Investigating the Role of the Stringent Response in Lipid Modifications during the Stationary Phase in E. coli by Direct Analysis with Time-of-Flight-Secondary Ion Mass Spectrometry

Abstract

Escherichia coli is able to rapidly adjust the biophysical properties of its membrane phospholipids to adapt to environmental challenges including starvation stress. These membrane lipid modifications were investigated in glucose starved E. coli cultures and compared to a ΔrelAΔspoT (ppGpp(0)) mutant strain of E. coli, deficient in the stringent response, by means of time-of-flight-secondary ion mass spectrometry (TOF-SIMS). Recent advances in TOF-SIMS, through the implementation of gas cluster ion beams (GCIBs), now permit the analysis of higher mass species from native, underivatized, biological specimen, i.e., intact bacterial cells. Cultures in stationary phase were found to exhibit a radically different lipid composition as compared to cultures in the exponential growth phase. Wild-type E. coli reacted upon carbon starvation by lipid modifications including elongation, cyclopropanation, and increased cardiolipin formation. Observations are consistent with variants of cardiolipins (CL), phosphatidylglycerols (PG), phosphatidylethanolamines (PE), phosphatidic acids (PA), and fatty acids. Notably, despite having a proteomic profile and a gene expression profile somewhat similar to the wild-type during growth, the ppGpp(0) mutant E. coli strain was found to exhibit modified phospholipids corresponding to unsaturated analogues of those found in the wild-type. We concluded that the ppGpp(0) mutant reacts upon starvation stress by elongation and desaturation of fatty acyl chains, implying that only the last step of the lipid modification, the cyclopropanation, is under stringent control. These observations suggest alternative stress response mechanisms and illustrate the role of the RelA and SpoT enzymes in the biosynthetic pathway underlying these lipid modifications.

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

“…In a previous study in our laboratory we observed an increase in cyclopropane containing lipids in carbon starved bacteria that was dependent on the presence of the global stress signaling compound ppGpp. 13 This increase in cyclopropane formation had previously been reported in GC-MS analysis but the SIMS experiments also suggested that the process occurred via the initial formation of the unsaturated fatty acids prior to cyclopropanation. These new results, showing a strong reduction in FA(cp19:0) upon loss of FabF, suggest that the FabF route to the formation of the FA(18:1) is strongly linked to the cyclopropane formation.…”

Localization of Cyclopropane Modifications in Bacterial Lipids via 213 nm Ultraviolet Photodissociation Mass Spectrometry

“…Although cyclopropane modifications may seem insignificant, they have significant biological implications. Cyclopropane fatty acids (CFAs) are generated by the addition of a methylene group across the double bonds of unsaturated fatty acids (UFAs) of bacterial phospholipids. , This methylene group is derived from S -adenosylmethionine, and the in situ reaction of UFA to CFA is catalyzed by CFA synthase. During this reaction, the CFA retains the original stereochemistry of the UFA, whether cis or trans. , This reaction occurs in response to adverse environmental conditions, and it is common in various Gram-positive and Gram-negative bacterial species including Escherichia coli (E.…”

On-Tissue Chemical Derivatization of Catecholamines Using 4-(N-Methyl)pyridinium Boronic Acid for ToF-SIMS and LDI-ToF Mass Spectrometry Imaging

“…The focus of the ion beam is continuously variable down to the minimum spot size, which for this GCIB is 2−3 μm as measured using the 20−80% signal rise or fall on the edge of a sharp feature in either SIMS or secondary electron images. 43 The ion gun has a series of selectable apertures that can be used to select current/spot size as required. The ion gun delivered 21 pA of DC current (measured in a Faraday cup on the sample holder) through a 100 μm aperture for the 30 μm per pixel imaging and approximately 2 pA through a 30 μm aperture for the 3 μm per pixel imaging.…”