Gene Transfer 11/4/13. Fredrick Griffith in the 1920s did an experiment. Not until 1944 was DNA shown to be the moveable element

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1 Gene Transfer Fredrick Griffith in the 1920s did an experiment. Not until 19 was DN shown to be the moveable element Dead pathogen cells able to make a capsule were able to pass this ability to the live cells NOT able to make a capsule. Organisms injected Living encapsulated cells Results Mouse dies The live, capsule-forming cells were able to infect Living non-encapsulated cells No effect Found that the material exchanged was DN Heat-killed encapsulated cells No effect This lead to the discovery of horizontal gene transfer (HGT). How does this work? Heat-killed encapsulated cells + Mouse dies Living non-encapsulated cells Living encapsulated cells isolated Gene transfer in bacteria There are three types of gene transfer 1. Transformation DN enters and is incorporated (cell imports it) 2. Conjugation DN moves from one bacterial cell to another during cell to cell contact. Transduction a virus injects DN into the bacterial cell 1

2 ll types of gene transfer Involve unidirectional transfer of information (donor-->recipient) Require the integration of newly acquired DN homologous recombination Increases genetic diversity Terms to remember Replicon DN containing an origin of replication (O.R.) This allows the DN to be copied. If no O.R. then it must be incorporated into the chromosome for replication. Homologous recombination DN has parts that match the DN in the chromosome, and bind to it to be incorporated into the chromosome. Competent bacterial cell that can take up DN from the environment is termed competent Naked DN outside of the cell, or virus 2

3 (a) Non-integrated DN fragment acterial chromosome DN fragment (no origin of replication) DN molecules without an origin of replication cannot replicate in a cell. Only one daughter cell will have a copy of the DN fragment. (b) Integrated DN fragment Homologous recombination DN fragment (no origin of replication) DN fragment integrated into a bacterial chromosome can be replicated and passed on to daughter cells. ll daughter cells will have a copy of the DN fragment. Transformation There are structures bacterial cells use to bind and import DN. These include the type pili aparatus, which are also used for making pili. Gene conferring Str S 1 2 Gene Conferring Str R Recipient chromosome Double-stranded DN binds to the surface of a competent cell. Single strand enters the cell; the other strand is degraded. The strand integrates into the recipient cell s genome by homologous recombination. Some bacteria take in any DN, while others stick to specific DN sequences and take in only DN they want. 5 Streptomycin-sensitive Streptomycin-resistant daughter cell daughter cell fter replicating the DN, the cell divides. Non-transformed cells (Str S ) die on streptomycin-containing medium, whereas transformed cells (Str R ) can multiply. ---acteria that take up DN from the environment often regulate this uptake. ---Environmental changes may cause them to become competent for taking in free DN. ---Or, they are competent all the time. acillus subtilis Senses N and C limitation and turns on competence genes lso uses quorum sensing must have a large number of cells first cinetobacter is competent all the time. (we used these in lab to test DN uptake) Neisseria gonorrhea is competent but only takes up DN with specific sequences from other Neisseria. Enables antigenic drift.

4 11//1 Conjugation Transfer of genes between 2 bacterial cells Gram negative cells use a sex pilus F(+) cells have F plasmid, F(-) lack F plasmid Conjugation between (F+) and F(-) cells First, the F pilus binds to specific receptor on the F- cell. Pilus retracts (gets shorter) and brings the cells closer. The F plasmid requires an Origin of Transfer we will refer to this as the OoT!! Without the OoT, DN can not be moved using the F pilus. The OoT is nicked to open the DN and one single strand of the plasmid moves through the pilus to the other cell. Then the complement of each of the ssdn plasmids is made, and voila! 2 F+ cells. F plasmid can move into the chromosome creating Hfr cell High frequency of recombination = large number of genes were incorporated

