11: Protein Metabolism of Unknowns

Learning Outcomes

By the end of this lab period, you will be able to:

• Describe the pattern of protein metabolism for each of your organisms for the tests that we completed
• Articulate how pH indicators such as phenol red, bromocresol purple, phenol red, etc. are used to track protein utilization.
• Explain how differences in the expression of specific enzymes (such as nitrate reductase, gelatinase, and urease, etc.) can be used to differentiate between bacteria species.

Introduction

In today’s lab, we will be continuing with a series of biochemical tests that will help you further narrow down which organisms are your Gram-positive and Gram-negative unknowns. The focus of today’s tests is on the metabolism and breakdown of proteins, and the metabolism of nitrogen.

The tests that we will be setting up today are

1. Decarboxylase tests
   1. Lysine
   2. Ornithine
2. Phenylalanine deaminase
3. SIM
   1. Sulfur
   2. Motility
   3. Indole
4. Nitrate reduction
5. Gelatin hydrolysis
6. Urea hydrolysis

Decarboxylase Tests

Amino acids are structurally composed of an amino group (NH₂), a “carboxy” or carboxylic acid group (COOH), and an “R” group, which is the part of the molecule that is variable. Enzymes that remove the carboxylic acid group are called “decarboxylases”. Determining whether or not a bacteria makes a specific decarboxylase can be useful in their identification. These tests are useful for helping us ID our Gram-negative bacteria, but not helpful for Gram-positive, so well only perform decarboxylase tests on our Gram-negative.

Today, we will be testing for two decarboxylase enzymes, ornithine decarboxylase, and lysine decarboxylase. Decarboxylase broths contain the pH indicator bromocresol purple which turns yellow in acidic conditions, and purple in basic solutions. After inoculation, a layer of mineral oil is overlaid onto the broth, creating anaerobic conditions and inducing the bacteria to ferment glucose. Initially, this will result in the production of acid and the media will turn yellow. However, for those organisms that produce the decarboxylase enzyme, the presence of the amino acid in the media will turn “on” the gene for the enzyme, and result in the production of amines. These amines accumulate and create a basic environment turning the media purple. If no decarboxylase is produced, the medium will remain yellow.

Figure \(\PageIndex{1}\): The decarboxylase test. A. The decarboxylation reaction showing how the carboxylic acid is removed, forming carbon dioxide. B. Left - a negative decarboxylase result. Right - a positive decarboxylase result. Photo Credit - Celeste Towner, used with permission.

Phenylalanine Deaminase

In addition to enzymes that remove the carboxylic acid group, some bacteria have enzymes that can remove the amino group from specific amino acids. Determining if an organism can remove the amino group from phenylalanine is also a useful test for identifying Gram-negative organisms.

The phenylalanine deaminase test is done by inoculating a phenylalanine slant. If the bacteria make the enzyme, it will react with the phenylalanine and produce phenylpyruvic acid. Phenylpyruvic acid is colorless but will react with ferric chloride (FeCl₃) to produce a forest green color.

Figure \(\PageIndex{2}\): Phenylalanine deaminase test. A. General amino acid deamination B. phenylalanine deaminase produces phenylpyruvic acid C. Right - positive deamination reaction. Left - negative result. Photo credits: Ron John Vargas, (negative result), I.H. (positive result), used with permission

SIM Tests

The SIM tests, again only performed on our Gram-negative bacteria, are actually three tests in one tube. In this tube, we will test for sulfur reduction, the production of indole, and bacterial motility. Both sulfur and indole production are the result of amino acid metabolism. The observation of motility is just a happy “extra” that we can often glean from this test.

SIMs media is a type of “deep” and is inoculated with a needle.

Sulfur reduction

The amino acid cysteine contains a sulfur atom in its “R” group. Some bacteria make an enzyme called cysteine desulfurase, which removes that sulfur atom in the form of hydrogen sulfide gas. The hydrogen sulfide gas will react with ferrous ammonium sulfate (FeSO₄) to produce a black precipitate, ferric sulfide (FeS).

There are other mechanisms by which bacteria can reduce sulfur. The enzyme thiosulfate reductase can reduce sodium thiosulfate to form hydrogen gas as well. This will also result in the formation of a black precipitate.

