Microbial GeneticsMicrobial Genetics

Department of MicrobiologyDepartment of MicrobiologyFK USU

GENETICS

• 1928 GRIFFITH - Dead bacteria could Transform livingg1941 AVERY - Showed that the transforming agent was DNA1940' BEEDLE & TATUM O• 1940's BEEDLE & TATUM - One gene, one enzyme Relationship between genes and enzymes

• 1950's WATSON & CRICK - Discovered the structure of1950 s WATSON & CRICK Discovered the structure ofDNA and the structure enabled the discovery of the functions of DNA1950' M CLINTOCK Fi t h d th f• 1950's Mc CLINTOCK - First showed the presence oftransposable genetic elements (transposons)

The Genetic MaterialThe Genetic Material

Frederick Griffith 1928Frederick Griffith, 1928studied Streptococcus pneumoniae, a

pathogenic bacterium causing pneumoniapathogenic bacterium causing pneumoniathere are 2 strains of Streptococcus:

- S strain is virulent- R strain is nonvirulent

Griffith infected mice with these strains hoping to understand the differencehoping to understand the difference between the strains

The Genetic MaterialThe Genetic Material

Griffith’s results:Griffith s results:- live S strain cells killed the mice

li R t i ll did t kill th i- 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 cells

killed the mice

The Genetic MaterialThe Genetic Material

Griffith’s conclusion:Griffith s conclusion:- information specifying virulence passed

from the dead S strain cells into the live Rfrom the dead S strain cells into the live R strain cellsG iffith ll d th t f f thi i f ti- Griffith called the transfer of this information transformation

The Genetic MaterialThe Genetic Material

Avery, MacLeod, & McCarty, 1944Avery, MacLeod, & McCarty, 1944repeated Griffith’s experiment using purified

cell extracts and discovered:cell extracts and discovered:- removal of all protein from the transforming material did not destroy itstransforming material did not destroy its ability to transform R strain cells- DNA-digesting enzymes destroyed allDNA digesting enzymes destroyed all transforming ability- the transforming material is DNAthe transforming material is DNA

The Genetic MaterialThe Genetic Material

Hershey & Chase 1952Hershey & Chase, 1952- investigated bacteriophages: viruses that

infect bacteriainfect bacteria- the bacteriophage was composed of only

DNA d t iDNA and protein- they wanted to determine which of these

molecules is the genetic material that is injected into the bacteria

The Genetic MaterialThe Genetic Material

- Bacteriophage DNA was labeled withBacteriophage DNA was labeled with radioactive phosphorus (32P)

- Bacteriophage protein was labeled withBacteriophage protein was labeled with radioactive sulfur (35S)

- radioactive molecules were trackedradioactive molecules were tracked - only the bacteriophage DNA (as indicated

by the 32P) entered the bacteria and wasby the P) entered the bacteria and was used to produce more bacteriophage

- conclusion: DNA is the genetic materialconclusion: DNA is the genetic material

DNA StructureDNA Structure

James Watson and Francis Crick 1953James Watson and Francis Crick, 1953– deduced the structure of DNA using evidence

from Chargaff Franklin and othersfrom Chargaff, Franklin, and others– proposed a double helix structure

DNA StructureDNA Structure

The double helix consists of:The double helix consists of:– 2 sugar-phosphate backbones

nitrogenous bases toward the interior of the– nitrogenous bases toward the interior of the molecule

– bases form hydrogen bonds with– bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backboneg p p

DNA StructureDNA Structure

The two strands of nucleotides areThe two strands of nucleotides are antiparallel to each other

one is oriented 5’ to 3’ the other 3’ to 5’– 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.

Organization of genesOrganization of genes

• Gene: unit of heredity/ a segment of DNAGene: unit of heredity/ a segment of DNA that carries in its nucleotide sequence information for a specific biochemical or pphysiologic property.

