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Keywords:

  • Streptococcus mutans;
  • glucose;
  • xylose;
  • metabolism;
  • thin layer chromatography

Abstract

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

The main objective of this class experiment is to show the time-dependent consumption of a fermentable sugar (glucose) in the presence and absence of a non-fermentable sugar (xylose) by bacteria in anaerobic conditions. The observation of the pH decrease due to acid production and the turbidity increase as a result of cell multiplication, both in function of time, enables an interesting discussion regarding the metabolic pathways and the products of anaerobic fermentation of carbohydrates. Also the time-dependent disappearance of glucose due to its metabolic consumption is compared with the non-disappearance of xylose and permits a discussion of the function of glycolysis and pentose phosphate pathways as metabolic routes.

This work was supported by the Universidade de Uberaba, Fundaçao de Amparo à Pesquisa do Estado de São Paulo, and Conselho Nacional de Pesquisas (Brazil).

The diverse fuels available in nature are metabolized by different organisms in different ways. For instance, glucose may be utilized aerobically, in which case this carbohydrate is transformed in CO2 and water giving a high energetic yield. Alternatively in anaerobic use the oxidation is incomplete, and the energetic yield is considerably lower. In the case of anaerobic use, by-products are released, and many of these by-products are of great commercial importance, such as ethanol (present in fuel and alcoholic beverages) and lactic/acetic acid (for industrial and domestic use) among others.

Class experiments for an introductory biochemistry course, which aims to demonstrate the metabolic diversity present in different organisms, require both the choice of an adequate biological model and the knowledge of its advantageous features and limitations. The bacteria Streptococcus mutans is present in the oral cavity and is one of the main organisms responsible for causing cavities. S. mutans is fast growing in a low cost and easy prepared culture medium, has a metabolic versatility reflected by the utilization of different carbon sources, and finally is readily studied biochemically [1]. S. mutans is an aerotolerant bacterium that does not have a respiratory chain and produces ATP by anaerobic glycolysis. In contrast to the aerobic organisms, the F0F1-ATPase in S. mutans is a proton-extruding enzyme and not an ATP synthesizer by H+ influx [2, 3].

The main objective of this experiment is to discuss the anaerobic utilization of glucose by S. mutans in a medium either supplemented exclusively with this sugar or mixed with a non-fermentable carbohydrate as well as the consequential acid production under these cultivation conditions.

BACKGROUND THEORY AND PRE-LABORATORY PREPARATION

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

D-Glucose is the major fuel of most organisms and occupies a central position in metabolism. Moreover it is also a remarkably versatile precursor capable of supplying a wide array of metabolic intermediates for biosynthetic reactions. A bacterium such as Escherichia coli can use glucose to obtain the carbon skeletons for every amino acid, nucleotide, co-enzyme, fatty acid, or other metabolic intermediate needed for growth. In a general way, glucose has three major fates.

  • It may be stored (as a polysaccharides or as sucrose),

  • oxidized to a three-carbon compound (pyruvate) via glycolysis, or

  • oxidized to pentoses via the pentose phosphate (phosphogluconate) pathway.

In glycolysis, a molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate. During the sequential reactions of glycolysis in the presence of oxygen, some of the free energy released from glucose is converted to ATP and NADH. For an integrated vision of aerobic glucose metabolism and of mitochondrial ATP formation, see Hanson [4] or Nicholson [5], respectively.

In anaerobic fermentation, glucose or other organic nutrients are degraded to obtain energy, which is conserved as ATP and occurs without the net production of NADH. Because living organisms first arose in an atmosphere without oxygen, anaerobic breakdown of glucose is probably the most ancient biological mechanism for obtaining energy from organic fuel molecules. In the course of evolution, the chemistry of this reaction sequence has been completely maintained, and both the amino acid sequence and three-dimensional structures of the glycolytic enzymes of vertebrates are closely similar. Glycolysis differs among species only in the details of its regulation and the subsequent metabolic fate of the pyruvate formed. Depending on the organism, lactate or ethanol can be formed anaerobically [6].

