Henrik Siegumfeldt

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

ABSTRACT We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright

Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts.

Physiological aspects of the response of Listeria monocytogenes to acidic conditions and effect of glucose availability were studied by fluorescence ratio-imaging microscopy (FRIM) as compared with traditional viable counts. Three types of experiments were conducted: (i) static with measurements of intracellular pH (pHi) at extracellular pH (pHo) values ranging from pH 3.0 to 6.0 at 0.5 pH unit intervals; (ii) kinetic with monitoring of bacterial responses to changes in the pHo from the value of 6.0 to 4.0 or 3.0; (iii) survival experiments studying bacterial recovery in response to a shift to favourable conditions after a treatment at low pH. All the experiments were performed at three levels of glucose in the medium (0, 1, and 10 mM). Both survival and pHi were greatly affected by pHo and glucose availability with the highest values for CFU and pHi at highest glucose concentration and pHo values in the medium in all trials. A high correlation (R2 = 0.995) between pHi and CFU counts was observed. The pH gradient started to collapse at pHo 4 and below for trials with glucose in the medium and at pHo 5.5 and below without glucose. A recovery step was proposed after the apparently lethal treatment to assess cell viability by FRIM.

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

ABSTRACT Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H+ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H+ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pHo) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pHi) and net H+ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pHi remained above 7.0 within a pHo range of 4 to 6 and decreased below pHo 4. Above pHo 4, H+ extrusion increased correspondingly, with the maximum value at pHo 5.5, and below pHo 4, a net H+ influx was observed. Without glucose in the medium, the pHi decreased, and a net H+ influx was observed below pHo 5.5. A high correlation (R = 0.75 to 0.92) between the pHi and net H+ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pHi homeostasis in L. monocytogenes subjected to acid stress.

Rapid assessment of cell viability of Lactobacillus delbrueckii subsp. bulgaricus by measurement of intracellular pH in individual cells using fluorescence ratio imaging microscopy.

The aim of this study was to investigate if the measurement of intracellular pH (pHi) of individual cells by fluorescence ratio imaging microscopy (FRIM) could be utilized as a rapid method for determining the bacterial viability, using Lactobacillus delbrueckii subsp. bulgaricus as a model organism. Five different standardized cultures with equal cell densities but varying viability were prepared on a trial-to-trial basis by combining aliquots of frozen and lyophilized cells with a 50-fold difference in viability, determined by the ability to form colonies on solid growth media. The acidification of milk and Acidification Power Test were used to determine the activity of these cultures. As expected, the cultures containing a higher proportion of viable cells acidified milk faster and performed better in the Acidification Power Test. All cells were fluorescent after staining with carboxyfluorescein diacetate succinimidyl ester but frozen cells reached higher fluorescence intensities than lyophilized cells. The number of strongly fluorescent cells determined by flow cytometry exceeded the number of viable cells determined by CFU. Analysis of pHi of individual cells by FRIM at an extracellular pH of 6.0 revealed two populations of cells with an average pHi of 6.9 +/- 0.1 and 6.1 +/- 0.1. As the number of cells maintaining a pH-gradient of 0.9 +/- 0.1 correlated well with CFU, we suggest that FRIM can be used as a rapid method for the determination of viability of L. delbrueckii subsp. bulgaricus. Measurement of pHi on a single cell basis is expected to provide accurate prediction of the fermentation performance in a wider range of industrial fermentation.

„Early” protein synthesis of Lactobacillus delbrueckii ssp. bulgaricus in milk revealed by [35S]methionine labeling and two-dimensional gel electrophoresis

The proteomes of exponentially growing and stationary cells of Lactobacillus delbrueckii ssp. bulgaricus grown in rich medium (MRS) were separated by two‐dimensional polyacrylamide gel electrophoresis (2‐DE) and quantified after Coomassie staining. Stationary cells grown in MRS were inoculated in reconstituted skim milk, and “early” protein synthesis during the first 30 min of fermentation in milk was monitored by [35S]methionine labeling and 2‐DE. In contrast to exponentially growing or stationary cells, the predominant “early” proteins were small (< 15 kDa) and of low pI (< 5.3). Quantification of the proteome of the “early” lag phase based on 47 “spots” revealed that only three “early” proteins accounted for more than 80% of the total label. They were identified as pI 4.7 and 4.9 isoforms of the heat‐stable phosphoryl carrier protein (HPr) with 45.2 and 9.4% of total label, respectively, and an unknown protein called EPr1 (“early” protein 1) with 26.6% of total label. Although an N‐terminal sequence of 19 amino acids was obtained, no homologs to EPr1 could be found. De novo synthesis of the 10 and 60 kDa heat shock proteins (GroES and GroEL) was considerably lower (0.04 and 0.9% of total label, respectively), indicating only low levels of stress. Synthesis of triosephosphate isomerase (Tpi) as marker for glycolytic enzymes reached only 0.08% of total label. Our results demonstrate that inoculation in milk, resulting in a change from glucose to lactose as carbon source, imposes only little need for synthesis of stress or glycolytic enzymes, as sufficient proteins are present in the stationary, MRS‐grown cells. The high level of expression of the pI 4.7 isoform of HPr suggests a regulatory function of the presumed Ser‐46 phosphorylated form of HPr.

