Unit 10: Protein Catabolism

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Unit 10: Protein CatabolismBy Heather Fitzgerald, Karen Bentz and Patricia G. Wilber.Copyright Central New Mexico Community College, 2015

Introduction

The thought of rotting meat usually brings up a gagging sensation for most people. Similarly, the smell of a human cadaver is not associated with pleasant smells. Nonetheless, in part, the smell of the decaying cadaver comes from the aptly named molecules, cadaverine and putrescine (putrid!). Cadaverine is produced when microbes break down (catabolize) animal protein to acquire nutrients and energy for DNA replication and growth. Protein catabolism by microbes is certainly important so microbes can acquire energy from sources other than carbohydrates and fats, and it is also critical ecologically. If microbes did not break down animal proteins, the world would be littered with undegraded carcasses!

Breakdown of specific proteins requires microbes to contain specific genes for specific enzymes (DNA RNAprotein). While there are many different enzymes involved in converting proteins to other molecules, we will focus on a few commonly analyzed microbial enzymatic processes.

Proteins consist of amino acids covalently bonded together (See Figure 10-1). There are 21 naturally occurring (20 are common, one is rare) amino acids (e.g. tryptophan(TRP) and lysine(LYS)). Each amino acid contains a central carbon atom covalently bonded to both an amino group (NH2) and a carboxylic acid group (COOH) as well as a side chain that varies in structure (R; R stands for “the Remainder”). The side chain or R-group, is the unique part of each amino acid and is the unit that gives each of the 21 different amino acids their unique properties

Figure 10-1. Basic structure of proteins (amino acids joined via covalent bond).

Accessed 8/17/2015 from https://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg

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What part of a protein is targeted for breakdown?

Usually, when enzymes cut the protein chain, they work on a specific amino acid and are named for the particular bond site that they are able to break. Decarboxylases are enzymes that remove the carboxyl group from amino acids by breaking the bond between the central carbon and the carboxyl group. (See Figure 10-2 for examples of general enzyme cut sites.) The specific enzyme lysine decarboxylase, removes the carboxyl group from the amino acid lysine. Deaminases are enzymes that remove a molecule’s amine group. Phenylalanine deaminase removes the amino group from the amino acid phenylalanine. Other enzymes we will study in this lab react with particular atoms in the side chains of proteins. For instance, the cysteine desulfhydrase enzyme in the SIM test (described below) will remove the sulfur atom from the R-group in the amino acid cysteine and the tryptophanase enzyme will remove the indole group and the amino group from the amino acid tryptophan. Finally, we will study an enzyme that deals with urea. Urea is produced by humans (and other organisms) when proteins are metabolized for energy and nutrients. The amino group (which contains nitrogen) on the amino acids in proteins is toxic, so urea is produced and we excrete it in our urine. Some microbes produce the enzyme urease. The Urease enzyme will convert the urea found in urine to ammonia and allows the bacteria that produce urease to successfully colonize the urinary tract

While carbohydrate usage generally produces an acid or neutral waste, the break down products of protein digestion generally increase the pH of the environment by producing basic end products. Just as in the carbohydrate unit, pH indicators (that change color) are used to visualize the pH change. The changes in pH in the growth media of a bacterium can provide information about what type of catabolism has occurred.

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Where Deaminases cut

Where Decarboxylases cut

Cysteine desulfhydrase works on the R group of cysteine

Tryptophanase works on both the R group tryptophan and the amine group (tryptophan).

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Figure 10-2. A generalized structure of an amino acid and target sites for enzymes.

Accessed 8/15-15 from https://commons.wikimedia.org/wiki/File%3AAminoAcidball.svg. By GYassineMrabetTalk This vector image was created with Inkscape. (Own work). Modifications by H. Fitzgerald.

Specific Protein Catabolism Tests

Lysine Decarboxylase test.

The lysine decarboxylase test shows us if the bacteria produce the enzyme lysine decarboxylase (LDC). Lysine decarboxylase cleaves the carboxyl group from the amino acid lysine (located on a protein) and produces the breakdown product cadaverine. Cadaverine has a strong odor that is commonly associated with rotting animal carcasses (cadavers!).

