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Non‐invasive, ratiometric determination of intracellular pH in Pseudomonas species using a novel genetically encoded indicator

Arce‐Rodríguez, Alejandro, Volke, Daniel C., Bense, Sarina, Häussler, Susanne, Nikel, Pablo I.·2019·Microbial Biotechnology
DOI 10.1111/1751-7915.13439PMC PMC6559197
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Abstract

Summary: The ability of Pseudomonas species to thrive in all major natural environments (i.e. terrestrial, freshwater and marine) is based on its exceptional capability to adapt to physicochemical changes. Thus, environmental bacteria have to tightly control the maintenance of numerous physiological traits across different conditions. The intracellular pH ( pH i ) homoeostasis is a particularly important feature, since the pH i influences a large portion of the biochemical processes in the cell. Despite its importance, relatively few reliable, easy‐to‐implement tools have been designed for quantifying in vivo pH i changes in Gram‐negative bacteria with minimal manipulations. Here we describe a convenient, non‐invasive protocol for the quantification of the pH i in bacteria, which is based on the ratiometric fluorescent indicator protein PHP ( pH indicator for P seudomonas ). The DNA sequence encoding PHP was thoroughly adapted to guarantee optimal transcription and translation of the indicator in Pseudomonas species. Our PHP ‐based quantification method demonstrated that pH i is tightly regulated over a narrow range of pH values not only in Pseudomonas , but also in other Gram‐negative bacterial species such as Escherichia coli . The maintenance of the cytoplasmic pH homoeostasis in vivo could also be observed upon internal (e.g. redirection of glucose consumption pathways in P . putida ) and external (e.g. antibiotic exposure in P . aeruginosa ) perturbations, and the PHP indicator was also used to follow dynamic changes in the pH i upon external pH shifts. In summary, our work describes a reliable method for measuring pH i in Pseudomonas , allowing for the detailed investigation of bacterial pH i homoeostasis and its regulation.

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Figure 1 Benchmarking of the PHP indicator for the assessment of intracellular pH. A. Two separate expression plasmids were constructed for the calibrated expression of the synthetic genes encoding the pHluorin2 or PHP indicators. In both cases, the expression of the genes is driven by the constitutive PEM 7 promoter, and the backbone used for these constructs (vector pSEVA2513) carries the oriV(RSF1010) origin of replication, indicated in the scheme as a purple circle. The kanamycin‐resistance (KmR) determinant and the T1 and T0 transcriptional terminators are likewise highlighted in the plasmid maps. B. Flow cytometry exploration of the pHluorin2 and PHP indicators. Plasmid pS2513·pHluorin2 or pS2513·PHP was transformed in P. putida KT2440, and the cells were grown in LB medium until the mid‐exponential phase of growth. Cells were processed as indicated in Experimental Procedures , and the histograms show the distribution of fluorescence (in arbitrary units, A.U.) stemming from pHluorin2 or PHP (the bacterial populations positive for the indicators are indicated in each case). The light grey rectangle in each plot identifies the region considered negative for the fluorescence signal, which was assessed with P. putida carrying the empty pSEVA2513 vector and grown under the same conditions. C. Key performance parameters of the pH indicators. The signal‐to‐noise ratio was calculated from flow cytometry measurements of P. putida KT2440 carrying either plasmid pS2513·pHluorin2 or pS2513·PHP as indicated above. The bars represent the signal‐to‐noise ratio for each sensor and the 95% confidence interval calculated from four independent experiments. The coefficient of variation (CV) of the fluorescence signal was also calculated from these measurements.
