Growth Response and Recovery of Corynebacterium glutamicum Colonies on Single-Cell Level Upon Defined pH Stress Pulses

Abstract

Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum.

How to Perform a Microfluidic Cultivation Experiment—A Guideline to Success

“…Consequently, we decided to make future pH adjustments with glass pipettes only. For a more detailed explanation of the results of this case study, the reader is referred to Täuber et al [82]. Example 4-altered morphology of cells: In this set of experiments, we cultivated C. glutamicum under constant CGXII medium supply, but with varying pH values at 30 °C to investigate its pH stress response [82].…”

“…For a more detailed explanation of the results of this case study, the reader is referred to Täuber et al [82]. Example 4-altered morphology of cells: In this set of experiments, we cultivated C. glutamicum under constant CGXII medium supply, but with varying pH values at 30 °C to investigate its pH stress response [82]. For each experiment, pH was adjusted to the desired pH value between pH 6 and 8.…”

“…Consequently, we decided to make future pH adjustments with glass pipettes only. For a more detailed explanation of the results of this case study, the reader is referred to Täuber et al [82]. Example 5-no growth on-chip: In this experiment, we cultivated C. glutamicum with BHI (complex) medium supply at 30 °C in perfusion to analyse the colony growth rate.…”

“…Example 1—increased lag phase of cells: In this series of experiments, Corynebacterium glutamicum was cultivated under constant CGXII minimal medium [ 27 , 81 ] conditions at 30 °C in an open-box monolayer cultivation chamber (2D chamber) [ 82 ]. After cell loading of our MSCC chip, we observed a distinct lag phase of all cells (see Figure 5 ).…”

“…Therefore, the cause of our problem was a compromised PCA solution. A new PCA stock solution finally resulted in regular growth on-chip (µ ≈ 0.6 h −1 ) [ 82 ]. For a more detail explanation of the results of this case study, the reader is referred to Grünberger et al [ 22 ] and Täuber et al [ 62 ].…”