The differential agent in the LDC test is the amino acid lysine and the pH indicator is Bromocresol purple. When LDC is present conditions become basic (high pH) and Bromocresol purple becomes purplish (a positive test result). When LDC is absent, the media turns yellow (pH less than 6.8; low pH). Uninoculated media is a brownish/purple color. Glucose and peptones are also in the media.

The LDC enzyme requires an acidic environment to become activated, so placing mineral oil over the broth and capping the tube lids tightly reduces oxygen and encourage fermentation of the glucose to produce the acidic environment. When this occurs the tube will first turn from brownish/purple to yellow. If the tubes remains yellow this indicates the bacteria DO NOT contain the LDC enzyme. If the enzyme is produced by the bacteria being tested, the enzyme will be activated by the low pH. When LDC cleaves the carboxyl group off the lysine, this generates cadaverine and other basic (high pH) end products which will raise the pH of the broth back above pH 6.8 and turn the pH indicator

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Photo Accessed 8/17/15 from http://www.microbelibrary.org/library/laboratory-test/3006-biochemical-test-media-for-lab-unknown-identification-part-1.

Lysine DecarboxylaseLysine Cadaverine

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(bromocresol purple) back to purple. Therefore a true purple result is a positive reaction indicating the presence of the LDC, and a yellow or original brownish/purple color is a negative result indicating growth of the bacteria, but the lack of LDC. See Figure 10-3.

Figure 10-3. Example results of Decarboxylase test. The leftmost tube shows a positive decarboxylase test, while the two right most tubes show negative results. The browinish tube in the middle shows the color of an uninoculated tube, while the bright yellow tube indicates a true negative result.

Phenylalanine Deaminase test.

This test will help distinguish among bacteria by determining whether or not they contain the enzyme phenylalanine deaminase. In particular, this test helps to distinguish Proteus species (which contain this enzyme) from bacteria that do not. Bacillus megaterium is also very weakly positive in this test. This enzyme cleaves off the amino group from the amino acid Phenylalanine. In doing so, the major products formed are phenylpyruvic acid and ammonia.

This test is performed using an agar slant medium. The differential material is the amino acid phenylalanine and after incubation a reagent called ferric chloride (FeCl3)(10%) is added to detect if the phenylpyruvic acid was produced, or not. If phenylpyruvic acid was produced, a light to dark green, color will appear, and we know the organism produces the enzyme phenylalanine deaminase. . The medium also contains yeast extract (for carbon and nitrogen. See Figure 10-4 for examples of positive and negative results after the addition of the ferric chloride solution.

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Accessed 8/17/15 from .

Phenylalanine DeaminasePhenylalanine Phenylpyruvic acid, Ammonia

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Figure 10-4. Examples of positive and negative results in the phenylalanine deaminase test.

Urease Test

Ureases are enzymes that will hydrolyze urea to ammonia and carbon dioxide. Urea is a nitrogen containing waste product found in animal urine. Detection of urease activity (which allows survival of bacteria in the gastric and/or urinary systems) is a key diagnostic for potential human pathogens. The ulcer-causing bacteria, Helicobacter pylori uses urease to produce ammonia and help de-acidify its local stomach environment. Some Proteus species utilize urease to increase the alkalinity of the urine. This allows the bacteria to survive in the urea-rich environment of the urinary tract. Infection with Proteus species can lead to inflammation and contribute to urinary tract stone formation. Thus urease producing bacterial species may be pathogens.

The urease test is done using an agar-based slant medium. The slant contains the differential material urea, and the pH indicator, phenol red. Phenol red turns yellow when the pH is less than pH 6.8 (acidic), and is a bright pink at a pH ≥ 8.4 (basic/alkaline). Since ammonia is a product of urease activity and ammonia is basic, a bright pink slant is a positive result, revealing that the bacteria produces urease. Peptone and other essential growth factors are also in the media.

If at least 80% of the slant and butt, is pink after 24 hours, there is rapid positive urease activity. If there is pink only along the slant after 24 hours, this is considered weak or delayed activity. These tubes may become pinker over time. A yellowish colored tube that remains roughly the same color as uninoculated media indicates a negative urease result. See Figure 10-5 for examples.