Other pathways, like the pentose phophate pathway (PPP),11 are also involved in the carbohydrate utilization. The PPP has two phases, called oxidative and non-oxidative, involved in supplying ribose for nucleic acid synthesis and in reducing potential (NADPH) for biosynthetic reactions, respectively. For more details, it is recommended that the student reads biochemistry textbooks [3, 6].

The simple experiment proposed here permits the discussion of essential aspects of anaerobic glucose metabolism and microbial growth. To profit from this practice, the students should know the basics of these topics.

GENERAL NOTES

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

In this class experiment, we describe a methodology adapted to the use of S. mutans, which can be acquired commercially. There is the possibility of isolating a lineage of S. mutans by inoculating a saliva sample in the selective culture medium MSBS (Mitis Salivarius Bacitracin Sucrose) [7]. Alternatively other acid-forming microorganisms could be used as well as other variations in the culture medium with the substitution of glucose by other sugars such as maltose or sucrose. Agents that inhibit specific enzymes of the glycolytic way, such as fluoride, can be added to the medium (5 μM final concentration), allowing the visualization of a delay and compromising the S. mutans growth.

Generally this class experiment uses some hazardous reagents such as sulfuric acid, methanol, and pyridine. These reagents should be manipulated with gloves and care in a fume cupboard. Their disposal should be made in accordance with local safety instructions.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

Strain—

The ATCC 25175 strain of S. mutans is available from the André Tosello Foundation, Campinas-SP (Brazil) (www.bdt.fat.org.br/). The lineage is stored at −20 °C in 40% (v/v) glycerol.

Liquid Medium—

The liquid medium used to cultivate the S. mutans strains was tryptic soy broth (TSB), prepared according to the manufacturer's instructions, which results in 13 mM dextrose, 17 g/liter pancreatic digest of casein, 3 g/liter papain digest of soybean meal, 5 g/liter NaCl, 2.5 g/liter K2HPO4. The medium (4 ml) was distributed in 15-ml Falcon tubes, which were autoclaved at 121 °C for 15 min. For the growth test in the presence of xylose as the only carbon source and for the tests without carbon source, the complete medium described by Dhasper and Reynolds [8] was used.

Anaerobic Growth Conditions—

All the cultivations were done in a candle jar in an anaerobic atmosphere obtained after lighting a candle inside the jar to consume part of the oxygen. A pre-inoculate was prepared by inoculating 50 μl of the S. mutans stock in 4 ml of TSB medium at 37 °C for 24 h. The stock was submitted to three successive transferences in TSB medium (12-h incubation) to reestablish their normal metabolism; this will be called the pre-inoculate. For growth assays, the initial turbidity of the pre-inoculate was adjusted to A600 nm = 0.5, corresponding to ∼109 cells/ml (for details see Ref. 9). Tubes containing 4 ml of previously autoclaved TSB medium were inoculated with 15 μl of this cell suspension. Cultures were grown in a microbiological oven and incubated in microaerobiosis (as cited above) at 37 °C. For each point of the curve (2-h intervals), two tubes were collected to measure the pH and turbidity of the culture medium. After this, the media were centrifuged at 3,000 × g for 10 min to obtain a medium free of cells, which was used in glucose dosage tests and sugar quantitative analysis by silica plate chromatography (thin layer chromatography (TLC)).

In another growth assay, 26 μl of xylose (2 M) was added to each tube containing culture medium (TSB or complete medium) to obtain a final concentration of 13 mM. This assay was submitted to the same conditions and tests as described above.

Xylose Solution—

The stock xylose solution, used to complement the TSB culture medium or the complete medium, was prepared at a final concentration of 2 M and autoclaved at 121 °C for 15 min.