Figure \(\PageIndex{3}\): SIMs media - detection of H₂S production. A. Production of hydrogen sulfide from cysteine metabolism B. Production of hydrogen sulfide from reduction of thiosulfate C. Reaction of hydrogen sulfide with iron sulfate. D. Reduction of sulfur in SIM media. Right - negative control. Left, positive reaction. Photo credits: Ron John Vargas (positive result) and Maithy Nguyen (negative result), used with permission

Indole

The enzyme tryptophanase hydrolyzes the amino acid tryptophan to pyruvate, ammonia, and indole. Indole will react with p-dimethylamino benzaldehyde (a component of Kovak’s reagent) to form a cherry red color, detected as a layer on top of the deep.

Figure \(\PageIndex{4}\): A. Tryptophanase catalyzes the breakdown of tryptophan into pyruvate and indole. B. Indole can be detected by adding Kovac’s reagent to the SIMs deep, resulting in a cherry red layer on the top. Left - indole positive tube. Right - negative result. Maithy Nguyen (negative result), used with permission

Motility

It is frequently serendipitous that we can determine motility from SIMs media. However, it is only possible to do this when sulfur is not reduced and the tube does not turn black. Because H2S gas can diffuse rapidly in the media, its presence usually results in a blackening of the entire tube, obscuring any motility observation.

If your organism is negative for sulfur reduction, hold the tube up to the light and look for growth that radiates away from the initial inoculation. This indicates that the bacteria are able to move through the media. If the media in the tube becomes cloudy as compared to an uninoculated control, this indicates that the organism is motile.

Figure \(\PageIndex{5}\): Motility in SIMs media. The tube on the right illustrates the cloudy radiating growth typical of a motile organism. The tube on the left is typical of a non-motile organism. A doubt, CC BY-SA 4.0, and A doubt, CC BY-SA 4.0, both via Wikimedia Commons. Both images cropped at top, bottom, and sides.

Nitrate Reduction Test

Many bacteria make an enzyme nitrate reductase that can reduce nitrate (NO₃) to nitrite (NO₂), while other organisms can reduce nitrate (NO₃) further, producing a set of possible intermediates and final products including ammonia (NH₃) and nitrogen gas (N₂).

Figure \(\PageIndex{6}\): Possible results of nitrate reduction. Many organisms “stop” at the production of nitrite, however ammonia, and nitrogen gas are also made by different organisms. Ammonia can be used by bacteria for amino acid synthesis. Denitrifiers, which are important ecologically can reduce nitrate all the way to nitrogen gas.

We identify organisms that have reduced nitrate to nitrite by the addition of two reagents (Nitrate Reagent A, and Nitrate Reagent B). If nitrite has been produced the nitrate broth will turn a deep blood red. That is a positive result and can be recorded.

However, because some organisms further reduce nitrite, it’s possible that your organism DID reduce nitrate, but there is simply no nitrite left in the broth because it has all been converted to any of the further endproducts or their intermediates shown in Figure \(\PageIndex{6}\). So how can you tell whether there was NO nitrate reduction, or that reduction continued past the production of nitrite? We do this by adding some powdered zinc to the nitrate broth. Zinc will react with any remaining nitrate, converting it into nitrite which will then react with Reagents A and B that you added previously. If the tube turns red after the addition of zinc, this is a negative result. If the tube does not turn red, then there was neither nitrate nor nitrite left in the tube, and this would be recorded as positive for nitrate reduction.

Figure \(\PageIndex{7}\): Schematic diagram showing testing for nitrate reduction and possible outcomes. If after the addition of Nitrate Reagent A and Reagent B, the tube turns red, this is a positive result. Zinc can be used to differentiate between tubes where no reduction has occurred and tubes where nitrate has been further reduced. Photo credit: Ron John Vargas, used with permission

Gelatin Hydrolysis

Earlier in the semester when we learned about agar, we discussed how gelatin was once used (or its use was attempted) to create solid substrates for growing bacteria. We learned that gelatin is a terrible choice because so many bacteria secrete enzymes called gelatinases that break down and liquefies the gelatin. The gelatin hydrolysis test differentiates between organisms that produce gelatinases, and those that do not. The gelatin test is performed in a “deep” and is inoculated with an inoculating needle.

Figure \(\PageIndex{8}\): Left - Gelatinases break the bonds between individual amino acids in the gelatin protein. Right - The presence of gelatinase will result in liquefaction of the gelatin. Top tube - a negative result. Bottom tube - a positive result.

Urea Hydrolysis

A common byproduct of the decarboxylation of amino acids is the urea molecule. Urea is a metabolic waste product, but the nitrogen in urea can be recycled by some microbes that make the enzyme urease. This enzyme is also a virulence factor in the pathogen Helicobacter pylori - it uses this enzyme to generate urea which neutralizes the acid in your stomach where it lives and reproduces.

