Protein Utilization in Bacteria

Bacteria can use a wide variety of substrates present in the environment as
sources of nutrition; these include simple or complex carbohydrates, lipids, nucleic acids
and proteins. The oxidation of proteins for energy production requires more complex
enzymatic processes than the oxidation of carbohydrates because proteins are
structurally more complex than carbohydrates and are too large to be transported
across the cytoplasmic membrane. Because bacteria cannot transport proteins they often
produce exoenzymes to partially or completely degrade these proteins into molecules
(small peptides and amino acids) that can easily be transported from the environment
into the cell. Once these molecules have been transported into the cell cytoplasm, the
bacteria produce endoenzymes to further degrade and then metabolize these molecules.
Media that contain peptides and free amino acids can be used to detect the presence of
endoenzymes in bacteria that metabolize these molecules. An example of this media is
Kligler Iron Agar (KIA), which is used to detect the utilization of the sulfur containing
amino acids (cysteine and methionine) by desulfhydrase activity. Another example is
tryptone broth, which is used to detect for the presence of tryptophanase.
In this exercise you will assess the ability of Staphylococcus aureus, Bacillus
subtilis, Enterobacter aerogenes, Serratia marcescens, Shigella flexneri, Pseudomonas
aeruginosa, Klebsiella pneumoniae and Citrobacter freundii to utilize protein based
nutritional sources for energy in Tryptone broth and KIA media.
TRYPTONE BROTH: Tryptone broth consists of proteins that have been degraded to
peptides and amino acids by trypsin. This medium contains high levels of the amino
acid tryptophan as well as other amino acids. To utilize tryptophan, a bacterium must
produce tryptophanase. Tryptophanase catalyzes the hydrolysis of tryptophan to
ammonia, pyruvate and indole. As indole is produced as an end product that is unique to
bacteria that produce tryptophanase, one can determine if a given bacteria has
tryptophanase activity by testing for the presence of indole. To this end, one adds
Kovac’s reagent; Kovac’s reagent contains chemicals that react with indole to produce a
red-ring at the top of the broth.
There are two possible reactions one might observe after the addition of
Kovac’s reagent:
indole positive (tryptophanase positive)—a red ring is formed at the top of the
indole negative (tryptophanase negative)—a green or yellow ring is formed at
the top of the broth.
KLIGLER IRON AGAR (KIA): KIA agar slants contain beef extract, peptones,
lactose and glucose in a 10:1 ratio, the pH indicator—phenol red and iron sulfate. This
complex media is used to identify bacteria that use a specialized enzyme to degrade
proteins and to identify bacteria that can ferment simple sugars. Bacteria that use
desulfhydrases in the proteolysis proteins for the metabolism of cysteine or methionine
produce hydrogen sulfide gas (H2S) as an end product of this reaction. If H2S is
produced in the KIA medium, it will react with the iron sulfate to produce ferrous
(iron) sulfide (FeS). The FeS appears as a black precipitate in the butt (or bottom)
of the tube.
KIA agar slants also contain the carbohydrates, lactose and glucose in a 10:1
ratio and phenol red (pH indicator) to detect for fermentation of one or both of these
sugars to produce metabolic acids or metabolic acids AND carbon dioxide gas.
Lactose is a disaccharide consisting of a glucose and galactose monomer linked by a
glycosidic bond; bacteria that can utilize lactose produce lactase or beta-galactosidase
to cleave the glycosidic bond; the resulting simple sugars, glucose and galactose are
transported into the cell’s cytoplasm and metabolized. Therefore, bacteria that ferment
lactose can also ferment glucose; however, not all bacteria that ferment glucose can
ferment lactose.
In KIA agar the amount of lactose available as a carbon or energy source is ten
times greater than the amount of glucose that is present. Therefore, organisms that can
ferment lactose as well as glucose have enough carbohydrate to support their growth. As
metabolic acids are produced during the fermentation of these sugars, the pH of the
media is reduced and the medium present in the butt and the slant of the tube turns
yellow. Therefore a yellow butt/yellow slant is indicative of both lactose and glucose
Not all bacteria can utilize lactose, however, many bacteria readily utilize glucose.
Therefore, the relatively small amount of glucose present in the KIA agar slants is rapidly
consumed. The fermentation of glucose as a sole carbohydrate source produces
significantly less acid than the fermentation of glucose AND lactose; therefore, the pH
of the KIA media does not drop as low if the medium is inoculated with bacteria that can
only ferment glucose as it does if the bacteria can ferment glucose and lactose.
