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NITROGEN AND SULFUR METABOLISM
Reported By: Claire April M. Macabidang
METABOLISMBiochemical reactions that take place inside a cell
Reactions involved: Anabolism – building up process of
energy Catabolism – breaking down process
of energy
NITROGENEssential element of biological molecules
Constituent of amino acids, nucleic acids, nucleotides, and enzymes
GENERAL VIEW OF NITROGEN METABOLISM
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Protein Digestion
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Oxidative Deamination
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Reductive Deamination
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Decarboxylation
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Transamination Reactions
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Nitrification
PATHWAYS INVOLVED IN NITROGEN UTILIZATION
Denitrification
SULFUREssential component of all living cells
Constituent of cysteine, methionine, glutathione, and several coenzymes
TEST PERFORMEDA. Utilization of Organic and Inorganic
NitrogenB. Urea HydrolysisC. Indole Production from TryptophanD. Phenylalanine Deamination TestE. Lysine Decarboxylase TestF. Nitrate ReductionG. Hydrogen Sulfide (H2S) Production
Utilization of Organic and Inorganic Nitrogen
Purpose
Determines the type of nitrogen (inorganic or organic) a bacterium
can utilize
Medium UsedNitrogen Test Media A
Potassium nitrate (KNO3) Inorganic nitrogen source Mineral niter and natural solid nitrogen
source Used in fertilizers, as a source of nitrogen
and potassium (2 of the macronutrients for plants)
Growth indicates that the organism is capable of using INORGANIC nitrogen
Medium UsedNitrogen Test Media A
These organisms are called NITROGEN – FIXING BACTERIA
Nitrogen is reduced to ammonia by the enzyme nitrogenase
Medium UsedNitrogen Test Media B
Sodium glutamate or “Vetsin” Organic nitrogen source Exists naturally in all living things One of the building blocks of proteins Found in "free form“, not bound to
other molecules
Growth indicates that bacteria can metabolize ORGANIC nitrogen
Urea Hydrolysis
Purpose
Detects presence of urease enzyme that catalyze the hydrolysis of urea
Medium UsedUrea Broth
Urea – nitrogen sourcePotassium phosphates – bufferYeast extract – vitamins sourcePhenol red – pH indicator
Principle Urea produces ammonia when acted upon by
urease
Ammonia increases the pH of the medium Phenol red: Orange to pink or red color
pH 6.8 to 7 – orange pH 8.1 – pink or red
Results
Color change from orange to dark pink or red
Proteus vulgaris (+)Escherichia coli ()
Results
Indole Production from Tryptophan
Purpose
Test for the ability of the organism to degrade tryptophan with the aid of
the enzyme tryptophanase
Medium UsedTryptone Broth
an enzymatic digest of casein used as nitrogen source
Tryptone – source of tryptophan
Reagent UsedKovac’s Reagent-dimethylaminobenzaldehyde – reacts with indole
Amyl alcohol (Butanol) – acts as solvent
Hydrochloric acid (HCl) – hasten extraction of indole
Principle The enzyme tryptophanase can
degrade tryptophan into indole, ammonia, pyruvic acid
Ammonia and pyruvate is metabolized Indole is not metabolized and stay in
the medium
Principle Upon addition of Kovac’s reagent, the
amyl alcohol (butanol) component dissolves the indole which then reacts with p-dimethylaminobenzaldehyde, producing red rosindole dye
Results
Formation of red colored ring at the surface of the medium after the
addition of Kovac’s reagent
Escherichia coli (+)Proteus vulgaris (+)
Enterobacter aerogenes ()
Results
Phenylalanine Deamination
Purpose
Test for the presence of the enzyme deaminase or amino acid oxidase
PurposeDeamination – oxidation of an amino acid
Principle: Before an amino acid can be used by the cell as an energy source, the amino group must be removed
Medium UsedPhenylalanine Agar
A.K.A. Phenylalanine deaminase medium
Used to differentiate members of the genera Proteus, Morganella (which were originally classified under the genus Proteus), and Providencia from other Enterobacteriaceae
Medium UsedPhenylalanine Agar
DL – phenylalanine Yeast extract – nutrient and vitamin
source for growth Sodium chloride (NaCl) – supplies
essential electrolytes for transport and osmotic balance
Dipotassium hydrogen phosphate (K2HPO4)– buffer
Agar – solidifying agent
Reagent Used10% Ferric chloride (FeCl3)
Oxidizing reagent Acts as an chelating agent (a substance used to reduce the concentration of free metal ion in solution by complexing)
Principle Phenylalanine in the medium is
deaminated by phenylalanine deaminase to phenylpyruvic acid
