4.1 Biologische Materialschädigung und Referenzorganismen
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Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future. It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4+ limitation shapes phenotypic heterogeneity in N2 fixation. In turn, the N2 fixation rate of single cells during NH4+ limitation correlates positively with their growth rate after a shift to NH4+ depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges - nutrient limitation and fluctuations - that many microorganisms face. Currently, we use NanoSIMS to develop a new approach that defines functionally-relevant, phenotypic biodiversity in microbial systems. In the last part of my presentation, I will highlight why the concept of phenotypic diversity is relevant for the understanding of antimicrobial resistance.
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained. In this contribution, the latest progress on biofilm characterisation by NAP-XPS will be presented, and measurement capabilities and limitations will be discussed.