Figure 1 Benchmarking of the PHP indicator for the assessment of intracellular pH. A. Two separate expression plasmids were constructed for the calibrated expression of the synthetic genes encoding the pHluorin2 or PHP indicators. In both cases, the expression of the genes is driven by the constitutive PEM 7 promoter, and the backbone used for these constructs (vector pSEVA2513) carries the oriV(RSF1010) origin of replication, indicated in the scheme as a purple circle. The kanamycin‐resistance (KmR) determinant and the T1 and T0 transcriptional terminators are likewise highlighted in the plasmid maps. B. Flow cytometry exploration of the pHluorin2 and PHP indicators. Plasmid pS2513·pHluorin2 or pS2513·PHP was transformed in P. putida KT2440, and the cells were grown in LB medium until the mid‐exponential phase of growth. Cells were processed as indicated in Experimental Procedures , and the histograms show the distribution of fluorescence (in arbitrary units, A.U.) stemming from pHluorin2 or PHP (the bacterial populations positive for the indicators are indicated in each case). The light grey rectangle in each plot identifies the region considered negative for the fluorescence signal, which was assessed with P. putida carrying the empty pSEVA2513 vector and grown under the same conditions. C. Key performance parameters of the pH indicators. The signal‐to‐noise ratio was calculated from flow cytometry measurements of P. putida KT2440 carrying either plasmid pS2513·pHluorin2 or pS2513·PHP as indicated above. The bars represent the signal‐to‐noise ratio for each sensor and the 95% confidence interval calculated from four independent experiments. The coefficient of variation (CV) of the fluorescence signal was also calculated from these measurements.CC BY · PubMed Central
Figure 2 Assessment of intracellular pH (pH i) in different Gram‐negative bacteria. A. Fluorescent microscopy from exponentially growing cultures of E. coli DH5α, P. putida KT2440 and P. aeruginosa PA14 transformed with plasmid pS2513·PHP and grown in LB medium. Scale bars are indicated in each case. B. Fluorescence excitation spectrum of the PHP indicator in P. aeruginosa PA14/pS2513·PHP cultured in LB medium. The intracellular pH (pH i) of the cells was collapsed to the extracellular pH as outlined in Experimental Procedures . The mean values obtained from three independent experiments are shown. A.U., arbitrary units. C. Calibration curve of the PHP indicator in P. aeruginosa PA14/pS2513·PHP cells cultured in LB medium supplemented with 20 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP). The data for the calibration curve were obtained as the ratio between the excitation peaks at λexcitation = 405 nm and 485 nm (R 405/485) plotted against the pH values of equilibrated cells (blue circles). These values were fitted using the Boltzmann sigmoid best‐fitting curve to obtain the calibration curve (orange triangles), which was used to calculate the pH i values in P. aeruginosa PA14 during exponential growth (red crosses). The lowest and highest pH values tested (pH = 5.5 and 8.5) are highlighted. Data points are representative of independent triplicates, and the red crosses (corresponding to six individual determinations) are shown in the curve to indicate the narrow dispersion of experimentally calculated pH i values.
Figure 2 Assessment of intracellular pH (pH i) in different Gram‐negative bacteria. A. Fluorescent microscopy from exponentially growing cultures of E. coli DH5α, P. putida KT2440 and P. aeruginosa PA14 transformed with plasmid pS2513·PHP and grown in LB medium. Scale bars are indicated in each case. B. Fluorescence excitation spectrum of the PHP indicator in P. aeruginosa PA14/pS2513·PHP cultured in LB medium. The intracellular pH (pH i) of the cells was collapsed to the extracellular pH as outlined in Experimental Procedures . The mean values obtained from three independent experiments are shown. A.U., arbitrary units. C. Calibration curve of the PHP indicator in P. aeruginosa PA14/pS2513·PHP cells cultured in LB medium supplemented with 20 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP). The data for the calibration curve were obtained as the ratio between the excitation peaks at λexcitation = 405 nm and 485 nm (R 405/485) plotted against the pH values of equilibrated cells (blue circles). These values were fitted using the Boltzmann sigmoid best‐fitting curve to obtain the calibration curve (orange triangles), which was used to calculate the pH i values in P. aeruginosa PA14 during exponential growth (red crosses). The lowest and highest pH values tested (pH = 5.5 and 8.5) are highlighted. Data points are representative of independent triplicates, and the red crosses (corresponding to six individual determinations) are shown in the curve to indicate the narrow dispersion of experimentally calculated pH i values.CC BY · PubMed Central
Figure 3 The cytoplasmic pH of P. putida is altered by the pathway used for glucose consumption. Wild‐type P. putida KT2440 and its isogenic mutants in the enzymes glucokinase (Δglk) and glucose 2‐dehydrogenase (Δglk) were transformed with plasmid pS2513·PHP and grown in M9 minimal medium supplemented with glucose at 20 mM as the sole carbon source. The intracellular pH (pH i) was investigated during the exponential and stationary (24 h post‐inoculation) phase of growth. The pH i of wild‐type cells cultured in the presence of 20 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) is also shown in the figure. Independent triplicates of the experiments were carried out, and the level of statistical significance in the pairwise comparisons indicated in the comparisons across experimental conditions is indicated as ** P < 0.01 and *** P < 0.001 (ANOVA).