As the bacteria that can only use glucose rapidly use up their supply of
carbohydrate, they use the proteins and peptones present in the medium as a secondary
source of nutrients. Protein utilization occurs most readily in the oxygen rich slant
region of the tube and less readily in the anaerobic butt region of the tube. The
process of protein utilization leads to the production of alkaline end products that raises
the pH of the slant portion of the tube. Therefore, a yellow butt/red slant is indicative
of an organism that can ferment glucose but cannot ferment lactose.
One can also use this media to determine if an organism ferments either glucose
or glucose and lactose to produce metabolic acids and ALSO produces carbon dioxide
gas. Carbon dioxide production is indicated by the presence of small bubbles between
the medium and the glass tube or by the presence of cracks in the agar.
There are several possible outcomes that one might observe when different
bacteria are grown on KIA Agar slants—not all are mutually exclusive:
1. Black precipitate—indicative of desulfhydrase activity and hydrogen sulfide
2. Cracks or bubbles in agar—indicative of carbon hydroxide production during
fermentation of glucose or glucose and lactose.
3. Red slant/yellow butt—indicative of glucose fermentation.
4. Yellow slant/yellow butt—indicative of glucose AND lactose fermentation.
5. Red slant/red butt—indicative of no glucose or lactose utilization but
utilization of protein as a sole carbon source
Laboratory Period 1:
Tryptone broth: protein utilization will be tested on the following bacteria:
Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes, Serratia marcescens,
Shigella flexneri, Pseudomonas aeruginosa, Klebsiella pneumoniae and Citrobacter
freundii. Each student will be assigned ONE of these bacteria. The student should
aceptically transfer bacteria from an overnight culture into the Tryptone broth. These
cultures will be incubated for 24-48 hours at 37 degrees Celsius and saved for analysis at
the next laboratory session.
Kligler Iron agar (KIA) slants: Each student will be supplied with one slant containing
Kligler Iron agar and ONE of following bacteria: Staphylococcus aureus, Bacillus
subtilis, Enterobacter aerogenes, Serratia marcescens, Shigella flexneri, Pseudomonas
aeruginosa, Klebsiella pneumoniae and Citrobacter freundii. Each student should streak
the bacteria onto the surface of the slant with a loop using the following method.
1. Flame the ENTIRE length of the wire of the inoculating loop.
2. use the cooled loop to transfer some of the culture from the supplied test tube
onto the KIA agar slant.
3. Using a gentle zig-zag motion along the surface of the slant, streak incoculate
the a tube starting at the bottom of the slant. IT IS VERY IMPORTANT that
you do not use too much bacteria to inoculate this media. A SMALL loopful
of bacteria (approximately the amount of bacteria that you would smear on a
microscope slide) from the liquid broth or plate is sufficient!!!
4. Without re-flaming or re-inoculating the loop, stab the loop through the center
of the agar in the tube to within 1 centimeter from the bottom of the tube. Do
NOT stab to the very bottom of the tube!!
5. Re-sterilize your loop.
These tubes will be incubated at 37 degrees Celsius for 24-48 hours; the tubes will
be analyzed at the next lab period
Laboratory Period 2: Analysis of results for protein utilization by bacteria.
Tryptone broth: vigorously shake or vortex the broth containing your bacteria before
testing for trytophanase activity and the presence of indole. Put on protective gloves
before handling the Kovac’s reagent. Add 6-8 drops of Kovac’s reagent to your tube.
Observe your tube for the appearance of a ring at the top of the broth. Use a (+) or(–) in
the table below to indicate whether the bacteria can uses tryptophanase in the utilization
of proteins and the production of indole. Describe the color of the ring at the top of your
Enterobacter aerogenes
Tryptone Broth (indole production) ring color
Staphylococcus aureus
Serratia marcescens
Bacillus subtilis
Shigella flexneri
Pseudomonas aeruginosa
Klebsiella pneumoniae
Citrobacter freundii
Kligler Iron Agar Slants: In the table provided below, record the results for the KIA
slants as follows: Glucose or Glucose/Lactose to indicate the type of carbohydrate
utilization. Protein to indicate protein utilization as the sole carbon source. CO2 to
indicate carbon dioxide production and H2S if desulfhydrase activity is detected. In the
adjacent column describe the characteristics of the agar slant that helped you to
reach these conclusions.
Protein and or
Utilization in KIA slants
Enterobacter aerogenes
Staphylococcus aureus
Serratia marcescens
Bacillus subtilis
Shigella flexneri
Klebsiella pneumoniae
Citrobacter freundii
Characteristics of
Media after growth
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