DNA BasicsDNA Basics

Bacterial Cell Bacterial Cell WallsWalls

(or Gram Stain “without (or Gram Stain “without the mess”)the mess”)

Bonnie Ownley and Robert TrigianoBonnie Ownley and Robert TrigianoDepartment of Entomology and Plant Department of Entomology and Plant

PathologyPathologyThe University of Tennessee, KnoxvilleThe University of Tennessee, Knoxville

PurposePurpose

To visualize DNA by breaking open To visualize DNA by breaking open bacterial cells on a slide in an alkaline bacterial cells on a slide in an alkaline solution (KOH) and releasing a thread solution (KOH) and releasing a thread of DNA that can be lifted from the of DNA that can be lifted from the slide surface with a toothpick. slide surface with a toothpick.

To study differences in the To study differences in the composition of bacterial cell walls composition of bacterial cell walls because only Gram-negative bacteria because only Gram-negative bacteria lyse in 3% KOH; Gram-positive lyse in 3% KOH; Gram-positive bacteria do not.bacteria do not.

BBackground informationackground information

Students need to be familiar Students need to be familiar with basic cell structure with basic cell structure including cell walls and the including cell walls and the presence of deoxyribonucleic presence of deoxyribonucleic acid (DNA) in the cell. acid (DNA) in the cell.

DNA is the basis of life DNA is the basis of life because it contains the because it contains the genetic code for all living genetic code for all living organismsorganisms

The genes contained in an The genes contained in an organism’s DNA make up the organism’s DNA make up the blueprint for that organism, blueprint for that organism, and determine its appearance and determine its appearance and all of its functionsand all of its functions

Deoxyribonucleic acid Deoxyribonucleic acid = DNA= DNA

DNA is a very long, DNA is a very long, double-stranded, double-stranded, helical molecule made helical molecule made of many nucleotide of many nucleotide unitsunits

Replication of DNA Replication of DNA occurs through the occurs through the pairing of nucleotide pairing of nucleotide bases to form new bases to form new strandsstrands

Each nucleotide Each nucleotide unit is composed ofunit is composed of a sugar a sugar

(deoxyribose)(deoxyribose) a phosphate groupa phosphate group a nitrogenous basea nitrogenous base

Molecular structure of DNA Molecular structure of DNA base pairsbase pairs

Adenine (A) Adenine (A) always pairs always pairs with thymine with thymine (T)(T)

Cytosine (C) Cytosine (C) always pairs always pairs with guanine with guanine (G)(G)

Pairing of nucleotide bases Pairing of nucleotide bases in DNAin DNA

T - - - AT - - - AC - - - GC - - - GG - - - CG - - - CA - - - TA - - - TA - - - TA - - - TT - - - AT - - - AC - - - GC - - - G

DNA mRNA proteinDNA mRNA protein

““transcripttranscription”ion”

““translation”translation”

tRNAtRNA ++ ribosomribosomeses

[chains of amino [chains of amino acids]acids]

Transcription and Transcription and translation of a genetic translation of a genetic message into a proteinmessage into a protein

Diagram of transcription and Diagram of transcription and translationtranslation

Chromosomes are Chromosomes are composed primarily of composed primarily of long, thin strands of DNAlong, thin strands of DNA

In eukaryotic organisms, In eukaryotic organisms, (plants, animals and (plants, animals and fungi) chromosomes are fungi) chromosomes are contained in a membrane-contained in a membrane-bound nucleusbound nucleus

In prokaryotes, such as In prokaryotes, such as bacteria, the single bacteria, the single chromosome is in the chromosome is in the cytoplasm and may be cytoplasm and may be accompanied by smaller accompanied by smaller pieces of circular DNA pieces of circular DNA called plasmidscalled plasmids

The first step in molecular The first step in molecular biology and genetic biology and genetic engineering is to isolate DNA engineering is to isolate DNA from cellsfrom cells

Because the genetic code of Because the genetic code of DNA is nearly universal, any DNA is nearly universal, any gene can potentially be gene can potentially be transferred from a donor transferred from a donor organism and function in a organism and function in a related or unrelated recipient related or unrelated recipient organismorganism

Rapid and simple Rapid and simple visualization of the visualization of the stringing effect of DNA from stringing effect of DNA from lysed bacterial cells may lysed bacterial cells may help attract the interest of help attract the interest of students to these subjectsstudents to these subjects

Bacterial cell with a single large chromosome and an

extra-circular piece of DNA known as a plasmid

Agrobacterium tumefaciensAgrobacterium tumefaciens, the , the crown gall bacterium, a natural crown gall bacterium, a natural

genetic engineer genetic engineer

Release of chromosomal and Release of chromosomal and plasmid (arrows) DNA from plasmid (arrows) DNA from an unidentified bacteriuman unidentified bacterium

Bacterial Cell Bacterial Cell WallsWalls

Gram-stain reactionGram-stain reaction

Gram-stain reactionGram-stain reaction

In 1884, Hans Christian Gram In 1884, Hans Christian Gram found that bacteria could be found that bacteria could be divided into two groupsdivided into two groups cells that retained crystal violet stain cells that retained crystal violet stain

solution (Gram-positive bacteria)solution (Gram-positive bacteria) cells that did not (Gram-negative)cells that did not (Gram-negative)

This differential stain reaction This differential stain reaction reflects a difference in cell wall reflects a difference in cell wall structurestructure

G+G+ and and G-G- cell cell wall wall

differencesdifferences

Gram-positive bacteria have a cell wall with a very thick layer of peptidoglycan

Gram-negative bacteria have a cell wall with several thin layers of peptidoglycan, protein and lipopolysaccharide

The Gram-stain reaction can The Gram-stain reaction can identify bacteria as Gram-identify bacteria as Gram-positive or Gram-negative, positive or Gram-negative, but messy stains and but messy stains and expensive microscopes are expensive microscopes are neededneeded

The KOH test is a faster and The KOH test is a faster and simpler method to determine simpler method to determine the same reactionthe same reaction

The KOH method was The KOH method was developed by a Japanese developed by a Japanese scientist named Ryu in 1938.scientist named Ryu in 1938.

