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