Multiplexed intracellular single-cell sensing strategies of microorganisms integrated for cultivation in microfluidic devices
- Intrazelluläre Multiplex-Messmethoden für Einzelzellen von mikrofluidisch kultivierten Mikroorganismen
Bucher-Krämer, Christina Erna Maria; Wiechert, Wolfgang (Thesis advisor); Blank, Lars M. (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2020
Intracellular sensing in microorganisms is challenging due to their fast response to environmental changes and the possibilities of unbiased intracellular probes for reliable live single-cell analysis. The submitted PhD thesis presents implementation of multiplexed fluorescence real time imaging concepts using conventional fluorescent fusion protein expression and novel non-toxic fluorescence in situ stainings (FISS) in a controlled microfluidic environment. Stressful growth conditions with subsequent recovery phases were simulated in microfluidic cultivation devices to study behaviour of cells and their descendants by time and single-cell resolved microscopy. Firstly, the sporadic promotor activities of the SOS cascade gene recA and of the induction of prophage CGP3 were determined under non-stressful conditions and under nutrient depletion. The involved promotor inductions have been analysed by spontaneous induced fusion protein fluorescences in a bacterium. Phenotypic minority formation and individual cell fates were visualized using eyfp, e2-crimson, and venus. Second, novel non-invasive, instantaneous sensing methods were developed without the requirement of genetic modification, to distinguish heterogeneous cell states of growing wild type bacteria using fluorogenic molecules. An innovative complementary approach to conventional cultivation-based methods is utilization of fluorogenic substrates with designed enzyme-labile moieties for live cell analysis. Hence, acetoxymethyl ester (AM) has been discovered as shuttle molecule moiety of aromatic compounds for C. glutamicum. This has been demonstrated for multiplexed imaging with six fluorogenic probe molecules coupled to AM to sense intracellular parameters (metabolic activity, dormancy, aging, cell lysis, apparent antibiotic tolerance, chemotoxicity, phototoxicity, spontaneous intracellular radical formation, and intracellular pH). Further, the protective function of the cell wall or cell membranes of native wild types have been analysed by novel live-cell sensing based on dynamic FISS with propidium iodide and PO-PRO-1. Loss of function of the cellular barriers, programmed cell death, survivor cells with evolving non-gene based antibiotic tolerance and resistance to gene based lytic toxin production were defined phenotypically by diffusive intrusion of probe molecules due to their specific phenotype development.