K.6 Microbial Genetics(k Lia)

8/13/2019 K.6 Microbial Genetics(k Lia)

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Microbial Genetics

R. Lia K. Iswara, Dr, MS, SpMK

Department of MicrobiologyFK USU


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GENETICS

1928 GRIFFITH- Dead bacteria could Transform living

1941 AVERY- Showed that the transforming agent was

DNA

1940's BEEDLE & TATUM- One gene, one enzyme

1950's WATSON & CRICK- Discovered the structure of

DNA and the structure enabled the discovery of the

functions of DNA

1950's Mc CLINTOCK- First showed the presence of

transposable genetic elements


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The Genetic Material

Frederick Griffith, 1928

studied Streptococcus pneumoniae, a

pathogenic bacterium causing pneumonia

there are 2 strains of Streptococcus:

- S strain is virulent

- R strain is nonvirulentGriffith infected mice with these strains

hoping to understand the difference

between the strains


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Griffiths results:

- live S strain cells killed the mice

- live R strain cells did not kill the mice- heat-killed S strain cells did not kill the

mice

- heat-killed S strain + live R strain cellskilled the mice


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Griffiths conclusion:

- information specifying virulence passed

from the dead S strain cells into the live R

strain cells

- Griffith called the transfer of this information

transformation


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Avery, MacLeod, & McCarty, 1944

repeated Griffiths experiment using purifiedcell extracts and discovered:

- removal of all protein from thetransforming material did not destroy itsability to transform R strain cells

- DNA-digesting enzymes destroyed alltransforming ability

- the transforming material is DNA


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Hershey & Chase, 1952

- investigated bacteriophages: viruses that

infect bacteria

- the bacteriophage was composed of only

DNA and protein

- they wanted to determine which of thesemolecules is the genetic material that is

injected into the bacteria


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- Bacteriophage DNA was labeled withradioactive phosphorus (32P)

- Bacteriophage protein was labeled with

radioactive sulfur (35S)- radioactive molecules were tracked

- only the bacteriophage DNA (as indicated

by the 32P) entered the bacteria and wasused to produce more bacteriophage

- conclusion: DNA is the genetic material


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DNA Structure

James Watson and Francis Crick, 1953

deduced the structure of DNA using evidence

from Chargaff, Franklin, and others

proposed a double helix structure


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DNA Structure

The double helix consists of:

2 sugar-phosphate backbones

nitrogenous bases toward the interior of the

molecule

bases form hydrogen bonds with

complementary baseson the opposite

sugar-phosphate backbone


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DNA Structure

The two strands of nucleotides are

antiparallelto each other

one is oriented 5 to 3, the other 3 to 5

The two strands wrap around each other to

create the helical shape of the molecule.


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Organization of genes

Gene: unit of heredity/ a segment of DNAthat carries in its nucleotide sequenceinformation for a specific biochemical or

physiologic property. DNA (deoxyribonucleic acid):

Four bases : A-T , G=C

RNA (Ribonucleic acid) : U-T , G=C Genome: totally of genetic information in

an organism

Mutation: genetic changes


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DNA = RNA=

Deoxyribonucleic acid Ribonucleic acid

Nucleotide components1. Phosphate2. Deoxyriboxe (a sugar)and

3. Bases as follows:a) adenine = A

b) guanine = G

c) cytosine = C

d) Thymine = T

DNA IS A DOUBLE HELIX

Nucleotide components:

1. Phosphate

2. Ribose

3. Basesa. Adenine = A

b. Guanine = G

c. Cytosine = C

d. URACIL = U and

replaces Thymine


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Genetics of Bacteria

Bacterial genome = One circular DNA

molecule

E. coli chromosome has 100 times more

DNA than in a typical virus, but much less

than a eukaryotic cell.

Packed into nucleoid region of cell

Plasmid = small circular extra piece of

DNA


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Bacterial genetics, mutations

and genetic transfer

STRUCTURE OF THE DNA (double helix,

bases, sugar and phosphate)

PROTEIN SYNTHESIS (RNA, transcription and

translation, expressed gene)

MUTATION (change in sequence of nucleotide

bases in DNA, AB resistance, mutagens,

carcinogens, radiation) TRANSFER (occurs with cell division,

transformation, transduction)


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Bacterial Genetic

Recombination What is the main source of genetic

recombination in bacteria?

Mutations

What are the other sources of

recombination?


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What causes mutations?

1. Errors during REPLICATION

2. Mutagenic agents such as chemical and

physical agents


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Mutation & Gene Rearrangement

Spontaneous mutation:

1. Base substitustions

2. Deletion3. Insertions

4. Rearrangement


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Why are mutations important?

