Chemical Changes On, and Through, The Bacterial Envelope in Escherichia coli Mutants Exhibiting Impaired Plasmid Transfer Identified Using Time-of-Flight Secondary Ion Mass Spectrometry

Abstract

Time-of-flight secondary ion mass spectrometry (ToF−SIMS) using a (CO 2 ) 6k + gas cluster ion beam (GCIB) was used to analyze Escherichia coli mutants previously identified as having impaired plasmid transfer capability related to the spread of antibiotic resistance. The subset of mutants selected were expected to result in changes in the bacterial envelope composition through the deletion of genes encoding for FabF, DapF, and Lpp, where the surface sensitivity of ToF−SIMS can be most useful. Analysis of arrays of spotted bacteria allowed changes in the lipid composition of the bacteria to be elucidated using multivariate analysis and confirmed through imaging of individual ion signals. Significant changes in chemical composition were observed, including a surprising loss of cyclopropanated fatty acids in the fabF mutant where FabF is associated with the elongation of FA(16:1) to FA(18:1) and not cyclopropane formation. The ability of the GCIB to generate increased higher mass signals from biological samples allowed intact lipid A (m/z 1796) to be detected on the bacteria and, despite a 40 keV impact energy, depth profiled through the bacterial envelope along with other high mass ions including species at m/z 1820 and 2428, attributed to ECA CYC , that were only observed below the surface of the bacteria and were notably absent in the depth profile of the lpp mutant. The analysis provides new insights into the action of the specific pathways targeted in this study and paves the way for whole new avenues for the characterization of intact molecules within the bacterial envelope.

Mass Spectrometric Imaging of Plasma Membrane Lipid Alteration Correlated with Amperometrically Measured Activity-Dependent Plasticity in Exocytosis

“…It is a surface-sensitive technique and allows label-free identification as well as relative quantification of multiple biomolecules down to the subcellular scale. Thus, ToF-SIMS has become an important tool in multiple research areas, especially in the biological field [15,16]. This technique has been applied to analyze changes of lipid species in cells and tissues caused by drug treatments, diseases, or injuries [17][18][19][20][21].…”

Interplay between Cocaine, Drug Removal, and Methylphenidate Reversal on Phospholipid Alterations in Drosophila Brain Determined by Imaging Mass Spectrometry

“…The use of a high energy gas cluster ion beam (GCIB) with ToF-SIMS makes it particularly well suited for imaging intact phospholipids owing to the reduced fragmentation that occurs during sputtering by large primary ion clusters which therefore preserves the molecular ions. 18,19 In this paper, we use ToF-SIMS imaging to test the hypothesis that the withdrawal of cocaine or addition of MPH after drug in Drosophila brain reverses the dramatic changes in the brain lipid composition caused by exposure to cocaine. We have investigated if this strategy can be used to rescue the adverse effects of cocaine on lipid composition and brain functions.…”

Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics

“…The ability to obtain molecular images of intact species depends upon ion beam characteristics such as the composition, the configuration of the ion optics, and the energy spread of the beam. , Ion beams composed of majority monatomic species readily provide subcellular resolution imaging capability, as they can be focused to below 50 nm. ,, However, the limitation for analysis of biological samples is that they generate significant fragmentation, therefore molecules are rarely desorbed (and consequently measured) intact . Gas cluster ion beams (GCIBs), which can be focused to <3 μm and are used on instruments including the Ionoptika J105, , have improved intact lipid imaging due to softer desorption processes that generate less fragmentation than the liquid metal ion guns (LMIG) typically used in SIMS. − Subcellular imaging of phosphocholine (PC) lipids in the brain, lipid visualization in 3D within bacterial envelopes, and de novo purine biosynthesis have been demonstrated at a single cell scale with (CO 2 ) n /Ar n gas clusters. − Recently, high energy water clusters have been shown to improve ion yields for single cell imaging with SIMS . Due to SIMS introducing significant fragmentation, instruments with MS 2 capabilities are desired as they aid assignment of fragment ions (produced by molecule fragmentation) to their corresponding parent ions in a fragment rich SIMS spectra. , However, many instruments only employ a simple TOF-multichannel plate detector, which limits the mass resolving power to <10000, making structural assignments nontrivial .…”