Lecture 13: Heritable Traits continued

Transformation: the uptake of naked (histone free) DNA in the form of

- Linear DNA
- Plasmid DNA
o Extrachromosomal dsDNA
o Circular
o Replicate autonomously from host chromosome
o Routinely encodes up to 30 genes the confer a selective advantage
 Antibiotic resistance
 Virulence factors
 Carbon source utilization

In nature, transformations can occur naturally but rare

- Only less than 1% bacteria can take up naked DNA Gram negative
o Staphylococcus, Streptococcus, Baccillus
- Only occurs between closely related species
- Uptake of DNA depends on the expression of competence proteins
(proteins that interact with incoming DNA) Gram positive
o Span cell wall and membrane
o Recognize specific DNA sequences and permit linear DNA uptake
o DsDNA interacting with protein, single strand interacts with N
(nuclease: breaks phosphodiester backbone) degrades second strand of DNA as it
enters cell, only one strand gets into cell

In the lab, strains can be made competent that are not competent in nature (ex. E. coli)
- How?
o CaCl2 treatment  causes transient pore formation in membrane that allows DNA
uptake
o Electroporation  exposure of cell to transient high electric field
o Biolistics  shoot at high velocity inert beads coated with DNA

Transformation of bacteria with linear (extracellular) DNA

In nature there are competence proteins
In the lab: artificial means to create competent cells also work

- Extracellular ssDNA can enter bacterium

- Endogenote, exogenote  merozygote

Two fates
Recombination can occur
Degradation of DNA (endonuclease)  unsuccessful
transformation

Bacterial Transformation Movie

Transformation of bacteria with plasmid DNA
- Artificial uptake
o Plasmid must be lost in subsequent generations if there is no selection
pressure
- Stable extrachromosomal DNA

Transformation of Bacteria
- Linear DNA integrates into recipient genome at regions of high sequence similarity
- 1 – more than 1000 copies (depends on the type) of the plasmid will be present in the
transformed strain
- The plasmid can also integrate if it has special sequences associated with it called insertion
sequences

Conjugation
- Another mechanism of non-reciprocal recombination
- Recipient receives donor DNA
- Therefore, conjugation requires cell to cell contact
o Contact point = pilus
- DNA from donor (plasmid DNA) and sometimes some of the donor chromosomal DNA gets
transferred during conjugation
- The plasmid DNA is the conjugative plasmid
o Encode 30 genes in the tra operon (transfer)
- Operon: DNA sequences that contain one or more structural genes and the operator
controlling their expression
o Encode the genes to allow pilus assembly and DNA transfer
o Make up the F factor or fertility factor

Conjugation by the F factor

- F factor in conjugative plasmid and donor chromosome in same
cell F+ cell (donor)
- Other bacterium  F- cell (recipient)
- Linking donor and recipient together is the pilus
- Pilus shortening brings cells into closer proximity

- A protein complex called the relaxosome cuts the F factor DNA to

be transferred at the origin of transfer
- Accessory proteins of the relaxosome are released, but relaxase
remains attached to donor DNA and together with coupling factor
brings it to the exporter region of the pilus
- Only single stranded DNA is transferred
- Relaxase used to join the two ends of DNA  DNA is replicated

Bacterial Conjugation Movie

F factor
- If F factor inserts into the chromosome of host cell, recombinant cell is known as Hfr strain
(High frequency of recombination)
 o Bacterial strain the donates its genes with a high frequency to a recipient cell during
conjugation because F-factor is integrated into bacterial chromosome

Conjugation of an Hfr Strain with an F- Strain

- F factor is integrated into chromosome of donor cell
- Pilus attached the cells and pulls them closer together Hfr F-
- New strand is synthesized by rolling circle replication
- Host chromosome transferred first
- Recombination

Transduction: transfer of genes between bacterial or archaeal cells by viruses

- Donor DNA is carried to recipient cell via an intermediate = bacterial virus
= bacteriophage (phage)
- Direct contact of the phage and recipient cell is required

Bacteriophage
- Phage injects its own phage DNA into recipient cell
- New phages can be generated in the recipient cell
- Bacterial cells can lyse releasing the phages

Bacteriophage infections
Host bacteria can be infected in two ways
Lytic infection
- Phage contacts bacterium
- Injects phage DNA
- Phage DNA is transcribed and
replicates
- Host DNA is degraded
- New phage particles are assembled
- Host cell lyses

Lysogenic infection
- Phage injects host bacterium
- Phage DNA incorporated into host
chromosome (=prophage)
- Phage DNA replicates with host bacterium
- Results in phage DNA excision
- Lytic cycle can ensue

Bacteriophages
- Virulent bacteriophages = lytic phage
- Temperate bacteriophages = lysogenic cycle
- Lysogeny will not persist indefinitely
o Exposure of host bacterium to stress (UV light exposure, dessication, nutrient
starvation)
o Lytic cycle can be reinstated
Mechanism of generalized transduction

host DNA degraded

Some phage accidentally incorporates host DNA and can infect another bacterium

Recombinant

One plasmid can influence human disease

- The EAF (E. coli adherence factor) plasmid
- Huge plasmid over 10,000 base pairs
- Required for full disease to be caused by pathogenic E. coli
- Needed for adhesion
- Encodes bfp (bundle forming pili)
- Has tra operon
- When deleted from bacteria, only 2/9 volunteers had diarrhea, mild diarrhea