ABSTRACT
AIMS: Using a flow cytometry (FC)-based approach in combination with four selected fluorescent probes, the biochemical pathway activated following the adaptation of Cronobacter spp. to lethal heat stress was investigated. This approach assessed the physiological changes induced in four strains of Cronobacter spp. METHODS AND RESULTS: Using the commercially available live/dead viability assessment fluorescence probes, live, injured or dead bacterial cells were studied. Cellular respiration and membrane potential were evaluated using the dye-labelled probe 3,3'-dihexylocarbocyanine iodide, metabolic activity was evaluated using a fluorescein diacetate (FDA) probe, intracellular pH changes were measured using a carboxy-fluorescein diacetate succinimidyl ester probe, and reactive oxygen species were measured using a hydroethidine fluorescent probe. Adaptation to lethal heat stress induced physiological changes that potentially improve the survival of Cronobacter spp. CONCLUSIONS: These data showed that in situ assessment of physiological behaviour of lethally stressed cells using multiparameter FC is a useful, rapid and sensitive tool to study and assess the viability and physiological state of Cronobacter cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that FC is a valuable tool in the study of physiological aspects of increased survival because of sublethal adaptation to heat.
Subject(s)
Cronobacter sakazakii/physiology , Flow Cytometry , Hot Temperature , Adaptation, Physiological , Cronobacter sakazakii/metabolism , Fluorescent Dyes , Hydrogen-Ion Concentration , Membrane Potentials , Microbial Viability , Reactive Oxygen Species/analysis , Stress, PhysiologicalABSTRACT
In yeast, three AAA superfamily metalloproteases (Yme1p, Afg3p and Rca1p) are localized to the mitochondrial inner membrane where they perform roles in the assembly and turnover of the respiratory chain complexes. We have investigated the function of the proposed human orthologue of yeast Yme1p, encoded by the YME1L gene on chromosome 10p. Transfection of both HEK-293EBNA and yeast cells with a green fluorescent protein-tagged YME1L cDNA confirmed mitochondrial targeting. When expressed in a yme1 disruptant yeast strain, YME1L restored growth on glycerol at 37 degrees C. We propose that YME1L plays a phylogenetically conserved role in mitochondrial protein metabolism and could be involved in mitochondrial pathologies.