• DNA (deoxyribonucleic acid): ( y )Four bases : A-T , G=C

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

an organismg• Mutation: genetic changes

DNA = RNA= D ib l i id Rib l i idDeoxyribonucleic acid Ribonucleic acid

Nucleotide components Nucleotide components:Nucleotide components 1. Phosphate2. Deoxyriboxe (a sugar) and

Nucleotide components:1. Phosphate2. Riboseand

3. Bases as follows:a) adenine = Ab) i G

3. Bases :a. Adenine = A

b) guanine = Gc) cytosine = Cd) Thymine = T

b. Guanine = Gc. Cytosine = Cd URACIL U dd) Thymine T

DNA IS A DOUBLE HELIX

d. URACIL = U and replaces Thymine

Bacterial Genome:Bacterial Genome:• Main Chomosome + Plasmid ( small (

genetic elements capable of independent replication)p )

• Transposons : genetic element that contain several kbp of DNA, including thecontain several kbp of DNA, including the information necessary for their migration from one genetic locus to another.from one genetic locus to another.

Genetics of BacteriaGenetics 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 cellPacked into nucleoid region of cell• Plasmid = small circular extra piece of DNA• The small size of plasmid renders them

amenable to genetic manipulation, and after alteration, may allow their intoduction into cells.

• Plasmid were identified as small genetic elementPlasmid were identified as small genetic element capable of independent replication in bacteria and yeast

Bacterial genetics, mutations and genetic transfer

• STRUCTURE OF THE DNA (double helix, bases, sugar and phosphate)PROTEIN SYNTHESIS (RNA t i ti d• PROTEIN SYNTHESIS (RNA, transcription and translation, expressed gene)MUTATION (change in sequence of nucleotide• MUTATION (change in sequence of nucleotide bases in DNA, AB resistance, mutagens, carcinogens radiation)carcinogens, radiation)

• TRANSFER (occurs with cell division, transformation, transduction)transformation, transduction)

Bacterial Genetic Recombination

• What is the main source of geneticWhat is the main source of genetic recombination in bacteria?

• Mutations• Mutations• What are the other sources of

bi ti ?recombination?

What causes mutations?What causes mutations?

• 1. Errors during REPLICATION

• 2. Mutagenic agents such as chemical and physical agents

Mutation & Gene RearrangementMutation & Gene Rearrangement

• Spontaneous mutation:Spontaneous mutation:1. Base substituations2 D l ti2. Deletion3. Insertions4. Rearrangement

Why are mutations important? Because the transfer of genetic material from a donor into a recipient followed by recombinationdonor into a recipient followed by recombination is a source of genetic diversity that may result in:

• resistance to antibiotics• resistance to antibiotics(Drug Resistance - Chromosomal mutations and R Plasmids) )

• increase in virulence

Recombination occurs after the transfer of genetic material g

DONOR DNA gets into the RECIPIENT in three ways O O gets to t e C t ee ays

A. TransformationB. Transduction

Life cycle of a lytic phage---->generalized transductionLife cycle of a temperate phage---->specialized transduction

C. Conjugationf fTransfer of F plasmid

Hfr and transfer of chromosomal genes

Transfer of DNAM h i f R bi ti /G T f• Mechanisms of Recombination/Gene Transfer:

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

2. Transduction: donor DNA is carried in a phage coat d i t f d i t th i i t b h iand is transferred into the recipient by mechanism

used for phage infection.

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

ConjugationConjugation

Hfr ConjugationHfr Conjugation• When it exists as a free

plasmid the F plasmid canplasmid, the F plasmid can only transfer itself. This isn’t all that useful for genetics.

• However, sometimes the F l id bplasmid can become

incorporated into the bacterial chromosome, by a crossover between the F plasmid and the h Th ltichromosome. The resulting

bacterial cell is called an “Hfr”, which stands for “High frequency of recombination”.

• Hfr bacteria conjugate just like F+ do, but they drag a copy of the entire chromosome into the F- cell.

TransductionTransduction

Wh t i thWhat is the vector of

t d ti ?transduction?

