Bacterial Recombination

Deepak S HiremathDepartment of Biotech, BIET

Bacterial Recombination• Genetic recombination refers to the exchange of genes between two

DNA molecules to form new combinations of genes on a chromosome.

• Like mutation, genetic recombination contributes to a population‘s genetic diversity, which is the source of variation in evolution.

• In highly evolved organisms such as present-day microbes, recombination is more likely than mutation to be beneficial because recombination will less likely destroy a gene's function and may bring together combinations of genes that enable the organism to carry out a valuable new function.

• Vertical gene transfer – From parents to offspring.

• Horizontal gene transfer – From one microbe to another.

• Part of total DNA from Donor cell integrated into Recipient cell.

• Remaining amount of DNA from donor cell degraded.

• Recipient cell with DNA from donor is called Recombinant.

• 1% of population might undergo Recombination.

Bacterial Recombination

• Transformation.

• Conjugation.

• Transduction.

Bacterial Recombination

Transformation• Transfer of naked DNA from donor to recipient cell.

• Transformation experiment by Griffith showed that DNA is the genetic material and can be transferred between host and recipient DNA.

• E.coli cannot undergo transformation naturally, hence it is made competent in the lab.

• The process is called ‘Artificial Transformation’.

• Bacterial transformation done without mice.

• Broth containing non-encapsulated living bacteria, to which dead encapsulated bacteria were added.

• After incubation, encapsulated living virulent bacteria were found.

• This proves that non-encapsulated bacteria received genes from dead encapsulated ones and got genes for forming a capsule.

Transformation

• The material responsible for transmission of this character was not known.

• In 1944, Oswald T Avery, Colin M Macleod, Maclyn Mccarty proved that DNA is the genetic material.

Transformation

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Transformation

• After death, cell lysis leads to release of DNA from bacteria.

• Other bacteria take up DNA and integrate into their chromosomes by recombination.

• recA protein binds to donor and cells DNA and causes exchange of strands.

• Recipient cell with this combination of genes will now become a hybrid or recombinant.

• All its daughter cells will be recombinant.

Transformation

• Bacillus, Haemophilus, Streptococcus, Staphylococcus, Neisseria etc. undergo transformation in nature.

• Transformation works best when both donor and recipient are closely related.

• A small portion of DNA is transferred, which is still large to cross the cell wall and membrane in the recipient cell.

Transformation

• Physiological ability to take up DNA is called ‘Competence’ and such cells are Competent cells.

• E.coli cannot undergo transformation naturally, hence made competent in the lab.

• This procedure is comparatively easy and simple.

• Involves Calcium chloride or Electroporation.

Transformation

• Mechanism is unclear.

• Cells are incubated in a solution containing divalent cations like calcium in cold condition and a rapid heat shock is given.

• Surface of E.coli is negatively charged (Phospholipids, Lipopolysaccharides) as well as DNA is negatively charged.

Transformation by Calcium Chloride

• The divalent cation shields the negative charges and hence DNA adheres to cell surface.

• Divalent cations might also weaken cell surface making it more permeable to DNA.

• Heat shock creates thermal imbalance within the cell.

• DNA enters the cell either by pores on the surface or damaged cell wall.

Transformation by Calcium Chloride

by Calcium Chloride• Electric shock is given to the cells which creates holes in the pores

of the membrane.

• DNA enters through the pores.

• After the shock, pores are closed rapidly by repair mechanisms of cell membrane.

Transformation by Electroporation

Conjugation• Needs extra chromosomal elements called Plasmids.

• Plasmids replicate independently of chromosome.

• They carry non-essential genes for growth during normal conditions.

• They give advantage for cells during stress.

• Ex: Antibiotic resistance genes.

• Plasmids can be transferred from one cell to another ( Conjugative or transferable plasmids).

Conjugation vs. Transformation• Conjugations needs direct cell to cell contact.

• Conjugating cells must be of opposite mating type.

• Donor cells carry plasmids, recipient cells don’t.

• Gram negative bacteria produce sex pili which contacts both cells directly.

• Gram positive bacteria produce sticky surface molecules that bring two cells in contact.

• Single strand is transferred from donor to recipient.

• In the recipient the SS plasmid is replicated.

• In E.coli, Fertility (F) Factor was the first plasmid observed to be transferred.

• Donor cells with F factor are F+ cells, recipients without F factor are F- cells.

• Donor cells transfer F factor to recipient cell, hence recipient cells become F+ cells.

Conjugation

• In donor cells, F factor may integrate into the host chromosome becoming hfr (High Frequency of Recombination).

• Thus F+ cells become hfr cells.

• Conjugation between hfr and F- cells results in replication of the chromosome with F factor.

• A single parental strand is transferred from hfr cell to the F- cells.

Conjugation

• Complete transfer of the chromosome does not take place.

• Only a small piece of F factor leads the chromosomal genes into F- cells.

• The small strand containing chromosomal genes recombines with the DNA of F- cells.

• Thus F- cells receive only a part of chromosomal genes and hence do not get converted to F+ cells.

Conjugation

Transduction• Transduction is the process of moving bacterial DNA 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 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 virus particles.

Transduction

1. Phage attaches to the cell and injects its DNA.

2. Phage DNA replicates, and is transcribed into RNA, then translated into new phage proteins.

3. New phage particles are assembled.

4. Cell is lysed, releasing about 200 new phage particles.

Total time = about 15 minutes.

Transduction

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

into small pieces, and then package it into some phage particles instead of their own DNA.

• These chromosomal pieces are quite small.

• A phage containing E. coli DNA can infect a fresh host, because 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 an endogenote (circular DNA that is the host’s chromosome).

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

Generalized Transduction

• Some phages can transfer only particular genes to other bacteria.

• Phage lambda (λ) has this property. To understand specialized transduction, we need to examine the phage lambda life cycle.

• lambda has 2 distinct phases of its life cycle. The “lytic” phase : the phage infects the cell, makes more copies of itself, then lyses the cell to release the new phage.

Specialized Transduction

• The “lysogenic” phase of the lambda life cycle starts the same way: the lambda phage binds to the bacterial cell and injects its DNA.

• Once inside the cell, the lambda DNA circularizes, then incorporates into the bacterial chromosome by a crossover.

• Once incorporated into the chromosome, the lambda DNA becomes quiescent: its genes are not expressed and it remains a passive element on the chromosome, being replicated along with the rest of the chromosome.

• The lambda DNA in this condition is called the “prophage”.

Specialized Transduction

• After many generations of the cell, conditions might get harsh. For lambda, bad conditions are signaled when DNA damage occurs.

• When the lambda prophage receives the DNA damage signal, it loops out and has a crossover, removing itself from the chromosome. Then the lambda genes become active and it goes into the lytic phase, reproducing itself, then lysing the cell.

Specialized Transduction

Lysogenic life cycle

• lambda can only incorporate into a specific site, called 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.

• When this happens, a piece of the E. coli chromosome is incorporated into the lambda phage chromosome

Specialized Transduction

• These phage that carry an E. coli gene in addition to the lambda genes are called “specialized transducing phages”.

• They can carry either the gal gene or the bio gene to other E. coli.

Specialized Transduction