. chembiol. 2015
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3. Figure 1
Vancomycin Inhibits Growth of E. coli in a Temperature-Dependent Manner
(A) Top: Potency analysis of vancomycin against wild-type E. coli at 42°C, 37°C, 30°C, 25°C, 20°C, and 15°C. Light to dark shaded symbols represent increasing temperatures from 20°C to 42°C. Open circles represent cells grown at 15°C. Bottom: Potency analysis of vancomycin at 15°C against E. coli harboring pBAD30-vanHBX (filled circles) or empty pBAD30 plasmid (open circles). Black circles represent cultures induced with 0.2% arabinose. Gray circles represent no induction. All experiments were performed in duplicate and varied by <10%.
(B) Morphology of wild-type E. coli grown at 15°C in the presence of increasing concentrations of vancomycin. Cells were grown to early log phase in the presence of vancomycin and stained with FM4-64 prior to visualization.
(C) TEM of wild-type E. coli grown at 15°C in the presence of DMSO (left) or 4 μg/ml vancomycin (right). Arrows indicate regions where the outer membrane and inner membrane have detached. Scale bars represent 1 μm.
(D) Suppression of vancomycin activity at 37°C (red) and 15°C (blue) by titration of Ca2+ or Mg2+. See also Figures S1 and S2.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2
Genetic Perturbations in Outer Membrane Biosynthesis Cause Vancomycin Resistance
(A) Vancomycin activity against the E. coli Keio collection at 15°C. All growth data are normalized to 1 (see Table S2). Red circles show gene-deletion strains that grew 3σ or greater above the mean of the entire collection, as measured by integrated density. 41 strains were vancomycin resistant using this criterion (see Table S1). Inset shows replicate plot of normalized growth data.
(B) Classification of 41 vancomycin-resistant Keio strains based on clusters of orthologous groups.
(C) Potency analysis of vancomycin against the Y324* mutant E. coli at 42°C, 37°C, 30°C, 25°C, 20°C, and 15°C. Light to dark shaded symbols represent increasing temperatures from 20°C to 42°C. Open circles represent cells grown at 15°C. All experiments were performed in duplicate and varied by <10%.
(D) Morphology of the Y324* mutant E. coli grown at 15°C in the presence of increasing concentrations of vancomycin. Cells were grown to early log phase in the presence of vancomycin and stained with FM4-64 prior to visualization.
See also Figures S3 and S4.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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5. Figure 3
LPS Profiles of Vancomycin-Sensitive and -Resistant E. coli Strains
Cells were grown in LB medium at 37°C and 15°C until early log phase and equal amounts of Proteinase K-treated whole-cell lysates were subjected to SDS-PAGE followed by silver staining. WT, wild-type.
(A) A selection of vancomycin-sensitive E. coli Keio mutants. skp, periplasmic chaperone; lpxL/M, lipid A acylation; waaJ/L/O/P, LPS core biosynthesis.
(B) A selection of vancomycin-resistant E. coli Keio mutants. galU, pgm, UDP-glucose biosynthesis; lpcA, rfaE, waaB/F/G/Q/Y, LPS core biosynthesis.
(C) Y324* mutant E. coli grown at 37°C and 15°C.
(D) The structure of E. coli K-12 core OS and genes involved in its biosynthesis. Inner core is shown in red and the outer core is shown in blue. Asterisks highlight biosynthetic genes that are functionally interdependent. The solid black line defines the distal region of the outer core where loss-of-function mutations do not affect vancomycin susceptibility.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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6. Figure 4
Modifications in LPS Chemistry Affect Vancomycin Activity in E. coli
Charge-deconvoluted negative-ion ESI mass spectra of core OS isolated from E. coli strains grown at 37°C (left column) and 15°C (right column).
(A) Wild-type (WT) E. coli fraction II.
(B) Y324* mutant fraction IIa.
(C) Y324* mutant fraction IIb.
All core OS fractions were found to be heterogeneous in composition to some extent, and differed in the presence of Kdo in normal and anhydro forms (Δm 18 u), as well as the content of phosphate (Δm 80 u, blue bars), phosphoethanolamine (Δm 123 u, orange bars), hexose (Δm 162 u, purple bars), and heptose (Δm 192 u, green bars). See also Figures S5 and S6.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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7. Figure 5
Proposed Structures of Core OS from Wild-Type and Y324* Mutant E. coli
(A) Core OS species found in fraction II in wild-type and fraction IIa in the Y324* mutant. Note that at 15°C, core species contained an additional one or two phosphate groups at currently unknown positions. Furthermore, the abundance of glycoform I is significantly reduced in the Y324* mutant at 15°C relative to wild-type (WT).
(B) Core OS species found in fraction IIb from the Y324* mutant. For all structures, the inner core is shown in red and outer core is shown in blue.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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8. Figure 6
Proposed Model for Vancomycin Activity in E. coli at Low Growth Temperature
(A) X-Ray structure of E. coli LPS isolated from PDB: 1FI1. Lipid A is shown in pink, Kdo is shown in blue, and core OS is shown in green. At 15°C, lipid A would contain a palmitoleate acyl chain at the location marked by the asterisk. Our results also show an additional one or two phosphates in core OS at currently unexplored locations. The maximum depth of core OS relative to lipid A is ∼20 Å (side view, right).
(B and C) Simplified depiction of the cell surface in wild-type (B) and vancomycin-resistant (C) E. coli. Gray circles represent divalent cations. Grey arrows represent vancomycin entry sites.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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