62 research outputs found
The Congo red derivative FSB binds to curli amyloid fibers and specifically stains curliated E. coli.
The Congo red derivative (E,E)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy) styrylbenzene (FSB) specifically stains the functional amyloid curli in Escherichia coli biofilms. FSB binds to curli with similar affinity as Congo red, yet exhibits much greater fluorescence upon binding to curli as compared to Congo red and does not exhibit undesired binding to the cellulosic component of the biofilm. Thus, FSB presents a powerful tool to identify and visualize curli in E. coli biofilms and also enables new biophysical investigations of curli
Peptide antibiotics in action: Investigation of polypeptide chains in insoluble environments by rotational-echo double resonance
AbstractRotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus
Nutrient-Dependent Structural Changes in <i>S. aureus</i> Peptidoglycan Revealed by Solid-State NMR Spectroscopy
The bacterial cell wall is essential to cell survival
and is a
major target of antibiotics. The main component of the bacterial cell
wall is peptidoglycan, a cage-like macromolecule that preserves cellular
integrity and maintains cell shape. The insolubility and heterogeneity
of peptidoglycan pose a challenge to conventional structural analyses.
Here we use solid-state NMR combined with specific isotopic labeling
to probe a key structural feature of the <i>Staphylococcus aureus</i> peptidoglycan quantitatively and nondestructively. We observed that
both the cell-wall morphology and the peptidoglycan structure are
functions of growth stage in <i>S. aureus</i> synthetic
medium (SASM). Specifically, <i>S. aureus</i> cells at stationary
phase have thicker cell walls with nonuniformly thickened septa compared
to cells in exponential phase, and remarkably, 12% (±2%) of the
stems in their peptidoglycan do not have pentaglycine bridges attached.
Mechanistically, we determined that these observations are triggered
by the depletion of glycine in the nutrient medium, which is coincident
with the start of the stationary phase, and that the production of
the structurally altered peptidoglycan can be prevented by the addition
of excess glycine. We also demonstrated that the structural changes
primarily arise within newly synthesized peptidoglycan rather than
through the modification of previously synthesized peptidoglycan.
Collectively, our observations emphasize the plasticity in bacterial
cell-wall assembly and the possibility to manipulate peptidoglycan
structure with external stimuli
CPMAS NMR platform for direct compositional analysis of mycobacterial cell-wall complexes and whole cells
Tuberculosis and non-tuberculosis mycobacterial infections are rising each year and often result in chronic incurable disease. Important antibiotics target cell-wall biosynthesis, yet some mycobacteria are alarmingly resistant or tolerant to currently available antibiotics. This resistance is often attributed to assumed differences in composition of the complex cell wall of different mycobacterial strains and species. However, due to the highly crosslinked and insoluble nature of mycobacterial cell walls, direct comparative determinations of cell-wall composition pose a challenge to analysis through conventional biochemical analyses. We introduce an approach to directly observe the chemical composition of mycobacterial cell walls using solid-state NMR spectroscopy. 13C CPMAS spectra are provided of individual components (peptidoglycan, arabinogalactan, and mycolic acids) and of in situ cell-wall complexes. We assigned the spectroscopic contributions of each component in the cell-wall spectrum. We uncovered a higher arabinogalactan-to-peptidoglycan ratio in the cell wall of M. abscessus, an organism noted for its antibiotic resistance, relative to M. smegmatis. Furthermore, differentiating influences of different types of cell-wall targeting antibiotics were observed in spectra of antibiotic-treated whole cells. This platform will be of value in evaluating cell-wall composition and antibiotic activity among different mycobacteria and in considering the most effective combination treatment regimens
Peptidoglycan and Teichoic Acid Levels and Alterations in <i>Staphylococcus aureus</i> by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance
Gram-positive
bacteria surround themselves with a multilayered
macromolecular cell wall that is essential to cell survival and serves
as a major target for antibiotics. The cell wall of <i>Staphylococcus
aureus</i> is composed of two major structural components, peptidoglycan
(PG) and wall teichoic acid (WTA), together creating a heterogeneous
and insoluble matrix that poses a challenge to quantitative compositional
analysis. Here, we present <sup>13</sup>C cross polarization magic
angle spinning solid-state nuclear magnetic resonance (NMR) spectra
of intact cell walls, purified PG, and purified WTA. The spectra reveal
the clear molecular differences in the two polymers and enable quantification
of PG and WTA in isolated cell walls, an attractive alternative to
estimating teichoic acid content from a phosphate analysis of completely
pyrolyzed cell walls. Furthermore, we discovered that unique PG and
WTA spectral signatures could be identified in whole-cell NMR spectra
and used to compare PG and WTA levels among intact bacterial cell
samples. The distinguishing whole-cell <sup>13</sup>C NMR contributions
associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl
α-carbons. WTA contributes carbons from the phosphoribitol backbone.
Distinguishing <sup>15</sup>N spectral signatures include glycyl amide
nitrogens in PG and the esterified d-alanyl amine nitrogens
in WTA. <sup>13</sup>C NMR analysis was performed with samples at
natural abundance and included 10 whole-cell sample comparisons. Changes
consistent with altered PG and WTA content were detected in whole-cell
spectra of bacteria harvested at different growth times and in cells
treated with tunicamycin. This use of whole-cell NMR provides quantitative
parameters of composition in the context of whole-cell activity
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