The enzyme phenylalanine deaminase will remove the amine group (NH2) from phenylalanine and release it as free ammonia (NH3)
Principle This leaves phenylpyruvic acid, which
can be detected by adding an oxidizing reagent such as ferric chloride
10% Ferric chloride (FeCl3) combines with phenylpyruvic acid to form green color
Results
Formation of intense green color upon the addition of 10% Ferric
chloride (FeCl3) solution
Proteus vulgaris (+)Escherichia coli ()
Enterobacter aerogenes ()
Results
Lysine Decarboxylation
Purpose
Test for the presence of the enzyme lysine decarboxylase which converts
lysine to amine (cadaverine)
Purpose Decarboxylation - removal of a carboxyl
group from an organic molecule
Medium Used Lysine Decarboxylase Broth
L – lysineDextrose – fermentable carbohydrate
Yeast extract – vitamins sourcePeptone – nitrogen and nutrient source essential for growth
Bromcresol Purple – pH indicator
PrincipleUnable to ferment dextrose = No color change
Able to ferment the dextrose = Formation of acidic by – products and yellowing of the medium
Low pH and presence of the amino acid will stimulate decarboxylation
Principle Lysine is converted to cadaverine Increase in the pH of the medium
changes its color from yellow back to purple
Results
Red or purple color of the medium
Escherichia coli (+)Proteus vulgaris ()
Results
Nitrate Reduction
Purpose
Detection for the presence of the enzyme reductase
Medium UsedNutrient Broth with 0.1% Potassium Nitrate (KNO3) Nitrate source Nutrients
Reagent UsedNitrate Reagent A
Acetic acid and Sulfanilic acid
Nitrate Reagent B Acetic acid and Alpha – naphthol
Principle If reductase is present in the bacteria,
nitrate present in the broth is reduced to nitrite and furthermore into nitric oxide, nitrous oxide or N2
Denitrification – process of nitrate reduction
Principle Gas inside the Durham tube – mixture of
carbon dioxide (CO2) and nitrogen (N2) released from the reduction of nitrate
Principle The presence of nitrite is detected by the
addition of Nitrate Reagent A and Nitrate Reagent B
Principle The Nitrate Reduction Test is based on
the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid (Nitrate Reagent A) to form a complex (nitrite – sulfanilic acid) which then reacts with a – naphthylamine (Nitrate Reagent B) to give a red precipitate (Prontosil)
Zinc dust catalyses the reduction of nitrate to nitrite
Results
Results
Results
Results Gas Production – reduction of nitrate
Upon addition of Nitrate Reagent A and Nitrate Reagent B – red color Presence of nitrite
*** If no color change: Nitrate is not reduced to nitrite Nitrate is reduced to a different form
other than nitrite
ResultsUpon addition of Zinc dust – no color change N2 is present Nitrate is reduced
*** If red color was observed: Nitrate is still present and reduced
by zinc
Results
Hydrogen Sulfide (H2S) Production
Purpose
Detection for the presence of the enzyme cysteine desulfonase which
helps in synthesizing hydrogen sulfide from the peptones in the
medium
Medium Used Lead Acetate Agar
Lead acetate – reacts with hydrogen sulfide (H2S)
Peptone – source of organic sulfur-containing compound cysteine
DextroseSodium thiosulfate – inorganic sulfur source
Agar – solidifying agent
Principle Hydrogen sulfide is produced when
amino acids containing sulfur are metabolized by microorganisms
If the medium contains metallic ions (lead, bismuth , or iron), the hydrogen sulfide formed during growth combines with the metallic ions to form metal sulfide that blackens the medium
Principle Lead acetate in the medium reacts with
H2S forming black precipitate (lead sulfide)
Results
Formation of black or brown precipitate in the medium
Proteus vulgaris (+)Escherichia coli ()
Results
STUDY QUESTIONS
QUESTIONWhat controls the ability of an organism to
utilize certain types of nitrogen sources?
Bacteria vary widely in their ability to utilize various sources of nitrogen for synthesis of proteins
Enzymes present in a bacteria Genetically determined
REFERENCES http://www.mesacc.edu/~johnson/labtools/Dbi
ochem/urea.jpg http://biosci.usc.edu/courses/2002-fall/docum
ents/bisc300-lab_Urea_Hydrolysis.jpg http://lh3.ggpht.com/-cC8H3nZwCuU/TDT7FSI
Z0kI/AAAAAAAAALw/dw9IhYM9dsA/INDOLE.JPG
http://academic.missouriwestern.edu/jcbaker/images/PD.jpg
http://www.google.com.ph/#hl=en&gs_nf=1&pq=k2hpo4&cp=9&gs_id=14&xhr=t&q=chelating+agent&pf=p&biw=1280&bih=691&sclient=psy-ab&oq=chelating&aq=0&aqi=g4&aql=&gs_sm=&gs_upl=&gs_l=&pbx=1&bav=on.2,or.r_gc.r_pw.r_qf.,cf.osb&fp=43e7b3983c4556bc
http://en.wikipedia.org/wiki/Metabolism http://www.cdc.gov/std/gonorrhea/lab/tests/nit
rate.htm