Urea broth contains only a tiny amount of yeast extract for nutrition and strong buffers that prevent a color change from occurring unless enough ammonia is rapidly produced. Phenol red is the pH indicator, which is pink above pH 8.4. An orange or yellow result is negative.

Figure \(\PageIndex{9}\): A. The hydrolysis of urea results in the production of ammonia, which makes the broth more basic (raises the pH). B. A positive result is shown on the left. A negative result is shown on the right. Photo credit - I.H., used with permission

Materials

Day 1

• One Lysine decarboxylase broth (for Gram-negative only)
• One Ornithine decarboxylase broth (for Gram-negative only)
• Sterile mineral oil in microcentrifuge tubes
• One Phenylalanine deaminase slant (for Gram-negative only)
• One SIM deep (for Gram-negative only)
• Two nitrate broths
• Two nutrient gelatin deeps
• Two urea broths

Day 2

• 12% ferric chloride solution
• Kovac’s reagent
• Nitrate test reagents A and B
• Zinc powder
• Ice

Experiment

Day 1

1. Inoculate the lysine decarboxylase and ornithine decarboxylase broths with your Gram-negative organism. Overlay both tubes with 3 to 4 mm sterile mineral oil. Incubate at 37^oC for 96 hours (up to one week).
2. Inoculate the phenylalanine deaminase slant with your Gram-negative organism.
3. Use an inoculation needle to inoculate a SIM tube with your Gram-negative organism. Insert the needle into the agar to within 1 cm of the bottom of the tube. Be careful to remove the needle along the original insertion line.
4. Inoculate 2 nitrate broths, one with your Gram-positive and one with your Gram-negative organism.
5. Use an inoculation needle to stab inoculate the nutrient gelatin medium.
6. Inoculate 2 urea broths, one with your Gram-positive and one with your Gram-negative organism.
7. Place all freshly inoculated cultures in the 37^oC incubator, and incubate for 48 hours. If this is a 5-day incubation, incubate at 30^oC to avoid cultures growing past stationary phase into death phase.
8. Each week during the ID project you should be making fresh subcultures of your organisms and verifying purity by gram staining. Depending on how old your TSA slants are this can be done on Day 1 OR Day 2.

Day 2

1. Remove all of your cultures from the incubator. Carefully inspect each decarboxylase broth, and note whether the result was positive (purple), negative (yellow or no change of color), or indeterminate. Compare the colors with the uninoculated control tubes. Sometimes a result is weakly positive - make a note of this! Decarboxylation tests may need to be incubated for up to one week. Return the broth tubes back to the 37^oC incubator, and make one more observation in the next lab period if the tube is yellow or indeterminate.
2. For the phenylalanine deaminase test, add a few drops of 12% ferric chloride solution to the slant and observe for color change (green). (Note: this color may fade quickly, so record your results immediately).
3. For the SIM test, examine the tube for spreading from the stab line and formation of black precipitate in the medium. Record any H₂S production and/or motility. Add Kovac’s reagent to each tube to a depth of 2 mm - 3 mm. After several minutes, observe the formation of red color in the reagent layer and record the Indole result.
4. For the nitrate reduction test, examine each tube for evidence of gas production. Record your results. Proceed to the addition of reagents to all tubes as follows:
   1. Add 8 drops of reagent A and 8 drops of reagent B to each tube. Mix well, but carefully, and let the tubes stand undisturbed for 10 minutes. Record the tube that has red color as nitrate reduction-positive.
   2. For any tube that did not change color, add a pinch of zinc dust. Mix well, and let the tubes stand for 10 minutes. If the color turns red, record the organism as nitrate reduction-negative. If there is no color change after adding zinc, record the organism as nitrate reduction-positive.
5. For the gelatin hydrolysis test, place all tubes in the ice bath until the control tube is solid. Examine the inoculated medium for gelatin liquefaction. Tip over the tubes to take photos of your test tubes, be careful not to spill liquefied gelatin out the top. Record your results. The gelatin test may need to be incubated for up to one week. Keep the gelatin tubes, and make one more observation in the next lab period.
6. Observe the color of the urease test tubes, and record any tube that has turned pink as urease-positive.

Data

Your primary goal as you leave lab today is to have a complete and well-documented record of the results you collected today. Be sure to update your test table with the new data! Continue to develop your dichotomous keys and identify your unknowns.