Figure 3 The cytoplasmic pH of P. putida is altered by the pathway used for glucose consumption. Wild‐type P. putida KT2440 and its isogenic mutants in the enzymes glucokinase (Δglk) and glucose 2‐dehydrogenase (Δglk) were transformed with plasmid pS2513·PHP and grown in M9 minimal medium supplemented with glucose at 20 mM as the sole carbon source. The intracellular pH (pH i) was investigated during the exponential and stationary (24 h post‐inoculation) phase of growth. The pH i of wild‐type cells cultured in the presence of 20 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) is also shown in the figure. Independent triplicates of the experiments were carried out, and the level of statistical significance in the pairwise comparisons indicated in the comparisons across experimental conditions is indicated as ** P < 0.01 and *** P < 0.001 (ANOVA).CC BY · PubMed Central
Figure 4 Monitoring the dynamic changes of cytoplasmic pH upon external pH shifts. Exponentially growing cells of (A) E. coli MG1655/pS2513·PHP or (B) P. putida KT2440/pS2513·PHP cultured in M9 minimal medium containing 20 mM glucose were aliquoted into a 96‐well microtiter plate and the cytoplasmic pH was continuously monitored, before and after a shift in the extracellular pH was induced by the addition of the same culture medium buffered at pH = 5.5 or pH = 8.5 as indicated in the Experimental Procedures , or sodium benzoate (permeant acid). Changes in cytoplasmic pH were monitored every 10 s during the first 5 min of incubation. Data shown in each case indicate mean values from independent triplicates ± SD.
Figure 4 Monitoring the dynamic changes of cytoplasmic pH upon external pH shifts. Exponentially growing cells of (A) E. coli MG1655/pS2513·PHP or (B) P. putida KT2440/pS2513·PHP cultured in M9 minimal medium containing 20 mM glucose were aliquoted into a 96‐well microtiter plate and the cytoplasmic pH was continuously monitored, before and after a shift in the extracellular pH was induced by the addition of the same culture medium buffered at pH = 5.5 or pH = 8.5 as indicated in the Experimental Procedures , or sodium benzoate (permeant acid). Changes in cytoplasmic pH were monitored every 10 s during the first 5 min of incubation. Data shown in each case indicate mean values from independent triplicates ± SD.CC BY · PubMed Central
Figure 5 Monitoring the changes of intracellular pH (pH i) in P. aeruginosa PA14 upon treatment with different antibiotics. Exponentially growing cultures of P. aeruginosa PA14 transformed with plasmid pS2513·PHP and grown in LB medium were challenged with one‐fifth (0.2×) and full (1×) minimal inhibitory concentrations of (A) ceftazidime (CAZ), (B) amikacin (AMK) and (C) ciprofloxacin (CIP) as indicated in the graphs. The data shown in each case indicate mean values of pH i from independent triplicates ± SD.
