Monitoring Surface Chemical Changes in the Bacterial Cell Wall

Abstract

Gram-negative bacteria can alter the composition of the lipopolysaccharide (LPS) layer of the outer membrane as a response to different growth conditions and external stimuli. These alterations can, for example, promote attachment to surfaces and biofilm formation. The changes occur in the outermost layer of the cell and may consequently influence interactions between bacterial cells and surrounding host tissue, as well as other surfaces. Microscopic analyses, fractionation of bacterial cells, or other traditional microbiological assays have previously been used to study these alterations. These methods can, however, be time consuming and do not always give detailed chemical information about the bacterial cell surface. We here present an analytical method that provides chemical information on the outermost portion of bacterial cells with respect to protein, peptidoglycan, lipid, and polysaccharide content. The method involves cryo-x-ray photoelectron spectroscopy analyses of the outermost portion (within ∼10 nm of the surface) of intact bacterial cells followed by a multivariate curve resolution analysis of carbon spectra. It can be used as a tool for characterizing and monitoring variations in the chemical composition of bacterial cell walls or of isolated outer membrane vesicles, variations that result from e.g. mutations or external stimuli. The method enabled us to predict accurately the alterations in polysaccharide content and surface chemistries of a set of well characterized Escherichia coli LPS mutants. The described approach may moreover be applied to monitor surface chemical composition of other biological samples.

Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae

“…The deletions mutants that were the least susceptible to ciclopirox, Δ rfaF , Δ rfaG , and Δ rfaP , encode proteins that synthesize the inner LPS core [41]. RfaF adds the first glucose group and RfaG adds heptose II to the developing inner LPS core, and deletions of these genes results in no outer core [41]. If no outer core is formed, then there is less demand for UDP-glucose or UDP-galactose.…”

Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

“…The percent hydrophobicity was calculated using the following formula: % hydrophobicity  =  [1− (OD 600 after vortex/OD 600 before vortex)] ×100. XPS analysis of bacterial samples were performed as described previously [41], as an alternative hydrophobicity assay. Bacterial samples were collected from fresh colonies on plates grown at 37°C for 17 hrs.…”

A multivariate approach to correlate bacterial surface properties to biofilm formation by lipopolysaccharide mutants of Pseudomonas aeruginosa

“…The amount of polysaccharide was estimated by the phenol sulfuric acid method [35]. Surface composition analyses of bacteria, grown overnight (24 h) on agar plates made with different types of growth medium, were done using the X-ray photoelectron spectroscopy (XPS) method described previously [36]. The elemental surface composition of 96-well plates (inside bottom of wells) was analyzed in a similar way but at room temperature.…”