Introduction - Central New Mexico Community College | CNM
Unit 10: Protein CatabolismBy Heather Fitzgerald, Karen Bentz, Patricia G. Wilber and Andrea Peterson, 2022Creative Commons Attribution-Noncommercial 4.0 International License.IntroductionThe breakdown of proteins provides amino acids which are required by bacteria for growth. Bacteria produce a variety of enzymes to help degrade proteins in their environment. These enzymes cut the protein chain at a specific amino acid and are named for the particular bond site that they are able to break. Figure 10-1. A generalized structure of an amino acid and target sites for bacterial enzymes. Accessed 8/15-15 from . By GYassineMrabetTalk This vector image was created with Inkscape. (Own work). Modifications by H. Fitzgerald.While carbohydrate usage produces an acid or neutral waste, the break down products of protein digestion are basic. Just as in the carbohydrate unit, pH indicators (that change color) are used to visualize the pH change when protein digestion has occurred.Specific Protein Catabolism TestsLysine 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. The LDC enzyme requires an acidic environment, so placing mineral oil over the broth reduces oxygen and encourages fermentation to produce an 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 LDC is produced, it will cleave the carboxyl group off the lysine, generating cadaverine and other basic (high pH) end products which will raise the pH and turn the bromocresol purple indicator back to purple. See Figure 10-2 for examples of positive and negative results.Figure 10-2. Example results of Decarboxylase test. The leftmost tube shows a purple positive decarboxylase test, while the two right most tubes show negative results. The brownish tube in the middle shows the color of an uninoculated tube, while the yellow tube indicates a true negative result.Photo Accessed 8/17/15 from Deaminase test. This test will help distinguish among bacteria by determining whether or not they contain the enzyme phenylalanine deaminase. 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 phenylalanine deaminase. See Figure 10-3 for examples of positive and negative results after the addition of the ferric chloride solution.Figure 10-3. Examples of positive and negative results in the phenylalanine deaminase test.The left tube shows no color change after the addition of ferric chloride, which is a negavite result. The tube on the right shows a green color after the addtion of ferric chloride, which is positive.Accessed 8/17/15 from . Urease TestUrea is a nitrogen containing waste product found in animal urine. The enzyme urease hydrolyzes urea to ammonia and carbon dioxide. Detection of urease activity (which allows survival of bacteria in the gastric and/or urinary systems) is a key diagnostic test for potential human pathogens. The ulcer-causing bacteria, Helicobacter pylori uses urease to produce ammonia, which is basic, and helps de-acidify its local stomach environment. Some Proteus species utilize urease to thrive 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 uses 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. If at least 80% of the slant and butt are 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. A yellowish colored tube that remains roughly the same color as the uninoculated medium indicates a negative urease result. See Figure 10-4 for examples.Figure 10-4. Example results of a Urease test. (a) Uninoculated control, (b) Rapid positive urease; over 80% pink, (c) Delayed positive urease; 50% pink, (d) Negative urease; no pink Accessed 8/17/15 from . Cultures and image description from Benita A. Brink, Adams State College, Alamosa, CO.Sulfur, Indole and Motility (SIM) test.The SIM tests analyze three different bacterial characteristics:The (S) or sulfur component of this test identifies the production of hydrogen sulfide (H2S). If a bacterial species produces hydrogen sulfide (H2S), the H2S will react with the iron ions in the ferrous sulfate (an ingredient in the medium) to produce a black color in the bottom of the tube. Bacteria produce H2S using the enzymes cysteine desulfhydrase and/or thiosulfate reductase. Cysteine and thiosulfate are differential ingredients in this medium. SIM media does not allow us to distinguish between these types of sulfide production. 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. Tryptophanase is an enzyme that both deaminates the amino acid tryptophan (the differential ingredient for this test) and also works on the R-group of tryptophan. Tryptophanase breaks down proteins that contain the amino acid tryptophan (an ingredient in the media) to produce pyruvate, indole and ammonia.