TLC—

The silica plate for TLC (Sigma, 20 × 20 cm) was activated at 100 °C for 2 h before use. 0.5 ml of 30% (w/v) trichloroacetic acid was added to aliquots (1 ml) of the growing medium free of cells, and after mixing with a Vortex for 1 min the aliquots were kept overnight at 4 °C. Samples were centrifuged at 3,000 × g for 15 min, and 50 μl of the supernatant was vacuum-concentrated (10-fold) and applied to the TLC plate. As standards, 3 μl of glucose, xylose (50 mg/ml), or a mixture of these sugar solutions (25 mg/ml of each) were applied. The mobile phase was composed of n-butyl alcohol:pyridine:water:methanol in a 7:3:1:0.45 proportion (by volume). After chromatography, the plate was dried at room temperature in the fume cupboard and then sprayed with a solution containing 0.2% (w/v) orcinol in sulfuric acid/methanol (v/v) and revealed in a oven at 150 °C for 5–10 min. For details about the TLC method, see Ref. 10.

Glucose Assays—

For the glucose dosage, a commercially available kit was used (Glucox 500, Doles Reagentes) that uses the enzymes glucose oxidase and peroxidase in the presence of 4-aminoantipyrine and p-hydroxybenzoate. This reaction is based on the oxidation of the glucose molecule with the consequent production of a compound (quinoneimine) that is then spectrophotometrically quantified using Lambert-Beer's law [11]. Alternatively other glucose dosage kits (e.g. Sigma) can be used since they use the same dosage principle. A standard curve was built with increasing quantities of glucose (0.05–0.5 mg/ml) and used to determine a conversion factor (absorbance at 510 nm/μg of glucose). The presence of xylose does not affect the glucose determination by this method.

Cell Disposal—

The cells that were not used or remained from the experiment were autoclaved at 121 °C for 20 min and then discarded.

STUDENT PITFALLS

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

This is a very easy class experiment, but some problems such as culture contamination may occur as a result of bad manipulation. Some doubts can arise in calculating the conversion factor. The conversion factor is calculated by linear regression of the standard curve and corresponds to its angular coefficient. An absorbance value multiplied by this factor gives the glucose concentration present in the tube, which should be used to calculate the amount of glucose present in the growth medium.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

S. mutans is a bacterium normally present in the buccal microbiota, and its relation to the appearance of caries is evident. This relation is due to its high growth capacity and production of organic acids such as lactic, propionic, and acetic [1].

Fig. 1A shows a growing curve of S. mutans in media without carbon source and with glucose-, xylose-, and glucose plus xylose-supplemented media in anaerobic conditions. In the first 5 growing h (lag phase), the cellular multiplication, as measured by A600 nm, is very low. In this phase, the cells adapt to the culture medium (with respect to the possible nutrients present) and prepare an enzymatic apparatus for the next step of the culture growth. In media supplemented with glucose or with a mixture of glucose plus xylose, the microorganism reproduces itself in exponential scale over the following 7 h, and the turbidity of the medium increases in a logarithmic form (log phase). Finally, after 12 h of cultivation, a phase with no substantial growth will start (stationary phase) due to nutrient depletion and/or to the accumulation of toxic metabolites, which may be the final products of the anaerobic cellular metabolism, such as organic acids. The addition of xylose to the culture medium containing glucose did not affect the growth kinetics as shown in Fig. 1A.

In media in which the only carbon source is xylose, a small increase in turbidity 2 h after the lag phase is observed that soon stabilizes and leads to a stationary phase (Fig. 1A). This suggests that S. mutans does not metabolize xylose because a similar growth curve is obtained when the cultivation is done in a medium with no carbon source. The limited growth in these two conditions shows that the microorganism has intracellular energy reserves, possibly in the form of polysaccharides, as previously described [12]. These reserves would be enough to start growth, but they would soon be depleted.

In Fig. 1B it may be observed that the most significant pH decrease of the culture media occurs during the log phase of the culture media supplemented with glucose or with the mixture of glucose and xylose. Nevertheless, in the media without a carbon source or supplemented only with xylose, the decrease is less significant. In all cases, the pH decrease is mainly due to lactic acid production as a result of the anaerobic metabolism of the glucose by S. mutans [9]. As in media without the addition of carbohydrate or supplemented only with xylose the fermentable quantity of sugar is small (restricted to the intracellular reserves), and the acidification is significantly smaller. Acid production in vivo by bacteria adhering to the dental surface induces the demineralization of the tooth enamel and consequently the appearance of cavities [13, 14].

