1. Plasmids Methods of DNA exchange 2 nd semester 17-2-2015

2. Bacteria can exchange DNA among them in three different ways. In every case the source cells of DNA are called DONORS and the cells that receive the DNA are called RECIPIENTS. In each case the donor DNA is incorporated into the recipients cell's DNA by the process of recombination. 2

3. Plasmids are pieces of DNA that exist separate from the chromosome. They contain an Origin of replication (ori) and, as such Replicate independently from the chromosome. Plasmid contain a variety other genes; antibiotic resistant gene, genes involved in causing disease, and genes for degradation of different carbon sources 3 Plasmids

4.  Because plasmids have a mechanism to replicate their DNA, ensuring that they are stably maintained in a cell population, they are also known as replicon. 4

5.  Low copy number plasmids (1-2 copy)  High copy number plasmids (50 copies) 5

6.  High copy no. plasmid do not have any mechanism of partitioning.  Low copy no. plasmids must have a mechanism to ensure their proper partitioning. 6

7.  parS : A specific site on low copy number plasmids required for proper and equal segregation.  ParA & parB proteins bind to parS sequence. * How parS, parA and parB protein complex function? Keep the plasmid molecule in the middle of the cell until daughter cell are clearly distinguished. 7

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10.  Maintenance of more than one plasmids in the cell can only occur if the plasmids can only carry different origins of replication.  The inability of two plasmids with the same origins to be maintained in the same cell is known as incompatibility. 10

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13.  Plasmid that can be replicated and maintained in many different bacterial species are known as broad host range plasmids. 13

14. RequirementContactGenes transferred in most Transformation Free donor DNA fragment Competent recipient cell no Most capsule gene, unlimited Conjugation Sex pili on donor Fertility plasmid in donor Live cells Closely related species yesDrug resistance, resistant genes to toxin, enzymes etc. Transduction Donor lysed by phage Defective phage carries donor DNA Live competent recipient cell no Toxins, drug resistance

16. Transformation: Is the process that allows bacteria to take up free or naked DNA from their surrounding environment.  Bacteria that have undergoes transformation are called transformants.  Transformation requires that the bacterium to be naturally competent to take up DNA from their surrounding environment.

17.  When do the bacterial cell become competent to take up DNA ?  the physiological state of the cell influences its ability to become competent.

18.  Based on Cell density (end of the exponential phase) Depletion carbon, nitrogen and phosphorus Transformation Sporulation

19. Two pathways activate the competence genes in B. subtilis. P1 sense the cell density, and when enough cells are present, signals the cells to induce the com genes. P2 monitors the available nutrients and when they become limiting, signals the cells to induce the com genes.

20.  B. subtillis can sense the cell density by monitoring a small peptide (comX) concentration in the surrounding medium, (use com X to sense the cell density and induce the com genes). ComX is produced in the cytoplasm as a larger precursor molecule, the comQ protein processes and modifies precursor comX to make a smaller active peptide. The active comx is excreted from the cell. How bacteria can sense the changes in cell density?

21.  By two component signal transduction systems. Bacteria use a membrane protein called sensor-kinase to sense an environmental signal. The sensor-kinase autophosphorylates on a specific histidine residue in the protein. The sensor- kinase transfer this phosphate to another protein in the cytoplasm called response regulator. This activates the response regulator so that it can induce and/ or repress specific genes. These genes allow a cell to respond to the original environmental signal in an appropriate way. How bacteria monitors nutrient concentration?

22.  ComP and comA comprise a two components signal transduction system in B. subtillis for the induction of the competency genes. (a) ComX binds to comP. (b) This binding induce autophosphorylation of comP. (c) The phosphate is relied to comA. (d) the phosphorylated comA activates the comS genes. (e) ComS leads to the induction of the competency genes.

23.  ComP, comA and comX depend on cell density therefore they called quorum sensing system.

24. Transformation in B. subtillis a- A pillus called IV pillus binds double stranded DNA. b- The DNA is transferred to the comEA receptor. c- An endonuclease comI recognizes the receptor with bound DNA. d- ComI degrade one strand of the DNA. e- The single stranded DNA is transported in to the cell and protected by a single stranded DNA binding protein.

25.  How DNA selected by the competent cell?  Some species that are naturally competent can only take up DNA that contains specific base sequence called USS or uptake signal sequence.

26.  Some species used a specialized structure called a transformasome to import DNA. The DNA most contain a USS and initially is double stranded. During the process of translocation, the DNA is degraded to a single – stranded molecule.

27.  Mapping genes requires that a piece of chromosomal DNA from one bacterium be introduced to another bacterium of the same species. Transformation as a molecular tool:  Introducing genes to many different bacteria.