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Urease Urea Ammonia + CO2

Accessed 8/17/15 from . Cultures and image description from Benita A. Brink, Adams State College, Alamosa, CO.

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Figure 10-5. Example results of a Urease test. A rapidly positive urease test by Proteus mirabilis (b) is indicated by a color change to bright pink (fuchsia) throughout the urea agar slant as compared to the uninoculated control (a). A delayed positive reaction by Klebsiella pneumoniae is indicated by a color change only along the slant (c). A negative reaction by Escherichia coli is indicated by the yellow coloration of the media (d). All samples were incubated at 37oC for 16 hours.

Sulfur, Indole and Motility (SIM) test.

The SIM tests analyze three different bacterial characteristics:

(S) The ability of bacteria to produce H2S using cysteine desulfhydrase or thiosulfate reductase (same as the TSI).

(I) The ability to split the indole group off of the amino acid tryptophan by the enzyme tryptophanase.

(M) The ability of the bacteria to move (be motile) in the media, due to the presence of flagella.

The SIM test is done using a semi-solid agar deep. The semi-solid consistency allows motile bacteria to move through the media, away from the stab line.

The SIM is a basic T-soy semisolid agar medium made with peptone (a source of cysteine) and beef extract, thiosulfate, and ferrous sulfate (a source of iron, Fe+2). For examples of expected results for each of the three components being assayed see Figure 10-6 below.

The (S) or sulfur component of this test identifies the production of hydrogen sulfide (H2S). If a bacteria produces hydrogen sulfide (H2S), it will react with the iron ions in the ferrous sulfate (an ingredient in the media) to produce a black color in the bottom of the tube.

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One means of producing H2S is using the enzyme cysteine desulfhydrase. The cysteine desulfhydrase enzyme reduces the sulfur in the amino acid cysteine (found in the peptone, an ingredient in the media) and produces the products of H2S, pyruvate and ammonia.

H2S can also be produced by organisms that produce thiosulfate reductase. Thiosulfate reductase reduces thiosulfate (an ingredient in the media) to H2S.

SIM media does not allow us to distinguish between these types of sulfide production.

Once H2S is produced by either means, the H2S reacts with the ferrous sulfate in the media to produce a black color in the butt of the media. The black can obscure the motility results, but fortunately, organisms that produce a black in the SIM are also motile.

The black in the SIM is produced by the same mechanisms as the black in the TSI.

The black indicates that the organism is a potential pathogen.

The indole (I) component of this test analyzes bacteria for the presence of the enzyme tryptophanase. Tryptophanse is a deaminase enzyme that breaks down proteins that contain the amino acid tryptophan (an ingredient in the media) to produce pyruvate, indole and ammonia (amino group). Some studies suggest that indole is a chemical signaling molecule that helps with bacterial multiplication and possibly contributes to biofilm formation. Among organisms that infect the respiratory tract, such as Haemophilus influenzae (the bacteria, not the flu virus), the ability to produce tryptophanase and break down tryptophan to produce indole is positively correlated with higher pathogenicity.

The production of indole is observed by the reaction of the indole end product with Kovak’s reagent. After incubation, Kovac’s is applied to the top of the deep. If a red ring occurs then the bacteria is positive for the production of indole and thus positive for tryptophanase. If the ring remains clearish or yellow, the result is negative for indole production and thus negative for tryptophanase.

The (M) test for motility indicates the presence of flagella (see Unit 5) on bacteria. If bacteria have flagella, then they are able to “swim” away from the inoculation site in a 360 degree manner and undergo binary fission. This produces a fuzzy haziness extending in all directions (three dimensionally) from the stab line. If whitish growth is only observed in only two dimensions along the stab line this is a negative result. Again, all known organisms that can produce H2S in the (S) test are also motile. Motility is often a sign of pathogenicity. Bacteria that produce flagella can more readily move from the site of infection in the body to other locations.

