Comparative Study of NAP-XPS and Cryo-XPS for the Investigation of Surface Chemistry of the Bacterial Cell-Envelope

Abstract

Bacteria generally interact with the environment via processes involving their cell-envelope. Thus, techniques that may shed light on their surface chemistry are attractive tools for providing an understanding of bacterial interactions. One of these tools is Al Kα-excited photoelectron spectroscopy (XPS) with its estimated information depth of <10 nm. XPS-analyses of bacteria have been performed for several decades on freeze-dried specimens in order to be compatible with the vacuum in the analysis chamber of the spectrometer. A limitation of these studies has been that the freeze-drying method may collapse cell structure as well as introduce surface contaminants. However, recent developments in XPS allow for analysis of biological samples at near ambient pressure (NAP-XPS) or as frozen hydrated specimens (cryo-XPS) in vacuum. In this work, we have analyzed bacterial samples from a reference strain of the Gram-negative bacterium Pseudomonas fluorescens using both techniques. We compare the results obtained and, in general, observe good agreement between the two techniques. Furthermore, we discuss advantages and disadvantages with the two analysis approaches and the output data they provide. XPS reference data from the bacterial strain are provided, and we propose that planktonic cells of this strain (DSM 50090) are used as a reference material for surface chemical analysis of bacterial systems.

Applying Cryo-X-ray Photoelectron Spectroscopy to Study the Surface Chemical Composition of Fungi and Viruses

“…Recently, we have been applying a cryogenic approach to XPS (cryo-XPS) where a biological sample is fastfrozen to liquid nitrogen temperatures prior to analysis and maintained at this temperature throughout the analysis time (Ramstedt and Shchukarev, 2016;Shchukarev and Ramstedt, 2017). Data from cryo-XPS has been shown to be in close agreement with data obtained from near-ambient pressure XPS for a reference bacterial strain (Kjaervik et al, 2021). Furthermore, we developed a model to predict the composition of lipid, polysaccharide and peptide (protein and/ or peptidoglycan) at the surface of the cell from the C 1s spectrum ("the Umeå method").…”

Spiers Memorial Lecture: prospects for photoelectron spectroscopy

“…81 In later studies of the bacterium Pseudomonas fluorescens , it has been shown that this difference can be minimised by using fast freezing in cryo-XPS. 83 Recently, the first NAP-XPS studies of radiation damage in fully hydrated double-strand DNA have been published. 84 DNA damage by ionising radiation is of fundamental importance to cancer therapy, but is difficult to study by XPS in realistic environments.…”

Cryo-XPS spectra from bacterial reference strain Pseudomonas fluorescens DSM50090

“…The cryo-XPS data set (n=9) reported good reproducibility between biological replicas with an average elemental composition +/-standard deviation of: 58.3 +/-1.7 at% C, 10.2 +/-1.0 at% N and 29.0 +/-1.6 at% O (Ref. 3). The variation in C components as ratio to total C were found to be 0.365 +/-0.011 at 285.0 eV, 0.419 +/-0.012 at 286.5 eV, 0.189 +/-0.012 at 288.1 eV and 0.026 +/-0.013 at 289.1 eV (Ref.…”

“…Recent work has shown that XPS spectra obtained from hydrated bacteria using near-ambient pressure XPS and cryo-XPS are comparable (Ref. 3). Furthermore, the compositional stability and reproducibility between cultures grown and subsequently analyzed using XPS in two different labs and locations led us to suggest that this strain could function as a reference sample for XPS analyses of Gramnegative bacterial cells (Ref.…”

“…Furthermore, the compositional stability and reproducibility between cultures grown and subsequently analyzed using XPS in two different labs and locations led us to suggest that this strain could function as a reference sample for XPS analyses of Gramnegative bacterial cells (Ref. 3). The cryo-XPS data set (n=9) reported good reproducibility between biological replicas with an average elemental composition +/-standard deviation of: 58.3 +/-1.7 at% C, 10.2 +/-1.0 at% N and 29.0 +/-1.6 at% O (Ref.…”