Lecture 3 Bacterial Genomes

• Genom = totality of genetic information in a cell
• Uses of a minimal genome (synthetic bacteria): make pigs produce insulin, engineer corn to resist herbicides, synthetically produce pharmaceuticals.
• Adv = large scale benefits for energy, renewable chemicals, sequestering carbon, create beneficial microbes to make them produce bio-products.
• How to get access into a cell? - projection into the cytoplasm, nucleoid structure into domain, translation/translation
  
• Replicatin - semi conservative, OriC, Ter/Dif, clusters of coding regions

Operon - lac, trp, ara 

Induction (lac) and repression (trp). Trp = advantage of cotranscription and horizontal gene transfer

• transfer of DNA in intra and inter species manner
• cause of increased drug resistance where one bacterial cell will acquire resistance and transfer resistance genes to other species e.g. S. aureus
  
• plays a role in the spread of virulence factors such as exotoxins and exoenzymes e.g. Shiga toxin spread to E. coli from Shigella
• Three types: Transformation, Transduction, Conjugation

• donor bacteria and recipient bacteria
• introduction, uptake and expression of foreign genetic material, forced or natural
• Natural = competent cells, only 40 species capable. Induced by: high cell density, nutritional limitation and conditions associated with stationary phase. Gram+/- differ in uptake. DNA will bind to surface of competent cells on a DNA recpetor, pass through membrane by DNA translocase. One strand can pass through and one strand is degraded by nuclease and the translocated DNA can then be integrated into the bacterial chromosomes by a Rec-A dependent process. Due to Gram-negative(extra membrane) needs secretins. Uptake of DNA is generally non-sequence specific.
• Forced = DNA can be taken from dead or lysed cells using heat shock or electroporation techniques. Chemically, cells are incubated in calcium chloride and heat-shocked.
• As the surface of bacteria is -ve charged and DNA is also negatively charged then the CaCl attempts to shield the charges. Incubation in CaCl occurs in cold conditions may change or weaken cell surface structure making it more permeable to DNA therefore when heat shock is applied, this creates a thermal imbalance, forcing the DNA to enter the cells through pores or the damaged cell wall.
• Electroporation works in a similar manner except with an electric field to create the pores, the holes are then closed by the cell membrane’s repair mechanisms. 

• DNA transferred from one bacterium to another by a virus where foregin  DNA is introduced into another cell by a viral vector.
• This method does not need physical contact between donor cell and recipient cells (like conjugation) and is DNAse resistant (unlike transformation).
• When bacteriophages infect a bacterial cells, reproduction occurs to control replicational, transcriptional and translational machinery to produce virions and virus particles
• Transduction occurs through the lytic or lysogenic cycle.
• If the lysogenic cycle is adopted, the phage is integrated (by covalent bonds) into the chromosome where it can remain dormant for generations. If induced through UV light for example, the phage genome is cut from the bacterial chromosome and initiates the lytic cycle resulting in cell lysis and release of phage particles.
• The lytic cycle skips the lysogenic phase and the new phage particles are released quicker. Generalised transduction is the process by which any bacterial gene can be transferred to another bacterium by a bacteriophage and only carries bacterial not viral DNA.
• Specialised transduction is the process by which a set of bacterial genes are transferred to another bacterium and occurs when the prophage is cut from the chromosome so the genes next to the prophage are included. This is then packaged into a new virus particle for delivery of the DNA to a new bacterium.

• Uses a sex pilus so recipient that received the plasmid can give it to the next bacteria, requires stable and extended contact & incorporates by homologous recombination.
• There are several types of plasmids - The F plasmid (F-factor) encodes all the functions needed for their transfer and the transfer of other DNA elements and mobilised plasmids into the recipient cell. These plasmids contain the Tra gene and Ori T sites.
• The second type- mobilizable plasmids which encode its transfer into the recipient cell only and requires the help of the F plasmid. The Tra gene composed of Dtr and mpf. Dtr prepares the plasmid for transfer and includes relaxases (site specific endonuclease which acts at OriT, the relaxosome complex (consists of groups of proteins around the OriT and helps bind relaxases to the OriT) and primase (synthesises primers to complete replication). Mpf holds the donor and recipient cell together to form a channel which DNA is transferred and signal Dtr to transfer. Composed of the pilus, channel and coupling protein. The pilus holds the donor and recipient cells together and projects out of the cell, the channel also encoded by Tra mediates the transfer and the coupling proteins signal the relaxases to initiate transfer also. The Ori T site is where the plasmid DNA transfer initiates in the donor cell and the recycling site in the recipient cell, recognised by relaxase.
• Conjugation occurs by firstly pilus formation, then physical contact between donor and recipient, transfer of the F-plasmid followed by complementary strand synthesis.