The amount of glucose present in the culture media was monitored during the growth period, and in Fig. 2 it is observed that during the time between the log phase (5–12 h) there is a sharp decrease in the amount of this sugar, which reaches minimum values at the beginning of the stationary phase (12 h) and is one of the factors that contributes to ending growth in this phase. The high consumption of glucose during the log phase clearly demonstrates the active metabolism of the bacterium, which is directed toward cellular activity maintenance such as active transport and cellular multiplication (Fig. 1A) and which also explains the high production of acid (Fig. 1B) during this phase. These observations are confirmed by TLC analysis of the culture media after different growth times (Fig. 3). The glucose is gradually consumed and disappears at the end of the log phase (lanes 1–4), while the xylose is detected during the whole period studied (lanes 5–8). Note that the mixture of glucose and xylose affects the migration of both carbohydrates (lanes 9–11). Thus, the progressive attenuation of spots 5–8 reflects the consumption of glucose, whereas the levels of xylose remain constant.

The lack of xylose consumption is interesting because this pentose could in principle be used through the PPP, whose major function is the production of reducing potential (NADPH) and the interconversion of sugars with carbon numbers between 3 and 7 [6]. However, S. mutans does not have the oxidative enzymes of the PPP and apparently uses ribose to supply carbon atoms for amino acid biosynthesis [15]. The lack of significant growth in the presence of xylose (Fig. 1) suggests that S. mutans is unable to transport this sugar inside the cell and/or convert this sugar in some PPP intermediate.

In conclusion, this class experiment shows that the anaerobic metabolism of S. mutans consumes fermentable sugars (glucose), resulting in acid production, and that the use of a given sugar in the environment depends on the enzyme and transport apparatus that the organism possesses.

LABORATORY TIME

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES

Our 2-year experience with different student classes shows that two periods of 8 h separated by an overnight interval are adequate to carry out the experiment as described. An assistant can prepare and autoclave the culture medium and inoculate 12 h in advance, leaving the students the inoculate of shorter times.

In addition to the study of glucose, the use of other sugars by bacteria can be subjected to the same analysis. Using other sugars does not increase the laboratory time. Another possibility is the addition of agents that affect the cellular metabolism such as antibiotics or fluoride in the culture medium. Fluoride will inhibit the glycolytic pathway, causing an increase of the lag phase of growth from 5 to ∼12 h. This effect is due at least in part to enolase inhibition [16, 17].

STUDY QUESTIONS

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES
  • What is the metabolic status of the cells in the lag, log, and stationary phases in the bacterial growth?

  • How is glucose transformed into lactic acid by lactic fermentation? How is the carbohydrate transported to the cell interior?

  • Locate in the pentose phosphate pathway the reactions involved in the reduction potential production and the reactions related to the sugar interconversion with three to seven carbons.

  • Which factors can stop the fermentation of a sugar by a given organism?

  • What is the reason for sugar separation through the TLC plate?

  • What is the role of fluoride in retarding the glucose metabolism?

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Figure FIGURE 1.. Growth curves of S. mutans showing the absorbance at 600 nm (A) and pH (B) variation as a function of cultivation time in TSB medium (—) or complete medium (− − − −) without carbon source (▵), with 13 mM glucose (○), with 13 mM xylose (▪), or with 13 mM glucose plus 13 mM xylose (▴). Each point is the mean of three experiments. For details of the assay, see “Experimental Procedures.”

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thumbnail image

Figure FIGURE 2.. Glucose concentration as a function of S. mutans growth time in TSB medium supplemented (▴) or not (○) with 13 mM xylose.Inset, standard calibration curve using the glucose assay kit. Each point is the mean of three experiments. For details of the assay, see “Experimental Procedures.”

