Bacterial RecombinationDeepak S HiremathDepartment of Biotech, BIET
Bacterial Recombination• Genetic recombination refers to the exchange of genes between twoDNA molecules to form new combinations of genes on achromosome.• Like mutation, genetic recombination contributes to a population„sgenetic 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 becauserecombination will less likely destroy a genes function and maybring together combinations of genes that enable the organism tocarry 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• Transfer of naked DNA from donor to recipient cell.• Transformation experiment by Griffith showed that DNA is thegenetic material and can be transferred between host and recipientDNA.• E.coli cannot undergo transformation naturally, hence it is madecompetent in the lab.• The process is called „Artificial Transformation‟.
• Bacterial transformation done without mice.• Broth containing non-encapsulated living bacteria, to which deadencapsulated bacteria were added.• After incubation, encapsulated living virulent bacteria were found.• This proves that non-encapsulated bacteria received genes fromdead encapsulated ones and got genes for forming a capsule.Transformation
• After death, cell lysis leads to release of DNA from bacteria.• Other bacteria take up DNA and integrate into their chromosomesby recombination.• recA protein binds to donor and cells DNA and causes exchange ofstrands.• Recipient cell with this combination of genes will now become ahybrid or recombinant.• All its daughter cells will be recombinant.Transformation
• Bacillus, Haemophilus, Streptococcus, Staphylococcus, Neisseriaetc. undergo transformation in nature.• Transformation works best when both donor and recipient areclosely related.• A small portion of DNA is transferred, which is still large to crossthe cell wall and membrane in the recipient cell.Transformation
• Physiological ability to take up DNA is called „Competence‟ andsuch cells are Competent cells.• E.coli cannot undergo transformation naturally, hence madecompetent 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 likecalcium 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 DNAadheres to cell surface.• Divalent cations might also weaken cell surface making it morepermeable to DNA.• Heat shock creates thermal imbalance within the cell.• DNA enters the cell either by pores on the surface or damaged cellwall.Transformation by Calcium Chloride
by Calcium Chloride• Electric shock is given to the cells which creates holes in the poresof the membrane.• DNA enters through the pores.• After the shock, pores are closed rapidly by repair mechanisms ofcell 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 ( Conjugativeor 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 cellsdirectly.• Gram positive bacteria produce sticky surface molecules that bringtwo 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 betransferred.• Donor cells with F factor are F+ cells, recipients without F factor areF- cells.• Donor cells transfer F factor to recipient cell, hence recipient cellsbecome F+ cells.Conjugation
• In donor cells, F factor may integrate into the host chromosomebecoming hfr (High Frequency of Recombination).• Thus F+ cells become hfr cells.• Conjugation between hfr and F- cells results in replication of thechromosome 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 withthe DNA of F- cells.• Thus F- cells receive only a part of chromosomal genes and hencedo not get converted to F+ cells.Conjugation
Transduction• Transduction is the process of moving bacterial DNA from one cellto 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 phageDNA.• The phage DNA then takes over the cell‟s machinery and replicatesmany virus particles.
1. Phage attaches to the cell and injects its DNA.2. Phage DNA replicates, and is transcribed into RNA, thentranslated 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 chromosomeinto small pieces, and then package it into some phage particlesinstead of their own DNA.• These chromosomal pieces are quite small.• A phage containing E. coli DNA can infect a fresh host, because thebinding to the cell surface and injection of DNA is caused by thephage proteins.
• After infection by such a phage, the cell contains an exogenote(linear DNA injected by the phage) and an endogenote (circularDNA that is the host‟s chromosome).• A double crossover event puts the exogenote‟s genes onto thechromosome, 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 specializedtransduction, we need to examine the phage lambda life cycle.• lambda has 2 distinct phases of its life cycle. The “lytic” phase : thephage infects the cell, makes more copies of itself, then lyses thecell 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 incorporatesinto the bacterial chromosome by a crossover.• Once incorporated into the chromosome, the lambda DNA becomesquiescent: its genes are not expressed and it remains a passiveelement on the chromosome, being replicated along with the rest ofthe chromosome.• The lambda DNA in this condition is called the “prophage”.Specialized Transduction
• After many generations of the cell, conditions might get harsh. Forlambda, bad conditions are signaled when DNA damage occurs.• When the lambda prophage receives the DNA damage signal, itloops out and has a crossover, removing itself from thechromosome. Then the lambda genes become active and it goes intothe lytic phase, reproducing itself, then lysing the cell.Specialized Transduction
• lambda can only incorporate into a specific site, called attλ. The galgene is on one side of attλ and the bio gene (biotin synthesis) is onthe other side.• Sometimes when lambda come out of the chromosome at the end ofthe lysogenic phase, it crosses over at the wrong point.• When this happens, a piece of the E. coli chromosome isincorporated into the lambda phage chromosomeSpecialized Transduction
• These phage that carry an E. coli gene in addition to the lambdagenes are called “specialized transducing phages”.• They can carry either the gal gene or the bio gene to other E. coli.Specialized Transduction