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Simple Experiments To Demonstrate Proton Flux in Pseudomonas after Alkaline or Acidic Stress Gabriela Previtali, Walter Giordano,† and Carlos E. Domenech* Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, 5800 Río Cuarto, Córdoba, Argentina; *
[email protected] We previously reported a laboratory experiment to familiarize undergraduate biochemistry students with techniques that are used in the quality control of medicines (1). We have designed a new set of experiments to further expand the students’ capabilities in the biological science. Students are challenged with techniques related to microorganism manipulation, sterile conditions, and growth parameters that are useful to study the adaptive response to environmental stimuli. Since bacterial habitats encompass a wide range of environmental conditions, bacteria are useful species for teaching and investigating the environmental adaptation of living things. These microorganisms have developed different systems to adapt to fluctuations in their habitat conditions, for example, pH, temperature, and osmolarity (2, 3). Members of the Pseudomonas genus are able to sense, respond, and adapt to different stress conditions, including extreme pH, found in the natural environment or as pathogens in their host environment (4). Changes in pH may be important in controlling solvolysis, ionization, and redox reactions of several cellular components (3). Pseudomonas are neutrophilic bacteria and under extreme extracellular pH (pHo) conditions, the regulation of intracellular pH (pHi ) is expected to be extremely important because virtually every biological process is pH sensitive. The mechanism for the pHi maintenance involves proton efflux or influx that can be accomplished by different transport systems localized in the plasma membrane, such as Na+兾H+ or K+兾H+ antiporters, H+-ATPases, the respiratory chain, et cetera (3). This article describes the growth curves of P. fluorescens; however, other bacteria of the Pseudomonas genus could also be used. The growing conditions initially at neutral, alkaline, or acidic conditions show that bacteria adapt to pH stress in different ways. Experiments with resting cells initially suspended in neutral, alkaline, or acidic conditions also show that bacteria adapt to pH stress. Experimental Procedure
Microorganisms and Growth Conditions Nonpathogenic Pseudomonas such as P. fluorescens A (ATCC 13525), P. putida A (ATCC 12633), or P. cichorii, isolated from lettuce, were grown at 30 ⬚C. Bacterial growth was carried out in a rotatory bath (150 rpm) in a rich medium (LB) containing 1% (w兾v) peptone, 0.5% (w兾v) yeast extract, and 0.5% (w兾v) NaCl. The cultures were incubated in 250-mL Erlenmeyer flasks containing 25 mL of medium † Current address: Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095-1606
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(or 10% of the flask volume). The pH of the culture medium was adjusted to pH 8.5, 6.5, or 5.0 by the addition of 1 N NaOH or 1 N HCl. A 1% (v兾v) of inoculum from an overnight LB culture was used to start the growth curves. Bacterial growth was assessed by the increase of optical density at 660 nm (OD660nm) and pH of the culture medium measured in 1-mL aliquots at one hour intervals for nine hours.
Experiments with Resting Cells Resting cells are viable or metabolically active bacteria that are not able to duplicate owing to the absence of nutrients. In this work they were prepared as follows: Bacteria from a LB overnight culture were diluted in the same medium to obtain an OD660nm near 0.2; they were allowed to grow until an OD660nm of approximately 0.6 was achieved. At this point, bacteria were harvested by centrifugation at 10,000 × g for 10 min. The pellet was washed with cold physiological solution and kept in an ice bath. Immediately before starting the experiment, the pellet was suspended in the same solution to obtain an OD660nm between 0.4 and 0.6. The cellular suspension, 15 mL, was maintained at 30 ⬚C in a 25-mL thermostatic chamber (Figure 1). The cell suspension was adjusted to an alkaline (8.5) or acid (4.5) pH by the addition of 1 N NaOH or 1 N HCl, respectively. Changes in the pH over time were monitored using a pH meter with a calomel electrode. Control experiments were carried out with bacteria suspended at pH 7. The influx or efflux of protons was calculated for the first 2 or 3 min of pH determination, which correspond to the linear portion of the curves in Figure 2.
cellular suspension water circulation at 30 °C
magnetic stirrer
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Figure 1. Thermostatic chamber used to register changes in pH in the cellular suspension. The internal diameter and height of the chamber were 3 cm and 5 cm, respectively. Bacteria were maintained in a homogeneous suspension during the assay by slow movement of a magnetic bar. Flow of water was maintained constant at 30 ⬚C.
Journal of Chemical Education • Vol. 80 No. 12 December 2003 • JChemEd.chem.wisc.edu
In the Laboratory
Experiments were also conducted in the presence of the pH indicators, phenol red or bromcresol green. In this case, 0.4 mL of the indicator was added to 20 mL of cellular suspension contained in the thermostatic chamber. Phenol red, with a visual transition interval from pH 8.2 (red) to 6.8 (yellow), or bromcresol green, with a visual transition interval from pH 3.8 (yellow) to pH 5.4 (blue), were used for alkaline or acid studies, respectively. To prepare the solutions of both indicators, 0.1 g of the free acid of each dye was dissolved in 28.2 mL or 14.3 mL of 0.01 N NaOH, respectively; the volume was brought up to 250 mL with water. If the sodium salt of each indicator was utilized, 0.1 g was dissolved in 250 mL of water. Protein concentration was measured by the Coomassie blue method described by Stoscheck (5) or by the Bradford method (6). Calculations are described in detail by Efiok (7) and additional chemical information was obtained from the CRC Handbook of Chemistry and Physics (8). Hazards No significant hazards are associated with this experiment since this laboratory experiment is carried out with nonpathogenic bacteria of the genus Pseudomonas.