TransductionTransduction• Transduction is the process of moving bacterial DNA p g

from one cell to another using a bacteriophage.• Bacteriophage or just “phage” are bacterial viruses.

They consist of a small piece of DNA inside a proteinThey 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 takes over the cell’s machinery and replicates many virusover the cell s machinery and replicates many virus particles.

• Two forms of transduction:1 li d i f th b t i l b– 1. generalized: any piece of the bacterial genome can be transferred

– 2. specialized: only specific pieces of the chromosome can be transferredtransferred.

General Phage Life CycleGeneral Phage Life Cycle• 1. Phage attaches to the

ll d i j t it DNAcell and injects its DNA.• 2. Phage DNA replicates,

and is transcribed into RNA th t l t d i tRNA, then translated into new phage proteins.

• 3. New phage particles are assembledare assembled.

• 4. Cell is lysed, releasing about 200 new phage particlesparticles.

• Total time = about 15 minutes.

Generalized TransductionGeneralized Transduction• Some phages, such as phage P1, break up the bacterial

h i t ll i d th k it i tchromosome into small pieces, and then package it into some phage particles instead of their own DNA.

• These chromosomal pieces are quite small: about 1 1/2 i t f th E li h hi h h t t lminutes 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 ofbecause the binding to the cell surface and injection of DNA is caused by the phage proteins.

• After infection by such a phage, the cell contains an exogenote (linear DNA injected by the phage) and anexogenote (linear DNA injected by the phage) and an endogenote (circular DNA that is the host’s chromosome).

• A double crossover event puts the exogenote’s genes• A double crossover event puts the exogenote s genes onto the chromosome, allowing them to be propagated.

Specialized TransductionSpecialized Transduction• Unlike the F plasmid that can incorporate anywhere in the E.

coli genome, lambda can only incorporate into a specific site, called attλ The gal gene is on one side of attλ and the bio genecalled attλ. The gal gene is on one side of attλ and the bio gene (biotin synthesis) is on the other side.

• Sometimes when lambda come out of the chromosome at the end of the lysogenic phase, it crosses over at the wrong point. This is very similar to the production of an F’ from an HfrThis 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 lambda p g y ggenes are called “specialized transducing phages”. They can carry either 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 trickdiploids) for any allele you like of gal or bio. Note that this trick can’t be used with other genes, but only for genes that flank the attachment site for lambda or another lysogenic phage.

TransformationTransformation

PlasmidsPlasmids• What is a plasmid?What is a plasmid?• Small circular, self replicating piece of

bacterial DNAbacterial DNA• Episomes = plasmids that can

reversibly incorporate into the bacterialreversibly incorporate into the bacterial chromosome Pl id d h• Plasmid genes are advantageous to the bacteria that has them

• Plasmids that confer resistance to antibiotics are called R plasmids

PlasmidPlasmid

• plasmid are genetic elements mostplasmid are genetic elements most frequently transferred by conjugation.

• Plasmid F (fertility)• Plasmid F (fertility)• Plasmid R (resistance)

Resistance plasmids = R plasmids made of RTF(Resistance Transfer Factor) and R (R i ) G(Resistance) Genes

R plasmids are promiscuousR plasmids are promiscuous

• R plasmids are promiscuousR plasmids are promiscuous• Klebsiella

E li dS l ll• E coli andSalmonella• Serratia• Shigella

TransposonsTransposons

• Jumping genesJumping genes• Does not depend on complementary base

pairing between homologous regions ofpairing between homologous regions of the chromosome.T t i th t th• Transposons move to regions that the gene has never been

• Transposons do not carry genetic information required to couple their own replication to cellrequired to couple their own replication to cell division,and their propagation therefore depends on their physical integration with a bacterial p y greplicon

• This association is fostered by the ability of transposons to form copies of themselves, which may be inserted within the same replicon

b i t t d i t th lior may be integrated into another replicon• Transposons often seems to insert in a random

pattern mutationspattern mutations

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