Intracellular pH as an indicator of viability and resuscitation of Campylobacter jejuni after decontamination with lactic acid

The aim of the study was to determine intracellular pH (pH(i)) as an indicator of the physiological state of two Campylobacter jejuni strains (603 and 608) at the single cell level after bactericidal treatment with lactic acid (3% v/v lactic acid, pH 4.0, 0.85% w/v NaCl) and during recovery and survival using Fluorescence Ratio Imaging Microscopy (FRIM). After exposure to lactic acid solution a decline in pH(i) to 5.5 (FRIM detection limit) was observed in the majority of cells (75-100%) within 2 min. The enumeration data revealed that after 2 min of lactic acid exposure, approx. 90% of the initial population became unculturable. In the following 10 min of exposure, a further decrease in the cell count was observed resulting in 3.53 and 3.21 log CFU/ml reduction of culturable cells at the end of the treatment. On the contrary, the FRIM results revealed that the subpopulations with pH(i)>5.5 increased between 2 and 12 min of exposure to lactic acid. Removing the acid stress and incubating the cells suspension under the more favourable conditions resulted in an immediate increase in cell population with pH(i)>pH(ex) for both C. jejuni strains. Further 24 h incubation at 37 degrees C resulted in increased pH(i) and colony count (recovery study). On the contrary, 24 h incubation at suboptimal temperature of 4 degrees C, showed pH(i) decrease to pH(ex)=6.0 (no pH gradient) in the whole population of C. jejuni cells. Rather than dying, cells exposed for longer time (72 and 120 h) to 4 degrees C increased the subpopulation of the cells with positive pH gradient, mostly comprised of the cells with DeltapH>0.5, indicating the ability of C. jejuni cells to regulate their metabolic activity under suboptimal conditions.

Survival of lactic acid and chlorine dioxide treated Campylobacter jejuni under suboptimal conditions of pH, temperature and modified atmosphere

As mild decontamination treatments are gaining more and more interest due to increased consumer demands for fresh foods, it is of great importance to establish the influence of decontamination treatments on the subsequent bacterial behaviour under suboptimal storage conditions. For this purpose Campylobacter jejuni cells treated with lactic acid (LA, 3% lactic acid, pH 4.0, 2 min) or chlorine dioxide (ClO(2), 20 ppm, 2 min) were inoculated in Bolton broth (pH 6.0) and incubated under 80% O(2)/20% N(2), 80% CO(2)/20% N(2), air or micro-aerophilic (10% CO(2)/85% N(2)/5% O(2)) atmosphere, at 4 degrees C during 7 days. Treatment with water served as a control. The most suppressive atmosphere for the survival of C. jejuni was O(2)-rich atmosphere, followed by air, micro-aerophilic and CO(2)-rich atmosphere. The survival of C. jejuni was dependent on the type of initial decontamination treatment, with water treated cells showing the greatest survival followed by LA and ClO(2) treated cells. Intracellular pH (pH(i)) of individual C. jejuni cells was determined using Fluorescence Ratio Imaging Microscopy (FRIM). At all tested conditions, different subpopulation of the cells could be distinguished based on their pH(i) values. The pH(i) response was independent on the surrounding atmosphere since similar distribution of the subpopulations was observed for all tested atmospheres. However, the pH(i) response was dependent on the initial decontamination treatment. The investigation of intracellular parameters gave an insight into pathogen behaviour under stressful conditions at intracellular level. The results obtained in this study highlighted the importance of combining decontamination technologies with subsequent preservation techniques to the control survival and growth of foodborne pathogens.

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains

The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30–35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pKa,i) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pKa,i close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pKa,i to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.

Effect of long- and short-term exposure to laser light at 1070 nm on growth of Saccharomyces cerevisiae

The effect of a 1070-nm continuous and pulsed wave ytterbium fiber laser on the growth of Saccharomyces cerevisiae single cells is investigated over a time span of 4 to 5 h. The cells are subjected to optical traps consisting of two counterpropagating plane wave beams with a uniform flux along the x, y axis. Even at the lowest continuous power investigated-i.e., 0.7 mW-the growth of S. cerevisiae cell clusters is markedly inhibited. The minimum power required to successfully trap single S. cerevisiae cells in three dimensions is estimated to be 3.5 mW. No threshold power for the photodamage, but instead a continuous response to the increased accumulated dose is found in the regime investigated from 0.7 to 2.6 mW. Furthermore, by keeping the delivered dose constant and varying the exposure time and power-i.e. pulsing-we find that the growth of S. cerevisiae cells is increasingly inhibited with increasing power. These results indicate that growth of S. cerevisiae is dependent on both the power as well as the accumulated dose at 1070 nm.