When Gram-negative bacteria When Gram-negative bacteria are placed in an alkaline are placed in an alkaline solution (3% KOH), the cells solution (3% KOH), the cells walls are destroyed, and the walls are destroyed, and the cell contents, including the cell contents, including the DNA, are released.DNA, are released.

The Gram stain is an important The Gram stain is an important diagnostic tool in human diagnostic tool in human medicine because some medicine because some antibiotics are effective against antibiotics are effective against only Gram-negative or Gram-only Gram-negative or Gram-positive bacteria.positive bacteria.

Bacteria also cause plant Bacteria also cause plant diseases. Most plant pathogenic diseases. Most plant pathogenic bacteria are Gram-negative.bacteria are Gram-negative.

Materials and MethodsMaterials and Methods

Materials needed for Materials needed for conceptual demonstration:conceptual demonstration:

hollow plastic eggs that hollow plastic eggs that open into two halves open into two halves

string or yarn string or yarn chopsticks chopsticks

Place a long piece of string (DNA) Place a long piece of string (DNA) inside a plastic egg (bacterial cell inside a plastic egg (bacterial cell wall).wall).

Simulate lysis (breaking open) of Simulate lysis (breaking open) of the bacterial cell by opening the the bacterial cell by opening the egg, which releases the string egg, which releases the string (DNA).(DNA).

Picking up the string with a Picking up the string with a chopstick is analogous to picking chopstick is analogous to picking up a string of viscous DNA with a up a string of viscous DNA with a toothpick.toothpick.

Prepare students for Prepare students for demonstrationdemonstration

Use plastic eggs (to represent Use plastic eggs (to represent the bacterial cells) containing the bacterial cells) containing string (to represent the DNA)string (to represent the DNA)

A chopstick (toothpick) could A chopstick (toothpick) could be used to pick up the string be used to pick up the string (DNA) only when the egg is (DNA) only when the egg is broken open, and the string is broken open, and the string is released.released.

For more advanced For more advanced classes..classes..

Technique is useful for Technique is useful for determining the Gram-determining the Gram-stain reaction of bacteria, stain reaction of bacteria, without the use of stains. without the use of stains.

The Gram stain is The Gram stain is essential in the essential in the identification and identification and classification of bacteria.classification of bacteria.

Materials and MethodsMaterials and Methods

Materials for the KOH test:Materials for the KOH test: Culture plates of Gram-negative Culture plates of Gram-negative

((Pseudomonas spp.Pseudomonas spp.) and Gram-) and Gram-positive (positive (Bacillus subtilisBacillus subtilis) ) bacteriabacteria

Flat wooden toothpicks Flat wooden toothpicks Glass microscope slides Glass microscope slides Dropper bottle containing 3% Dropper bottle containing 3%

(w/v) potassium hydroxide (KOH) (w/v) potassium hydroxide (KOH)

Transfer bacterial cultures to Transfer bacterial cultures to fresh media 24-48 hours before fresh media 24-48 hours before conducting the testconducting the test

Older cultures may give a Older cultures may give a Gram-variable reactionGram-variable reaction

Streak the culture plates Streak the culture plates generously to provide generously to provide sufficient quantities of bacteriasufficient quantities of bacteria

Pick-up a mass of bacteria Pick-up a mass of bacteria on the toothpick and on the toothpick and transfer it to a glass slide transfer it to a glass slide with 2-3 drops of 3% KOH with 2-3 drops of 3% KOH

Use the toothpick to agitate Use the toothpick to agitate the bacteria in the liquid the bacteria in the liquid with a rapid, circular with a rapid, circular motionmotion

Gram-negative bacteria will Gram-negative bacteria will break down in the 3% KOH, and break down in the 3% KOH, and the liquid will become viscous in the liquid will become viscous in 15-30 seconds 15-30 seconds

Continued agitation will increase Continued agitation will increase the viscosity of the liquidthe viscosity of the liquid

The DNA released from the lysed The DNA released from the lysed cells of Gram-negative bacteria cells of Gram-negative bacteria can be lifted from the slide can be lifted from the slide surface on the toothpick when it surface on the toothpick when it is drawn up slowlyis drawn up slowly

No viscosity will be No viscosity will be observed in the KOH observed in the KOH solution with Gram-solution with Gram-positive bacteria positive bacteria

Stringing DNAStringing DNA

For more ideas/resourcesFor more ideas/resources Go to the American Go to the American

Phytopathological Society website Phytopathological Society website http://www.apsnet.orghttp://www.apsnet.org

Contact info: Bonnie OwnleyContact info: Bonnie Ownley 865-974-0219865-974-0219 bownley@utk.edubownley@utk.edu

Clark, D.P., and Russell, L.D. 2000. Clark, D.P., and Russell, L.D. 2000. Molecular Biology: made simple Molecular Biology: made simple and fun. Cache River Press, St. and fun. Cache River Press, St. Louis, MO [ISBN 1-889899-04-6]Louis, MO [ISBN 1-889899-04-6]