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thumbnail image

Figure FIGURE 3.. TLC of reference carbohydrate and culture medium of S. mutans after growth in medium supplemented with 13 mM glucose (lanes 1–4) for 4, 8, 12, and 24 h, respectively, or with 13 mM glucose plus 13 mM xylose (lanes 5–8) for 4, 8, 12, and 24 h, respectively. Lanes 9, 10, and 11 contain glucose, xylose, and glucose plus xylose mixture standards, respectively. For details, see “Experimental Procedures.”

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  • 1

    The abbreviations used are: PPP, pentose phophate pathway; TSB, tryptic soy broth; TLC, thin layer chromatography.

REFERENCES

  1. Top of page
  2. Abstract
  3. BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
  4. GENERAL NOTES
  5. EXPERIMENTAL PROCEDURES
  6. STUDENT PITFALLS
  7. RESULTS AND DISCUSSION
  8. LABORATORY TIME
  9. STUDY QUESTIONS
  10. Acknowledgements
  11. REFERENCES
  • 1
    K. W. Stephen, I. G. Chestnutt, T. W. MacFarlane (1994) An in vitro investigation of the cariogenic potential of oral Streptococci, Arch. Oral. Biol. 7, 589593.
  • 2
    B. Alberts, D. Bray, J. M. Lewis, M. Raff, K. Roberts, J. D. Watson (1994) Molecular Biology of the Cell, 4th ed., Garland Publishing Inc., New York, pp. 792793.
  • 3
    J. D. Rawn (1989) Biochemistry, Neil Patterson Publishers, Burlington, NC, pp. 379406.
  • 4
    R. W. Hanson (2002) Metabolic minimaps of glycolysis and gluconeogenesis and their regulation, designed by Donald E. Nicholson, Biochem. Mol. Biol. Educ. 30, 35.
  • 5
    D. Nicholson (2002) Mitochondrial ATP formation, Biochem. Mol. Biol. Educ. 30, 221223.
  • 6
    D. L. Nelson, M. M. Cox (2000) Lehninger Principles of Biochemistry, 3rd ed., Worth Publishers, New York, pp. 542683.
  • 7
    J. Van Houte, G. G. Olga, H. V. Jordan (1973) A selective medium for Streptococcus mutans, Arch. Oral Biol. 18, 13571364.
  • 8
    S. G. Dashper, E. C. Reynolds (1990) Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutans Ingbritt, J. Bacteriol. 172, 556563.
  • 9
    D. S. Harper, W. S. Loesche (1984) Growth and acid tolerance of human dental plaque bacteria, Arch. Oral. Biol. 10, 843848.
  • 10
    M. F.Chaplin, J. F.Kennedy, Eds. (1987) Carbohydrate Analysis, a Practical Approach, IRL Press, Oxford, pp. 113.
  • 11
    D. C. Harris (1997) Qualitative Chemical Analysis, 5th ed., Freeman, New York.
  • 12
    R. B. Bridges (1977) Ribose biosynthesis in Streptococcus mutans, Arch. Oral Biol. 22, 139145.
  • 13
    M. R. Wegman, A. D. Eisenberg, M. E. Curzon, S. L. Handelman (1984) Effects of fluoride, lithium, and strontium on intracellular polysaccharide accumulation in S. mutans and A. viscosus, J. Dent. Res. 63, 11261129.
  • 14
    S. W. Leung, C. F. Schachtele (1975) Effect of sugar analogues on growth, sugar utilization, and acid production by Streptococcus mutans, J. Dent. Res. 3, 433440.
  • 15
    G. Rolla (1989) Why is sucrose so cariogenic? The role of glucosyltransferase and polysaccharides, Scand. J. Dent. Res. 97, 115119.
  • 16
    R. J. Doyle, S. D. M. Cox, M. O. Lassiter, B. S. Miller (1999) A new mechanism of action of fluoride on streptococci, Biochim. Biophys. Acta 1428, 415423.
  • 17
    G. N. Jenkins (1999) Review of fluoride research since 1959, Arch. Oral. Biol. 44, 985992.