NITROGEN AND SULFUR METABOLISM

Reported By: Claire April M. Macabidang

METABOLISM
Biochemical

reactions that take place inside a cell involved:

Reactions

Anabolism building up process of energy Catabolism breaking down process of energy

NITROGEN
Essential

element of biological molecules of amino acids, nucleic acids, nucleotides, and enzymes

Constituent

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

SULFUR
Essential

component of all living

cells
Constituent

of cysteine, methionine, glutathione, and several coenzymes

TEST PERFORMED
A. B. C.

D.
E. F. G.

Utilization of Organic and Inorganic Nitrogen Urea Hydrolysis Indole Production from Tryptophan Phenylalanine Deamination Test Lysine Decarboxylase Test Nitrate Reduction Hydrogen Sulfide (H2S) Production

Utilization of Organic and Inorganic Nitrogen

Purpose
Determines the type of nitrogen (inorganic or organic) a bacterium can utilize

Medium Used

Nitrogen 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 Used

Nitrogen Test Media A These organisms are called NITROGEN FIXING BACTERIA
Nitrogen

is reduced to ammonia by the enzyme nitrogenase

Medium Used
Nitrogen
Sodium

Test Media B
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 Used
Urea

Broth

Urea

nitrogen source Potassium phosphates buffer Yeast extract vitamins source Phenol 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 Used
Tryptone

Broth

an enzymatic digest of casein used as nitrogen source Tryptone source of tryptophan

Reagent Used
Kovacs

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 Kovacs 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 Kovacs reagent
Escherichia coli (+) Proteus vulgaris (+) Enterobacter aerogenes ()

Results

Phenylalanine Deamination

Purpose
Test for the presence of the enzyme deaminase or amino acid oxidase

Purpose
Deamination

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 Used
Phenylalanine

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 Used
Phenylalanine 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 Used
10%

Oxidizing reagent Acts as an chelating agent (a substance used to reduce the concentration of free metal ion in solution by complexing)

Ferric chloride (FeCl3)

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 lysine Dextrose fermentable carbohydrate Yeast extract vitamins source Peptone nitrogen and nutrient source essential for growth Bromcresol Purple pH indicator

Principle
Unable

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 Used
Nutrient

Broth with 0.1% Potassium Nitrate (KNO3) Nitrate source Nutrients

Reagent Used
Nitrate

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

Results

Upon 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 sulfurcontaining compound cysteine Dextrose Sodium 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

QUESTION
What 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/Dbiochem/urea

.jpg http://biosci.usc.edu/courses/2002fall/documents/bisc300-lab_Urea_Hydrolysis.jpg http://lh3.ggpht.com/cC8H3nZwCuU/TDT7FSIZ0kI/AAAAAAAAALw/dw9IhYM 9dsA/INDOLE.JPG http://academic.missouriwestern.edu/jcbaker/images/PD.j pg 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=12 80&bih=691&sclient=psyab&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=43 e7b3983c4556bc http://en.wikipedia.org/wiki/Metabolism http://www.cdc.gov/std/gonorrhea/lab/tests/nitrate.htm