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Figure 10-6. Example SIM test results. Tube A is uninoculated. Note that the stab line is not present. Tube B has growth from the stab line in a two dimensional pattern but no real change in haziness compared to Tube A, the uninoculated tube. The lack of haziness and the 2D growth pattern indicates a negative test for motility. Inoculated tubes should be compared to an uninoculated tube to discriminate between cloudiness (motility) and lack of motility. Tube B is also negative for indole. Tube C has a red color on the surface of the medium after addition of Kovac’s reagent. This indicates indole production and the production of tryptophanase by the bacteria. Tube C also has fuzziness/cloudiness spreading three dimensionally away from the stab. This indicates motility—the presence of flagella. Tube D is black, which indicates H2S production via the digestion of cysteine by cysteine desulfhydrase (or reduction of thiosulfate) and therefore is also positive for motility. D is negative for indole production.

.

Accessed 8/18/15 from http://www.microbelibrary.org/library/2-associated-figure-resource/3645-sim-medium. Image and results interpretation provided courtesy of Renee Wilkins, University of Mississippi Medical Center, Jackson, MS.

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TryptophanaseTryptophan Indole, ammonia Cysteine Desulfhydrase Cysteine H2S, pyruvate, ammonia

Thiosulfate reductaseThiosulfate H2S

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DAY ONE: INOCULATIONS

Lysine Decarboxylase https://www.youtube.com/watch?v=-RtPXdDQVjI Phenylalanine Deaminase https://www.youtube.com/watch?v=msBI0_Fb_nE SIM https://www.youtube.com/watch?v=tXNGV6MaNVw

Urease Test https://www.youtube.com/watch?v=1MCWEPhxLZU

 Videos created by Corrie Andries.

Materials

Media (per pair of students)o 2 Lysine broth tubeso 3 Phenylalanine slants o 3 SIM deep tubeso 3 Urea slant tubes

Bacteria Cultureso Escherichia coli (Ec)o Proteus vulgaris (Pv)o Klebsiella pneumoniae (Kp)o Bacillus megaterium (Bm)

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Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links: Video Links:

Ec or Kp Bm or Pv

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Procedure

A. Lysine Decarboxylase Test

Image created 2015 by H. Fitzgerald and K. Bentz

1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image above.

2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial species.

3. Dip the loop into the broth and swirl the loop around.

4. Resterilize the loop and place it back into the tools container on the lab bench.

5. Add around 2mm (about 5 drops) of mineral oil (make sure NOT to use the microscope objective oil for this) to the top of the broth.

6. Tightly (!!) cap the tubes and then place into the appropriate inoculation tray.

7. Repeat this procedure for each of the other three bacterial species.

Precautions: In order for the decarboxylases to be well activated the environment must first become acidic. This will only happen in low oxygen conditions so make sure to add the oil after you inoculate the tubes, and tighten the caps!!

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B. Phenylalanine Deaminase Test

1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image above.

2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial species.

3. Create fishtail streak on the surface of the slant.

4. Resterilize the loop and place it back into the tools container on the lab bench.

5. Loosely cap the Phenylalanine tube and place it into the appropriate inoculation tray.

6. Repeat this procedure for each of the other three bacterial species.

7. Remember that on day #2 you will need to add 10% ferric chloride solution to each tube.

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ChoicePvBm

Kp or Bm Pv Ec

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C. Urease test

Figure 10-8. Inoculation of Urea slants with four different bacteria.

Image created 2015 by Karen Bentz and modified by H. Fitzgerald

1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image above.

2. Use aseptic technique and a cooled, sterile metal loop to acquire a small amount of one bacterial species.

3. Make a fishtail streak on the surface of the slant.

4. Resterilize the loop and place it back into the tools container on the lab bench.

5. Loosely cap the Urea tube and place it into the appropriate inoculation tray.

6. Repeat this procedure for each of the other three bacterial species.

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D. SIM test

Figure 10-7. Inoculating each of three SIM semi-solid deeps with a different bacteria as shown

1. Label your tubes appropriately. Use one tube for each bacterial species, as indicated in the image above.

2. Use aseptic technique and a cooled, sterile metal needle to acquire a small amount of one bacterial species.

3. Stab the SIM deep tube about 2/3 of the way into the media. Put the needle in and out as straight as possible.

4. Resterilize the needle and place it back into the tools container on the lab bench.

5. Loosely cap the SIM deep tube and place into the appropriate inoculation tray.

6. Repeat this procedure for each of the other three bacterial species.

7. Remember that on Day 2, these tubes will to have Kovack’s reagent added to observe the indole result.

Precautions

If your needle wire is crooked, use a gloved hand to get it as straight as possible Be careful not to stab all the way to the bottom of the media.