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 adding Kovac’s reagent to the top of the tube after incubation. If a red ring forms 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 6) on bacteria. The semi-solid consistency (< 1% agar) of the SIM medium allows motile bacteria to move through the media, away from the stab line. This produces a cloudy media extending in all directions (3-dimensional) from the stab line. If growth is only observed along the stab line in a 2-dimensional pattern and the media remains clear, this is a negative result. All known organisms that can produce H2S in the (S) test are also motile, so bacteria that produce a black precipitate are also motile. Motility is often a sign of pathogenicity. Bacteria that produce flagella can move from the site of infection in the body to other locations. Figure 10-5. Example SIM test results. Tube A is uninoculated. Tube B has growth along the stab line in a 2D pattern while the rest of tube is clear like A, the uninoculated tube. This indicates no 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, indicating a positive test for indole.? Tube C is also cloudy in a 3D pattern, indicating motility. Tube D is black, which indicates a positive test for desulfhydrase/reductase and also a positive result for motility. Tube D is negative for indole.Accessed 8/18/15 from . Image provided courtesy of Renee Wilkins, University of Mississippi Medical Center, Jackson, MS.DAY ONE: InoculationsLysine Decarboxylase? Deaminase Test created by Corrie AndriesMaterialsMedia (per pair of students)2 Lysine broth tubes3 Phenylalanine slants 3 SIM deep tubes3 Urea slant tubesBacteria CulturesEscherichia coli (Ec)Proteus vulgaris (Pv)Klebsiella pneumoniae (Kp)Priestia megaterium (Pm)ProcedureLysine Decarboxylase TestFigure 10-6. Lysine inoculationsImage created 2022 by H. Fitzgerald, K. Bentz, P. Wilber1. Label your tubes. Use one tube for each bacterial species, as indicated in the image above.2. Use a sterile metal loop to acquire a small amount of one bacterial species. 3. Dip the loop into the lysine broth and swirl the loop around.4. Re-sterilize the loop and place it back into the tools container on the lab bench.5. Add around 5 drops (enough to cover the surface) of mineral oil to the top of the broth.6. Tightly (!!) cap the tube.7. Repeat this procedure for the remaining tube.Phenylalanine Deaminase TestFigure 10-7. Phenylalanine inoculations Image created 2022 by H. Fitzgerald, P.Wilber1. Label your tubes. Use one tube for each bacterial species, as indicated in the image above.2. Use a sterile metal loop to acquire a small amount of one bacterial species. 3. Create fishtail streak on the surface of the phenylalanine slant.4. Re-sterilize 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 rack for incubation.6. Repeat this procedure for the remaining tubes. Urease testFigure 10-8. Urease inoculationsImage created 2022 by Karen Bentz, H. Fitzgerald, P.Wilber1. Label your tubes. Use one tube for each bacterial species, as indicated in the image above.2. Use a sterile metal loop to acquire a small amount of one bacterial species. 3. Make a fishtail streak on the surface of the urease slant.4. Re-sterilize the loop and place it back into the tools container on the lab bench.5. Loosely cap the urease tube and place it into the appropriate rack for incubation.6. Repeat this procedure for the remaining tubes. SIM testFigure 10-9. SIM inoculations.Image created 2022 by H. Fitzgerald, P.Wilber1. Label your tubes. Use one tube for each bacterial species, as indicated in the image above.2. Use a 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. 4. Re-sterilize the needle and place it back into the tools container on the lab bench.5. Loosely cap the SIM deep tube and place it into the appropriate rack for incubation.6. Repeat this procedure for each of the remaining tubes. DAY TWO: Results and InterpretationProcedure:1. Collect the media you inoculated in the previous lab.2. Add 4 drops of Kovac’s reagent to your SIM tubes.3. Add 8 drops of FeCl3 reagent to each of your phenylalanine tubes. 4. Rock the tubes to make sure the reagent moves over the slant to ensure a reaction.5. Observe any color changes, growth patterns or other visible changes to the inoculation tubes. 6. Fill in your observations in Table 10-1 below. Be aware that P. megaterium will show a weak green in the PA tube after about 5-10 minutes with the reagent.Table 10-1. Results of Protein Catabolism testsBacteriaLysine Decarboxlase(+ or -)Include the colorPhenylalanine Deaminase(+ or -)Include the colorUrease(+ or -)Potential pathogen? Include the colorSIMSulfurdesulfhydrase/reductase(+ or -)Potential pathogen?Include colorIndole(+ or -)(breakdown product of tryptophan via Tryptophanase)Potential pathogen?Record appearanceMotility (+ or -)Potential pathogen?Record 2D or 3DEscherichia coli (Ec)Proteus vulgaris (Pv)Klebsiella pneumonia(Kp)Priestia megaterium(Pm)Insert photos of test results here:InterpretationComplete the following tables for the four bacteria that you tested for protein catabolism.Bacterial Species: ___________________________________Which, if any, enzymes does this bacteria produce?(Cysteine Desulfhydrase, Thiosulfate reductase, Tryptophanase, Urease, Phenylalanine Deaminase, Lysine Decarboxylase)Which media result(s) supports your enzyme choice(s)?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 your species has more than four enzymes.Bacterial Species: ___________________________________Which, if any, enzymes does this bacteria produce?