Results and Discussion Bacterial growth curves carried out at acid, neutral, and alkaline conditions are shown in Figure 3. Independent of the initial pH, in all cases the pHo was near 8 at the end of the experiment. Determination of the efflux or influx of protons was made with resting cells suspended in physiological solution initially at different pH levels. The increase or decrease of the pHo with time after incubation of bacteria at pH 4.5 or 8.5, respectively is shown in Figure 2. The results also show that the pH changes were more pronounced when the cells were subjected to alkaline stress. For example, the cell suspension initially adjusted to pH 8.5 decreased to pH 7.8 during the first 2 min; this is a change of 0.35 pH units in 1 min. Suspensions initially at pH 4.5 increased 0.25 pH units in 1 min. Resting cells suspended at pH 8.5 in physiological solution at an OD660nm between 0.5 and 0.6 excrete approximately 4–5 × 10᎑11 H+ min᎑1 (mg protein)᎑1 (see Supplemental Material for calculationsW). Measurement of pH changes using pH indicators is useful, because it allows visualization of the alkaline or acid bacterial adaptation in the conditions shown in Figure 2. In these
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Time / h Figure 3. Variation of (A) optical density and (B) pH over time of the culture during growth of Pseudomonas fluorescens in an LB medium adjusted to either an (ⵧ) alkaline, (䊊) a neutral, or ( 䉭) an acidic pH.
JChemEd.chem.wisc.edu • Vol. 80 No. 12 December 2003 • Journal of Chemical Education
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In the Laboratory
cases the visual transition of the pH indicators, red to yellow for the phenol red and yellow to blue for the bromcresol green, removes the need for a pH meter. The indicators showed the movement of protons to a large class with many students. In a laboratory class, the experiment was carried out in the same thermostatic chamber with both the electrode and indicator; it was noted that these chemicals did not affect the capacity of the bacteria to produce the efflux or influx of protons. The utilization of indicators only allowed an approximate calculation of the number of protons involved in the bacterial adaptation. The students were encouraged to compare the error introduced in this method to the error when using a pH meter. To calculate the output or input of protons the students considered the pH limit for the color change of each indicator. After these calculations were made, the results were discussed between the students and the teachers, and the students prepared a report that was structured like a scientific paper. The whole laboratory class experiment, including a preliminary explanation of the work, calculations, and discussion of the results, can be performed in two consecutive days. The growth study and adaptation of the microorganism under pH stress require eight hours of work on the first day. Experiments with resting cells, calculations, and the discussion of the results occur on the second day. If the growth curves are carried out separately and the laboratory class is performed only with resting cells from an overnight culture, the experiment can be completed in three hours. The experiment is relatively inexpensive and requires no sophisticated instruments. It has the advantage that the students, through the use of simple mathematical operations, learn about the quantity of protons that a cell utilizes to adapt to a hostile environment. Additionally, this laboratory class experiment may be carried out with other nonpathogenic bacteria of the genus Pseudomonas. Among others, we tested P. putida and P. cichorii and found similar results as those described for P. fluorescens. The advantage of using these bac-
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teria is that P. fluorescens and P. putida are saprophytic and nonpathogenic and P. cichorii, a plant pathogen, may be interesting to agronomy students. Students who are unfamiliar with handling microorganisms can utilize all the three bacteria with few risks. Acknowledgments We acknowledge Nancy Fujishige, University of California-Los Angeles, for help in the English preparation of the manuscript. WG and CED are Career Members of CONICET. W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online. Literature Cited 1. Giordano, W.; Domenech, C. E. Biochem. Educ. 1999, 27, 229–231. 2. Csonka, L. N. Microbiol. Rev. 1989, 53, 121–147. 3. Booth, I. R. Microbiol. Rev. 1985, 49, 359–378. 4. Pseudomonas: Molecular Biology and Biotechnology; Galli, E., Silver, S., Witholt, B., Eds.; ASM Press, American Society for Microbiology: Washington, DC, 1992. 5. Stoscheck, C. M. Methods in Enzymology. In Guide to Protein Purification; Deutscher M. P., Ed.; Academic Press, Inc.: San Diego, CA, 1990; Vol. 182, pp 50–68. 6. Protein Determination by the Bradford Method. http:// www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html (accessed Aug 2003). 7. Efiok, B. J. S. Basic Calculations for Chemical and Biological Analyses; AOAC International: Gaithersburg, MD, 1996; pp 31–42. 8. CRC Handbook of Chemistry and Physics, 78th ed.; Lide, D. R., Ed.; CRC Press: Boca Raton, FL, 1997–1998.
Journal of Chemical Education • Vol. 80 No. 12 December 2003 • JChemEd.chem.wisc.edu
