BACTERIAL TRANSFORMATION



SEQ CHAPTER \h \r 1BACTERIAL TRANSFORMATIONNomenclature1. Donor cell (DNA)2. Recipient Cell3. Transformant (recipient with donor DNA)Features1.low frequency event2.requires homologous recombination3. one of the best ways to introduce altered DNA into a cellDNA uptake can be greatly enhanced.(104 - 106 increase)If incubate cells under certain conditions - competent cellsNaturally Transformable1. About 40 species known2. Gram positive – Bacillus, Streptococcus pneumoniae3. Gram negative – Haemophilus influenzae, Neisseria gonorrheae4. Requires about 12 genesTwo Stages for Transformation:petence (Naturally Competent)1. conditions vary by species2. percent of cells also variesMechanism1.not well understood2.probably changes in the cell wall3.synthesis of CW material at a particular stage of growth4.receptors are formed or activated - responsible for initial binding of DNA(similar to ABS transporters)a. Bacillus subtilis – com genesb. Gram negative – competence systems similar to type IV pili formation of a “pseudopilus” that binds to DNA and pulls it into the cell5.number of receptors varies by species/genusReceptors only bind SS-DNAReceptors:Some strains uptake of any DNA - Most receptors bind any DNASome strains only uptake of own species’ DNA(own DNA contains “uptake sequences” – 10 bp in length)Factors that influence development of competence:a.type of mediab.stage of growth (log)c.degree of aeration6.Some cells secrete "competence factors" – small peptides excreted by cells as they multiply that activate the rest of the cells to become competent...probably bind to receptors.- species specific- factors trigger "competence factor" production - this is a form of quorum sensing“competence pheromones” – small peptides triggered by increased cell density in stationary phase examples:a. B. subtilis – 10% of all cells no matter how favorable the conditionsb. S. pneumoniae – CSP (competence-stimulating peptide, which activates 20 competence genes, allows cells to know presence of nearby cellsc. Genome sequencing reveals competence genes in other MO yet natural competence is not observed.B.DNA Uptake Possible results of DNA Uptake by a cell: 1. plasmid that circularlizes 2. SS-DNA – homologous recombination 3. DNA is degraded in the cellTwo Stages of DNA uptake1.brief, reversible binding stage2.longer, irreversible stage-resistant to DNase-requires energy-passes through cell membraneDNA must be DS to be attached to CM.DNA is denatured to SS-DNA by nuclease contained in a multi-protein complex in cell membraneOnce inside the cell the SS-DNA is coated by SSBLength SS-DNA that can be taken up: 8.5 -12 KBGram Positive TransformationUses com genes (competence)comG – codes for pseudopilus (like type IV pili used in conjugation)1. DS DNA at the cell surface cleaved by NucA endonucleasecan take in 6 – 15 Kb of DNA2. DNA brought through cell wall by retraction of the pseudopilus3. DS-DNA on the outside of the membranenuclease makes SS-DNA as goes through channel in membraneuses an ATP-dependent DNA translocasetransport rate – 80-100 nucleotides /second (10 KB in 2 minutes)Gram Negative Transformation2 fundamentally different systems/mechanismsA. similar system to type II secretion system/pseudopilus in Gm+ cellspore through the outer membranetype IV pili (long and thin, used for attachment) binds to DNA and pulls it into the cellexonucleases – DS-DNA to SS-DNAB. TransformasomesCapture DNA in membrane-associated vesicles, DS-DNA becomes SSthen transported through the inner membrane1. binding of the DS-DNA to the outer cell surface2. movement of DNA across cell wall/outer membrane3. degradation of one DNA strand so only SS-DNA enters cell4. translocation of SS-DNA across inner membrane to cytoplasmNatural vs. Induced TransformationInduced (Manipulated) 1. Calcium ion induction – reacts with cell wall and makes cell wall “leaky” 2. Electroporation – creates artificial porescan promote transinfection (uptake of phage DNA) 3. Protoplasting – PEG triggers membrane fusion events that trap DNA 4. Heat/cold shockRole of Natural Transformation1. Nutrition – cell death and cannibalismDNA used as a carbon or nitrogen source2. Repair – uptake of same species DNA can repair DNA damagemain role of homologous recombinationB. subtilis and S. pneumo – competence induces expression of RecA3. Recombination – increase genetic variabilityTransformation in bacteria may serve the same function as sex in eukaryotic cellsAllows assembly of new combinations of genes, increase speed of evolutionObservation – some cells “leak” DNA as they growNeisseria – type IV secretion system for active transport of DNA from cell to media ................
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