Growth response and recovery of Corynebacterium glutamicum colonies on single-cell level upon defined pH stress pulses

Bacteria respond to pH changes in their environment via pH homeostasis to keep the intracellular pH as constant as possible within a small range. A change of the intracellular pH value influences e.g., the enzyme activity, protein stability, solubility of trace elements and the proton motive force. Here, the species Corynebacterium glutamicum has been chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with an external pH between 5.5 and 9. In the 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 responding to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyse the physiological growth response of C. glutamicum upon precise pH stress pulses at a single-cell level. Analysis of the growth behaviour at the colony level by dMSCC exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in the viability with increasing stress duration. Colony regrowth was possible after increasing lag phases when stress durations were increased from 5 min to 9 h for all tested pH values. Furthermore, the single-cell analysis revealed heterogeneous regrowth of cells after pH stress, which can be distinguished into two distinct behaviours: firstly, cells continue to grow without interruption after the pH stress, and secondly, some cells rest for several hours after the pH stress before they start to grow again after this lag phase. This study provides the first insights into the single-cell response to acidic and alkaline pH stress adaptation of C. glutamicum.

Download Full-text

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.711893 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sarah Täuber ◽

Luisa Blöbaum ◽

Volker F. Wendisch ◽

Alexander Grünberger

Keyword(s):

Single Cell ◽

Corynebacterium Glutamicum ◽

Intracellular Ph ◽

Growth Response ◽

Physiological State ◽

Single Cell Level ◽

Cell Level ◽

Stress Pulse ◽

Ph Values ◽

Ph Stress

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.

Download Full-text

Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Applied and Environmental Microbiology ◽

10.1128/aem.02625-08 ◽

2009 ◽

Vol 75 (13) ◽

pp. 4550-4556 ◽

Cited By ~ 27

Author(s):

Vicky G. Kastbjerg ◽

Dennis S. Nielsen ◽

Nils Arneborg ◽

Lone Gram

Keyword(s):

Listeria Monocytogenes ◽

Single Cell ◽

Intracellular Ph ◽

Bacterial Population ◽

Single Cells ◽

Viable Cell ◽

Population Subdivision ◽

Single Cell Level ◽

Cell Level ◽

Cell Counts

ABSTRACT Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.

Download Full-text

Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2010.05.031 ◽

2010 ◽

Vol 141 ◽

pp. S188-S192 ◽

Cited By ~ 15

Author(s):

Francesca Gaggìa ◽

Dennis Sandris Nielsen ◽

Bruno Biavati ◽

Henrik Siegumfeldt

Keyword(s):

Single Cell ◽

Intracellular Ph ◽

Mycobacterium Avium ◽

Mycobacterium Avium Subsp ◽

Single Cell Level ◽

Antimicrobial Compounds ◽

Cell Level ◽

Mycobacterium Avium Subsp Paratuberculosis

Download Full-text

An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics

10.1101/2021.03.09.434608 ◽

2021 ◽

Author(s):

Caitlin E. T. Donahue ◽

Michael D. Siroky ◽

Katharine A. White

Keyword(s):

Single Cell ◽

Intracellular Ph ◽

Single Cells ◽

Cell Polarization ◽

Membrane Dynamics ◽

Cell Physiology ◽

Single Cell Level ◽

Cell Level ◽

Cell Behaviors ◽

Membrane Ruffling

AbstractIntracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and increased membrane dynamics (both protrusion and retraction events) compared to control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating the experiments required to determine if increased pHi is a driver of these cell behaviors at the single-cell level.

Download Full-text