Figure 5 Monitoring the changes of intracellular pH (pH i) in P. aeruginosa PA14 upon treatment with different antibiotics. Exponentially growing cultures of P. aeruginosa PA14 transformed with plasmid pS2513·PHP and grown in LB medium were challenged with one‐fifth (0.2×) and full (1×) minimal inhibitory concentrations of (A) ceftazidime (CAZ), (B) amikacin (AMK) and (C) ciprofloxacin (CIP) as indicated in the graphs. The data shown in each case indicate mean values of pH i from independent triplicates ± SD.CC BY · PubMed Central
Figure 6 General procedure for in vivo assessment of intracellular pH (pH i) in Pseudomonas species by means of the PHP indicator. Plasmid pS2513·PHP is introduced into the bacteria of interest by chemical transformation, mating or electroporation. The expression of PHP is driven by the constitutive PEM 7 promoter, and vector pSEVA2513 carries the oriV(RSF1010) origin of replication, indicated as a purple circle. The kanamycin‐resistance (KmR) determinant and the T1 and T0 transcriptional terminators are likewise highlighted in the plasmid. Cells are grown in the appropriate culture medium, and a small culture sample is harvested by centrifugation. The cell pellet is resuspended in M9 medium salts to a final optical density at 600 nm (OD 600) = 0.4–0.5. To establish the calibration curve, a second culture is prepared in the same culture medium supplemented with carbonyl cyanide m‐chlorophenyl hydrazine (CCCP). Cells from this culture are also harvested by centrifugation and equilibrated in M9 medium salts adjusted to different pH values (ranging from pH = 5.5–8.5, see Experimental Procedures ) to a final OD 600 = 0.4–0.5. A 200‐μl aliquot of each cell suspension is transferred into a 96‐well microtiter plate, and OD 600 and fluorescence (λexcitation = 405 nm and 485 nm; λemission = 515 nm) are measured in a plate reader. Finally, the R 405/485 values are plotted versus the pH values of pH‐equilibrated cells (calibration curve), and the pH i is calculated using a Boltzmann sigmoid best‐fitting equation.
Figure 6 General procedure for in vivo assessment of intracellular pH (pH i) in Pseudomonas species by means of the PHP indicator. Plasmid pS2513·PHP is introduced into the bacteria of interest by chemical transformation, mating or electroporation. The expression of PHP is driven by the constitutive PEM 7 promoter, and vector pSEVA2513 carries the oriV(RSF1010) origin of replication, indicated as a purple circle. The kanamycin‐resistance (KmR) determinant and the T1 and T0 transcriptional terminators are likewise highlighted in the plasmid. Cells are grown in the appropriate culture medium, and a small culture sample is harvested by centrifugation. The cell pellet is resuspended in M9 medium salts to a final optical density at 600 nm (OD 600) = 0.4–0.5. To establish the calibration curve, a second culture is prepared in the same culture medium supplemented with carbonyl cyanide m‐chlorophenyl hydrazine (CCCP). Cells from this culture are also harvested by centrifugation and equilibrated in M9 medium salts adjusted to different pH values (ranging from pH = 5.5–8.5, see Experimental Procedures ) to a final OD 600 = 0.4–0.5. A 200‐μl aliquot of each cell suspension is transferred into a 96‐well microtiter plate, and OD 600 and fluorescence (λexcitation = 405 nm and 485 nm; λemission = 515 nm) are measured in a plate reader. Finally, the R 405/485 values are plotted versus the pH values of pH‐equilibrated cells (calibration curve), and the pH i is calculated using a Boltzmann sigmoid best‐fitting equation.CC BY · PubMed Central

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Metadata

Authors
Arce‐Rodríguez, A., Volke, D. C., Bense, S., Häussler, S., & Nikel, P. I.
Affiliations
  • Department of Molecular Bacteriology Helmholtz Centre for Infection Research 38124 Braunschweig Germany
  • The Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark 2800 Kongens Lyngby Denmark
ORCID
Venue
Microbial Biotechnology
Publisher
Wiley
Published
2019-06-04
Volume
12
Issue
4
Pages
799-813
ISSN
1751-7915
DOI
10.1111/1751-7915.13439
PMC
PMC6559197
Cited by
40
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Novo Nordisk Fonden (NNF10CC1016517)
  • Horizon 2020 Framework Programme (814418)
  • Communities Foundation of Texas (SRC 017)
  • H2020 European Research Council (COMBAT 724290)

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