1459494
| Question | Answer |
| Introduction | Horizontal Gene Transfer: Donor forms MATING PAIR PILUS 2 Strands of plasmids separate similar to rolling circle replication Two single strands replicated concurrently with transfer |
| History | -Lederberg & Tatum: first observed "gene recombination in E. coli" -Mixed 2 different auxotrophic strains -> strains unlike either parent Possible explanations for "assortment of genes in new combinations": cell fusion, transforming factors in media, bacterial sex |
| "Fertility" (F) Plasmid | First conjugative plasmid discovered. Entire 100kb sequence is known, containing sequences from many different sources -tra genes are required for transfer -3 replicative origins: RepFIA, RepFIB, RepFIC (only RepFIA/oriV functional) -Partitioning system Selfish DNA Elements: -Toxin/anti-toxin systems for postsegregational killing -Genes to prevent induction of SOS response -Genes that block T7 phage development |
| Tra genes | -Mpf component = Mating Pair Formation -Dtr component = DNA transfer & conjugal replication |
| Mating Pair Formation | Channel that transfers DNA from donor to recipient -Pilus is made of many copies of PILIN protein. Retraction draws donor & recipient together to form MATING PAIR -Type IV Secretion Systems: Conserved in many organisms; multi-protein machine that makes synthesizes pilus. After retraction, serves as conduit for DNA or virulence factor (in eukaryotes) transfer |
| Relaxosome | Protein complex that binds @ oriV and prepares plasmid for transfer. Tral Relaxase: site-specific endonuclease that nicks & attaches to 5' end at oriT via transesterification. -Pilots strand into recipient cell -Recircularizes transferred strand via transesterification in recipient -TraY & host IHF -> essential accessory proteins for nicking -TraM & TraD (MPF gene) -> coordinate DNA processing with transfer apparatus -TraU -Helicase |
| Triparental Mating | Highly efficient. Uses 3 strains: 1. Carrying self-transmissible plasmid 2. Carrying mobilizable plasmid w/ gene(s) to be moved 3. Desired recipient strain I. Some self-transmissible plasmid will transfer -> strain #2 II. Quickly spread through strain with mobilizable plasmid because conjugation is depressed immediately after transfer III. Efficiently move mobilizable plasmid to recipient for same reason |
| Coupling Proteins | TraD: Dtr & Mpf components Signal contact w/ recipient cell to the Dtr machinery -Provide specificity (can only transfer some plasmids) -DNA translocator that pumps DNA into recipient is related to FtsK -Key component that turns type IV protein secreting machine -> DNA transfer device |
| DNA Transfer | 1. Form mating pairs 2. Coupling protein signals to relaxase 3. Relaxase & helicase make single-strand nick at *oriT* 4. Relaxase & helicase (bound to DNA) are translocated to recipient 5. Coupling protein pumps DNA out of donor cell. Relaxase seals nick in recipient cell 6. Replicate second strand in both cells 7. Separate mating pair -> Donor and Transconjugant |
| Regulation of Conjugation | Cell contacts through plasmid initiation -> 100% result in DNA transfer Plasmid transfer occurs at high rates in recent recipients but later, after initial MPF, it occurs only sporadically FinP & FinO regulate traJ expression -TraJ: transcriptional activator of tra genes -FinP: sRNA that inhibits traJ mRNA translation & stimulates degradation -FinO: Stabilizes & unfolds FinP |
| Conjugative Elements | Self-Transmissible: encode all functions needed to move amongst cells OR Mobilizable: Transferredby a self-transmissible plasmid in the same cell. Contains its own Dtr system, *mob genes*. Mobilized by a variety of tra systems. Relaxases of mobilizable plasmids evolved to communicate with many coupling proteins of many self-transmissible plasmids In practice, only *oriT* region is needed for plasmid to be mobilized by a compatible self-transmissible plasmid. |
| Conjugate Element: Mobilizable | Transferred by a self-transmissible plasmid in the same cell. Contains its own Dtr system, *mob genes*. Mobilized by a variety of tra systems. Relaxases of mobilizable plasmids evolved to communicate with many coupling proteins of many self-transmissible plasmids In practice, only *oriT* region is needed for plasmid to be mobilized by a compatible self-transmissible plasmid |
| Conjugative Element: Promiscuous | Can transfer DNA between unrelated species (Cyanobacteria, Gram + bacteria, plants) Important in evolution -> similar genes in different organisms Contributes to spread of antibiotic resistance: -Bacteria carrying R plasmids selected for in normal flora -Can be passed to pathogens |
| Antibiotic Resistance | {New Delhi metallobetalactamase (NDM) genes allows bacteria to become resistant to multiple antibiotics} Some bacteria acquire resistance genes through: Transformation {from exogenous DNA}, transduction {bact. -> phage -> bact}, conjugation {bact. -> bact.} |
| Evolution | Conjugative plasmids can transfer chromosomal DNA between bacteria; acquire new genes/mutations Through Hfr/chromo DNA -> recipient OR through imperfect excision of Hfr from the chromosome -> F' factors |
| Evolution: Hfr strains | Integrating into chromosome; insertion of F+ plasmid {oriT + tra genes} High Frequence of Recombination (Hfr) Forms many recombinants. Subsequent initiation of conjugation leads to transfer of plasmid & chromosome. {Integrated f in Hfr donor cell. It's conjugated to F- cell. Recombination occurs between donor copy & recipient chromosomes at a specific site on each} Lederberg & Tatum: Transfer of whole chromosome is rare since mating pairs break apart before this can happen. Transfer of entire plasmid rare since oriT in Hfr strain is in middle of integrated plasmid |
| Evolution: Imperfect excision of Hfr | ---IS1--IS2--F--IS2--IS1 Created through homologous recombination event that leads to conjugative plasmid + some chromosomal DNA = F' factor carrying chromosomal genes. Often through recombination between insertion sequence (IS) elements. Can be very large. {Causes deletion in chromosome} |
| Triparental Mating | Highly efficient. Uses 3 strains: 1. Carrying self-transmissible plasmid 2. Carrying mobilizable plasmid w/ gene(s) to be moved 3. Desired recipient strain I. Some self-transmissible plasmid will transfer -> strain #2 II. Quickly spread through strain with mobilizable plasmid because conjugation is depressed immediately after transfer III. Efficiently move mobilizable plasmid to recipient for same reason |
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