5 11//1 Hfr cell can also become a F cell Similar to when a virus takes some of the host DN out of the chromosome These F plasmids can move bacterial DN from one cell to another Formation of an Hfr cell Chromosome F plasmid The F plasmid sometimes integrates into the bacterial chromosome by homologous recombination, generating an Hfr cell; the process Integrated is reversible. F plasmid F pilus F+ cell Hfr cell Formation of an F cell Integrated F plasmid Chromosome Hfr cell Chromosomal DN n incorrect excision of the integrated F plasmid brings F plasmid along a portion of the chromosome, generating an F cell. F pilus F cell 1 Conjugation between Hfr cell and F(-) 1. The OoT is in the plasmid piece and this is the end that begins moving through the pilus. 2. ecause the whole chromosome is too big to move all the way over, only a piece goes, and the other half of the plasmid is NOT transferred. The intact OoT is lost.. This creates an F(-) recombinant cell Making contact Integrated F plasmid Chromosomal genes Origin of transfer Chromosome C a b Donor cell Hfr c Recipient cell F The F pilus contacts the recipient F 2 Transferring DN Origin of transfer Origin of transfer C a b c single strand of the donor chromosome begins to be transferred, starting at the origin of transfer. Gene, closest to the origin, is transferred first. DN synthesis creates complementary strands in both cells. Only half an OoT! Transfer ends C a b Note:figure # should have an Hfr cell and a F(-) recombinant cell C The donor and recipient cells separate, interrupting DN transfer. Integration of transferred DN C C Hfr cell F cell The donor DN is integrated into the recipient cell s chromosome by homologous recombination. Unincorporated DN is degraded. The recipient cell is still F. 5

6 11//1 Transduction Transfer of genes from a phage to bacterial cell Generalized transduction: occurs with lytic or lysogenic phage (section 8.7) Specialized transduction: occurs with lysogenic phage (section 1.) (a) Formation of a transducing particle (b) The process of transduction Phage Transducing particle bacteriophage 1 attaches to a specific receptor on a host cell. 1 acterial DN 2 The phage DN enters the cell. Enzymes encoded by the phage genome cut the bacterial DN Replaced host DN 2 transducing particle attaches to a specific receptor on a host cell. The bacterial DN is injected in to a cell. The injected bacterial DN inserts into the chromosome by Homologous recombination. Phage nucleic acid is replicated and coat proteins synthesized. Transducing particle During construction of 5 viral particles, bacterial DN mistakenly enters a protein coat. -- now a transducing particle acteria multiply with new genetic material. Replaced host DN is degraded. Specialized Transduction What type of phage is involved?--lysogenic Will it result in generation of new phage? 6

7 The Mobile gene pool In E. coli, 75% of genes are found in all strains Rest of the genome is made up of the mobile gene pool or mobilome Mobile gene pool made from plasmids, transposons, or phage DN Comparison of mechanisms of DN transfer Plasmids Found in many types of organisms as dsdn Provide the cell new characteristics 7

8 Plasmids-types Can be broad host range Or specific to particular species Some can be maintained within the same cell as others (rranged in groups by compatibility) Some can not, and are not compatible High copy number vs. low copy number Conjugative plasmids, carry genes needed for conjugation Mobilizable plasmids have an OoT, but not the conjugation genes. If conjugative and mobilizable are together, they can both be moved to a new cell. Resistance Plasmids (R plasmids) Transposons way to move genes between organisms Insertion sequence Mobile element Inverted repeat Transposase gene Inverted repeat 5 5 T C G T G C T C G G C T C...G T G C T 5 5 Composite transposon Mobile element Insertion sequence ntibiotic-resistance gene Insertion sequence 8

9 Vancomycinresistance gene (encoded on a transposon on a plasmid) Plasmid How did this S. aureus become Vancomycin resistant S. aureus (VRS)? Staphylococcus aureus sensitive to vancomycin Enterococcus faecalis plasmid transferred Enterococcus faecalis by conjugation resistant to vancomycin Transposon jumps from one plasmid to another. Plasmid from Enterococcus faecalis is destroyed. Vancomycin-resistant Staphylococcus aureus 9