Because the transfer of genetic material from adonor into a recipient followed by recombination

is a source of genetic diversity that may result in:

resistance to antibiotics

(Drug Resistance - Chromosomal mutations and R

Plasmids)

increase in virulence

Recombination occurs after the transfer of

genetic material


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DONOR DNA gets into the RECIPIENT in three ways

A. Transformation

B. Transduction

Life cycle of a lytic phage---->generalized transduction

Life cycle of a temperate phage---->specialized transductionC. Conjugation

Transfer of F plasmid

Hfr and transfer of chromosomal genes


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Transfer of DNA Mechanisms of Recombination/Gene Transfer:

1. Conjugation: The DNA is physically transferred tothe recipient cell, pass a bridge between the cells.(plasmid are genetic elements most frequentlytransferred by conjugation).

2. Transduction: donor DNA is carried in a phage coatand is transferred into the recipient by mechanismused for phage infection.

3. Transformation: the direct uptake of donor DNA by therecipient cell, may be natural or forced (induced inthe laboratory, fundamental to genetic engineering)


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Conjugation


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Hfr Conjugation

When it exists as a freeplasmid, the F plasmid canonly transfer itself. This isntall that useful for genetics.

However, sometimes the Fplasmid can become

incorporated into the bacterialchromosome, by a crossoverbetween the F plasmid and thechromosome. The resultingbacterial cell is called an Hfr,which stands for High

frequency of recombination. Hfr bacteria conjugate just likeF+ do, but they drag a copy ofthe entire chromosome into theF- cell.


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Transduction

What is the

vector of

transduction?


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Transduction

Transduction is the process of moving bacterial DNAfrom one cell to another using a bacteriophage.

Bacteriophage or just phage are bacterial viruses.They consist of a small piece of DNA inside a protein

coat. The protein coat binds to the bacterial surface,then injects the phage DNA. The phage DNA then takesover the cells machinery and replicates many virusparticles.

Two forms of transduction:

1. generalized: any piece of the bacterial genome can betransferred

2. specialized: only specific pieces of the chromosome can betransferred.


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General Phage Life Cycle

1. Phage attaches to thecell and injects its DNA.

2. Phage DNA replicates,and is transcribed intoRNA, then translated into

new phage proteins. 3. New phage particles

are assembled.

4. Cell is lysed, releasingabout 200 new phage

particles. Total time = about 15

minutes.


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Generalized Transduction

Some phages, such as phage P1, break up the bacterialchromosome into small pieces, and then package it intosome phage particles instead of their own DNA.

These chromosomal pieces are quite small: about 1 1/2minutes of the E. coli chromosome, which has a total

length of 100 minutes. A phage containing E. coli DNA can infect a fresh host,

because the binding to the cell surface and injection ofDNA is caused by the phage proteins.

After infection by such a phage, the cell contains an

exogenote (linear DNA injected by the phage) and anendogenote (circular DNA that is the hostschromosome).

A double crossover event puts the exogenotes genesonto the chromosome, allowing them to be propagated.


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Specialized Transduction

Unlike the F plasmid that can incorporate anywhere in the E. coligenome, lambda can only incorporate into a specific site, called att.The gal gene is on one side of attand the bio gene (biotinsynthesis) is on the other side.

Sometimes when lambda come out of the chromosome at the end ofthe lysogenic phase, it crosses over at the wrong point. This is very

similar to the production of an F from an Hfr. When this happens, a piece of the E. coli chromosome is

incorporated into the lambda phage chromosome

These phage that carry an E. coli gene in addition to the lambdagenes are called specialized transducing phages. They can carryeither the gal gene or the bio gene to other E. coli.

Thus it is possible to quickly develop merodiploids (partial diploids)for any allele you like of gal or bio. Note that this trick cant be usedwith other genes, but only for genes that flank the attachment site forlambda or another lysogenic phage.


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Transformation


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Plasmids

What is a plasmid?

Small circular, self replicating piece of

bacterial DNA

Episomes = plasmids that can

reversibly incorporate into the bacterial

chromosome

Plasmid genes are advantageous to the

bacteria that has them

Plasmids that confer resistance to

antibiotics are called R plasmids


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Plasmid

plasmid are genetic elements most

frequently transferred by conjugation.

Plasmid F (fertility)

Plasmid R (resistance)

Resistance plasmids = R plasmids made of

RTF(Resistance Transfer Factor) and R

(Resistance) Genes


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R plasmids are promiscuous

R plasmids are promiscuous

Klebsiella

E coli andSalmonella Serratia

Shigella


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Transposons

Jumping genes

Does not depend on complementary base

pairing between homologous regions of

the chromosome.

Transposons move to regions that the

gene has never been


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THANK YOU

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K.6 Microbial Genetics(k Lia)