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choicePvEc

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DAY TWO: Results and Interpretation

Procedure:1. Collect the media you inoculated in the previous lab.

2. Add 4 drops of Kovak’s reagent to your SIM tubes.

3. When you are ready to observe your phenylalanine tubes (PA), add 4 drops of FeCl3 reagent to each of your PA tubes.

4. Observe any color changes, growth patterns or other visible changes to the inoculation tubes. Fill in your observations in the tables below. Be aware that B. megaterium is likely to VERY WEAKLY green in the PA tube after addition of the FeCl3 reagent ONLY after about 5-10 minutes.

A. Lysine Decarboxylase Media Results (and a little interpretation)Name of Bacteria If the Bacteria Grew, What Color is the Media?

(purple=positive; not purple=negative)

Insert Photos of the Lysine Decarboxylase test results here:

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Insert photos of your Phenylalanine deaminase test results here:

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B. Phenylalanine Deaminase Media Results (and a little interpretation)

Name of Bacteria If the bacteria grew, after addition of ferric chloride, what color is the liquid in the media?

(green=positive; not green, clearish=negative)

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Insert Photos of the Urease test results here:

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C. Urease Media Results (and a little interpretation)

Name of BacteriaIf the Bacteria Grew, What Color is the Media?

(pink all over = strong positive; pink slant and yellow butt= delayed positive; yellow or original color all over=negatvie)

Slant color Butt color

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D. SIM Media Results (and a little interpretation)

Name of BacteriaSulfur

(black ppt.= positive; no black = negative)

Indole(red ring at top=positive;

no ring = negative

Motility(cloudiness or black ppt. away from the

stab line=motility)

Insert Photos of the SIM test results here:

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InterpretationBased on all of your protein catabolism results, explain what you know about the enzymes and motility of each bacterial species you tested. If you didn’t use the test for that species, leave rows blank

Bacterial Species: ___________________________________

Which, if any, Protein Degrading Enzymes does this Bacteria

Produce?(Cysteine Desulfhydrase, Thiosulfate reductase, Tryptophanase, Urease, Phenylalanine Deaminase, Lysine

Decarboxylase)

What evidence from which media supports your enzyme

choice?

Does this Bacterial Species have Flagella?

Evidence?

What is the Evidence (if any) for this Organism

being a Possible Pathogen?

Enzyme:

Enzyme:

Enzyme:

Enzyme:

Add more rows if you need more room.

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Bacterial Species: ___________________________________

Which, if any, Protein Degrading Enzymes does this Bacteria

Produce?(Cysteine Desulfhydrase, Thiosulfate reductase,Tryptophanase, Urease, Phenylalanine Deaminase, Lysine

Decarboxylase)

What evidence from which media supports your enzyme

choice?

Does this Bacterial Species have Flagella?

Evidence?

What is the Evidence (if any) for this Organism

being a Possible Pathogen?

Enzyme:

Enzyme:

Enzyme:

Enzyme:

Add more rows if you need more room.

Bacterial Species: ___________________________________

Which, if any, Protein Degrading Enzymes does this Bacteria

Produce?(Cysteine Desulfhydrase, Thiosulfate reductase,Tryptophanase, Urease, Phenylalanine Deaminase, Lysine

Decarboxylase)

What evidence from which media supports your enzyme

choice?

Does this Bacterial Species have Flagella?

Evidence?

What is the Evidence (if any) for this Organism

being a Possible Pathogen?

Enzyme:

Enzyme:

Enzyme:

Enzyme:

Add more rows if you need more room.

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Bacterial Species: ___________________________________

Which, if any, Protein Degrading Enzymes does this Bacteria

Produce?(Cysteine Desulfhydrase, Thiosulfate reductase,Tryptophanase, Urease, Phenylalanine Deaminase, Lysine

Decarboxylase)

What evidence from which media supports your enzyme

choice?