(Cysteine Desulfhydrase, Thiosulfate reductase, Tryptophanase, Urease, Phenylalanine Deaminase, Lysine Decarboxylase)Which media result(s) supports your enzyme choice(s)?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 your species has more than four enzymes.Bacterial Species: ___________________________________Which, if any, enzymes does this bacteria produce?(Cysteine Desulfhydrase, Thiosulfate reductase, Tryptophanase, Urease, Phenylalanine Deaminase, Lysine Decarboxylase)Which media result(s) supports your enzyme choice(s)?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 your species has more than four enzymes.Bacterial Species: ___________________________________Which, if any, enzymes does this bacteria produce?(Cysteine Desulfhydrase, Thiosulfate reductase, Tryptophanase, Urease, Phenylalanine Deaminase, Lysine Decarboxylase)Which media result(s) supports your enzyme choice(s)?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 your species has more than four enzymes.Post Lab QuestionsName: ________________________This Unit is about enzymes that break down ____________. What is a difference between the end products of protein catabolism vs. carbohydrate metabolism?Enzymes that we are testing for would be produced by the _________________, while the amino acid that is being catabolized is considered a _________________ material or ingredient.Indicate, with arrows, where each of the following enzymes breaks a bond in the amino acid.Phenylalanine DeaminaseLysine DecarboxylaseTryptophanase Cysteine DesulfhydraseKovac’s reagent is added to the __________ medium and will form a ______ ring at the top of the media if the result is positive. The enzyme that may be produced (or not) by the bacteria is _______________. The end product is called ___________. A positive result indicates a potential ______________.FeCl3 is added to the _______________________ medium and will turn a __________ color if the result is positive. A positive result indicated the enzyme _________________________ was produced by the bacteria.The pH indicator in the lysine test is ________________ _____________. A positive lysine test will be a ___________ color. If the result is purple, what enzyme did the bacteria produce? _________________________ The pH indicator in the urease medium is _________________ and a positive result will be a ____________ color. A positive urease test indicated a potential _______________.SIM stands for _________, ___________ and __________.Describe what a positive test for sulfur would look like:What enzymes, produced by bacteria, can create a positive S test?List two things have your learned about a species that is positive for sulfur. A positive indole test would have a _________ ____________ at the top of the tube.What enzyme, produced by the bacteria, would result in a positive indole reaction?What is the differential ingredient for the indole test?If a bacteria is motile, it will have one or more _________ and can move through the media away from the __________ _________. What is the stab line appearance when the organism lacks flagella?Table 10.2. Media used, major ingredients, results, interpretation and notesMedia IngredientsWhat Positive/Negative Results Look LikeInterpretation if Results are PositiveOf Special NoteSIM deepInoculate with a stab into the mediaT-soy semisolid agar plus:- beef extract- peptones (source of cysteine, differential ingredient)- thiosulfate, differential ingredient- ferrous sulfate as a source of iron to indicate H2S production1. S A black precipitate in the butt of the tube is positive2. I Presence of a red ring at the top of the media after Kovac’s reagent is added is positive3. M Cloudiness away from the stab line or BLACK is positive1. S The bacteria digest cysteine to H2S using the enzyme cysteine desulfhydrase 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 flagella1. S Black. If bacteria produce 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 slantUse a fishtail inoculation on the surface of the slantT-soy agar plus:- Urea, differential ingredient- Phenol red pH indicatorColor 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 negativeThe bacteria produce 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 brothUse a loop to inoculate the brothBasic nutrient broth plus:- peptone and glucose- lysine, differential ingredient- Bromocresol purple pH indicatorA color change to purple is a positive test.Yellow or any other color is negativeThe bacteria produce the enzyme lysine decarboxylase which digests lysine to produce cadaverine. This raises the pH and the Bromocresol purple turns purpleRemember to put 5 drops of mineral oil on the top of the broth after inoculation and close the cap of the tube tightly. Phenylalanine Deaminase SlantUse a fishtail inoculation on the surface of the slantT-soy agar plus:- yeast extract- phenylalanine is the differential ingredient.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. ................
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