Day_7_-_Transformation__Plasmids__Conjug

Day_7_-_Transformation__Plasmids__Conjug - Transformation...

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Unformatted text preview: Transformation Transformation is the process by which cells take up DNA directly from their environment. Terms are similar to those used to describe conjugation are used in discussions of transformation. DNA derived from a donor cell, which may or may not be a bacterium, is taken up by a recipient , which then is called a TRANSFORMANT . Bacterial cells that are in a state where they can take up DNA are described as being COMPETENT . 1. Cells are given a fixed amount of radioactive DNA. 2. The cells are then treated with Dnase and collected on a microfilter (or by centrifugation). 3. DNA in the cells is insensitive to DNase. Degraded DNA will pass through a filter (or remain in the supernatant). The asterisks refer to radioactively labeled DNA. Measuring the efficiency DNA uptake by cells Fig 6.4 1. Some bacteria are naturally competent and take up naked DNA at some phase of their life cycle, most commonly in the late log phase when cell density is high and nutrients become scarce. EXAMPLES: Bacillus subtilis (Gram positive, late log phase), Neisseria gonorrhoeae (Gram negative, possibly competent at all stages of growth). 2. Many bacteria DO NOT take up DNA unless a temporary state of competence is induced either by a chemical treatment , for example, exposure of the cells to 0.1 M CaCl 2 , or an electrical shock ( electroporation ). EXAMPLES: Escherichia coli (Gram ) Pseudomonas aeruginosa (Gram ). Natural and induced competence ATURALLY TRANSFORMABLE bacteria can take up DNA without special treatments . Most of the bacteria that are naturally competent develop this state late during their growth phase, usually shortly before the cells reach stationary phase. ATURAL COMPETENCE occurs in several genera of Gram positive and Gram negative bacteria, including Bacillus subtilis (soil bacterium), Hemophilus influenzae (spinal meningitis), Neisseria gonorrhoeae (guess what!), Streptococcus pneumoniae (throat infections), and species of cyanobacteria from the genera Synechococcus and Synechocystis . In natural settings, the source of transforming DNA until recently was assumed to be cells that incurred some form of unprovoked death . However, recent evidence indicates that transforming DNA may be produced by a) secretion (Neisseria gonorrhoeae, type IV system), b) induced autolysis of stationary cells (Neisseria), and fratricide of non-competent cells by competent cells through induction of bacteriocins and lytic enzymes by CSP (competence stimulating protein) in Streptococcus sp. Natural competence Development of competence in Bacillus subtilis A TWO-COMPONENT REGULATORY SYSTEM controls the development of competence . The ComP protein in the membrane senses high cell density through ComX pheromone concentration, and is autophosphorylated ( ComP~P ). The phosphate is then transferred to ComA ( ComA~P ), which activates the transcription of competence genes . The ComA ComA~P phosphorylation requires two competence pheromone peptides , one of...
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This note was uploaded on 09/14/2010 for the course MMG 431 taught by Professor Bertrand during the Fall '08 term at Michigan State University.

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Day_7_-_Transformation__Plasmids__Conjug - Transformation...

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