Does this Bacterial Species have Flagella?

Evidence?

What is the Evidence (if any) for this Organism

being a Possible Pathogen?

Enzyme:

Enzyme:

Enzyme:

Enzyme:

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Add more rows if you need more room.

Post-Activity Questions1. What is the unifying feature of the enzyme tests described in this unit?

2. Indicate with arrows where each of the following enzymes breaks a bond in the amino acid.

Lysine Decarboxylase

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Tryptophanase

Cysteine Desulfhydrase

Phenylalanine Deaminase

3. Kovac’s reagent is added to the __________ media and will form a ______ ring at the top of the media if the result is positive.

4. FeCl3 is added to the _______________________ media and will turn a __________ color if the result is positive.

5. The pH indicator in the lysine test is _____________ _____________. A positive lysine test will be a ___________ color.

6. SIM stands for _________, ___________ and __________.a. Describe what a positive test for sulfur would look like:

b. A positive indole test would have a:

c. If a bacteria is motile, it will have one or more _________ and can move through the media away from the ______________ _________.

7. The pH indicator in the Urease media is _________________ and a positive result will be a ____________ color.

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8. List the differential material(s) (if any) for each test.

S

I

M

Phenylalanine Deaminase

Lysine Decarboxylase

Urease

Table 10-1. Protein Catabolism

Media Ingredients What Positive/Negative Results Look Like

Interpretation if Results are Positive

Of Special Note

SIM deep

Inoculate with a stab into the media

T-soy semisolid agar plus:

- beef extract- peptones (source of cysteine)- thiosulfate- ferrous sulfate as a source of iron to indicate H2S production

1. S A black precipitate in the butt of the tube is positive

2. I Presence of a red ring at the top of the media after Kovac’s reagent is added is positive

3. M Cloudiness away from the stab line or BLACK is positive

1. S Black. The bacteria digest cysteine using the enzyme cysteine desulfhydrase to H2S or reduces thiosulfate using the enzyme thiosulfate reductase to produce H2S. H2S reacts with ferrous sulfate to produce the black precipitate.

2. I The bacteria digest tryptophan to indole, indicating the presence of tryptophanase (a deaminase that also works on the R-group)

3. M The bacteria produces one or more flagella

1. S&M If a bacteria produces a black precipitate, it also has flagella. Flagella allow motility and contribute to pathogenicity.

2. I Tryptophan is digested to indole, which reacts with Kovac’s reagent for form a red ring. Indole can contribute to pathogenicity.

Urease slant

Use a fishtail inoculation on the surface of the slant

T-soy agar plus:

- Urea- Phenol red pH indicator

Color change to bright pink (fuchsia) is positive. Fuchsia color throughout indicates rapid activity of urease, fuchsia color only on the top of the slant indicates weak or delayed activity of urease.

If the agar remains yellowish, the test is negative

The bacteria produce the enzyme urease that converts urea, a waste product of protein digestion, to ammonia and CO2. This raises the pH and the Phenol red indicator turns a bright pink.

A positive urease result suggests an organism can live in the urinary tract and thus may be pathogenic.

Lysine Decarboxylase broth

Use a loop to inoculate the broth

Basic nutrient broth plus:

- peptone and glucose- lysine- Bromocresol purple pH indicator

A color change to purple is a positive test.

Yellow or any other color is negative

The bacteria produce the enzyme lysine decarboxylase which digests lysine to produce cadaverine. This raises the pH and the Bromocresol purple turns purple

Remember to put 5 drops of mineral oil on the top of the broth after inoculation and close the cap of the tube tightly.

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Phenylalanine Deaminase Slant

Use a fishtail inoculation on the surface of the slant

T-soy agar plus:

- yeast extract- phenylalanine

When 10% ferric chloride is added to the top of the slant, a green color is positive.

A yellow color is a negative result.

The bacteria produce phenylalanine deaminase that digests phenylalanine into phenylpyruvic acid and ammonia.

The green color will form almost immediately if the bacteria is positive for phenylalanine deaminase.

The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Michael Jillson and Alex Silage for IT support, and our Dean, John Cornish .

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