* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Microbial Genetics - University of Montana
DNA damage theory of aging wikipedia , lookup
Transposable element wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Neocentromere wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Epigenomics wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genome evolution wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene expression programming wikipedia , lookup
Minimal genome wikipedia , lookup
Primary transcript wikipedia , lookup
Nutriepigenomics wikipedia , lookup
X-inactivation wikipedia , lookup
Molecular cloning wikipedia , lookup
Non-coding DNA wikipedia , lookup
DNA supercoil wikipedia , lookup
Genetic engineering wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Epigenetics of human development wikipedia , lookup
DNA vaccination wikipedia , lookup
Genome (book) wikipedia , lookup
Genomic library wikipedia , lookup
Point mutation wikipedia , lookup
Designer baby wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Microevolution wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Helitron (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Microbial Genetics MICB404, Spring 2008 Lecture #14 Conjugation: Mechanisms of plasmid-mediated gene transfer • Summaries, exam and quiz returned today • New quiz. Due Friday 03-07-08 • Supplemental reading posted on website (URL to be distributed). 1) Lanka and Wilkins (1995) DNA processing reactions in bacterial conjugation. Annu. Rev. Biochem. 64, 141-169. 2) Foley et al., (1998) A short noncoding viral DNA element showing characteristics of a replication origin confers bacteriophage resistance to Streptococcus thermophilus. Virology 250, 377387. Review ColE1 plasmids Mutation of RNA I = Mut of RNA II Different secondary structure Altered G + C content or distribution Foley et al., 1998 Article is, or will be, available on MICB404 website Plasmids containing phage ori Acts as an origin of replication driven by phage infection. Could a plasmid contain no genes? Conjugation • One bacterium (the "male" or donor cell) transfers DNA to another (the "female" or recipient cell) while the cells are in physical contact. • The recipient that has received DNA from a donor is called a “transconjugant” Conjugation • Lederberg & Tatum, 1947 – started with two strains of E. coli that had different nutritional deficiencies caused by mutations in genes that normally synthesize biotin, cysteine, leucine, phenylalanine, thiamine, and threonine • “. . . single nutritional requirements were established as single mutational steps under the influence of X-ray or ultra-violet. By successive treatments, strains with several requirements have been obtained” – strains were both triple auxotrophic mutants, as shown below, where the genes are listed alphabetically: Conjugation • Lederberg & Tatum, 1947 Strain Y10 bio + cys+ leu- phe+ thi- thr- Strain Y24 bio- cys- leu+ phe- thi+ thr+ – Neither auxotrophic strain grows on minimal medium – Mix 2 strains together, plate to minimal medium • Isolates recovered with prototrophic phenotype Conjugation • Lederberg & Tatum, 1947 Two strains exchanged genetic information; progeny had characteristics of both parents Conjugation • Genetic information shared during conjugation is plasmid DNA – Transfer is replicative – Process similar to rolling-circle replication • One single DNA strand remains in donor, one transferred to recipient • Both ssDNA are replicated Conjugation • Plasmids can be – self-transmissible • encode all functions required for conjugation – mobilizable • encode some functions required for transfer • rely on self-transmissible plasmid for remainder Conjugation • Transfer occurs via physical contact – Gram-negative bacteria: sex pilus • produced from genes on self-transmissible plasmid • tra genes – various types of transfer systems – correlate with Inc group to prevent transfer of plasmids to recipients already carrying plasmids incompatible with donor cell plasmid Conjugation • tra genes: 11 or more in this large selftransmissible plasmid – oriT, origin required for conjugation Conjugation • Transfer between strains of one species • Transfer between different bacterial species – Promiscuous plasmids • e.g. IncN, IncP, IncW • IncP: self-transmissible and capable of mobilization from E. coli to most other Gramnegative bacteria, and to others (g+, plants) – Evolutionary role: similar genes found in evolutionarily distant organisms – Spread of antibiotic resistance Mechanism of Conjugation • Overview I – Contact made between donor and recipient • pilus formation – Plasmid is nicked – One DNA strand attached to relaxase enzyme through ester bond • transesterification • plasmid duplex separated by strand displacement Mechanism of Conjugation • Overview II – DNA strand attached to relaxase transferred into recipient cell • replication of donor cell strand from 3’-OH of transferred strand – ssDNAs are recircularized – complementary strand synthesized in recipient Mechanism of Conjugation • tra genes – DNA transfer and replication (Dtr) component: functions required to prepare plasmid DNA for transfer – mating pair formation (Mpf) component: proteins involved in forming transfer structure • Membrane associated • Sex pilus Mechanism of Conjugation • DNA processing (Dtr) component proteins – Relaxase: endonuclease, introducing specific nick in plasmid • Protein bound to 5’-PO4 – Relaxase is then transferred into recipient cell along with DNA Mechanism of Conjugation • Relaxase – In recipient cell, enzyme recircularizes plasmid by breaking protein-DNA bond and ligating 5’-PO4 with 3’-OH Mechanism of Conjugation • Relaxosome – Multi-component complex containing relaxase • Accessory proteins (binding) • Regulatory proteins (coordinating Dtr and Mpf activities) • Helicase (strand separation at oriT) Mechanism of Conjugation • Primase – Synthesize RNA primers required for complementary strand synthesis of recipient cell strand – Translated in donor cell, transferred into recipient cell Mechanism of Conjugation • Mpf component proteins – Cell-cell attachment – Formation of channel through which transfer takes place – Regulate Dtr activity – Akin to virulence protein secretion systems & natural transformation factors Mechanism of Conjugation • Pilus – Composed of pilin protein molecules – Attaches donor to recipient – DNA transfer occurs through channel or pore, not pilus Mechanism of Conjugation • Coupling proteins – Sense contact with recipient cell – Switch on Dtr activity (signals relaxase) – Up-regulate what proteins are transferred to recipient Mechanism of Conjugation • tra gene regulation – Expressed immediately after transfer to recipient is completed • Subsequently repressed, with sporadic periods of expression – prevents exploitation of pilus as bacteriophage infection site • Despite low frequency of tra gene expression in individual cells, at population level conjugation leads rapidly to spread of plasmid among potential recipients – Transfer is nearly 100% efficient Mobilizable plasmids • Plasmids requiring tra gene functions from self-transmissible plasmids for cell-cell transfer – Minimum requirement for mobilizable plasmid is oriT sequence – In nature, encode Dtr component functions • mob genes • allow mobilizable plasmids to exploit tra functions without common oriT sequence • Dtr system of mobilizable plasmid must be able to respond to coupling protein of coresident self-transmissible plasmid Mobilizable plasmids • Donor cell has mobilizable and selftransmissible plasmids • Coupling protein of selftransmissible plasmids signals cell contact made • mob relaxase initiates transfer of mobilizable plasmid DNA strand • Mobilized plasmid replicated in recipient cell • Self-transmissible plasmid can be transferred too Chromosomal transfer • During conjugation, transfer of chromosomal DNA can sometimes occur – When plasmid has integrated into chromosome – When chromosome contain oriT sequence, e.g. after transposon insertion – When plasmid contains chromosomal DNA • Homologous recombination • transposition Chromosomal transfer • Hfr strains (High frequency recombination) – Plasmid integrates into chromosome, then during conjugation, chromosomal DNA carried along in transfer – Integration • recombination – Insertion Element (IS) transposons provide sequence homology • transposition Chromosomal transfer • Recombinational integration – One IS2 site in plasmid, 20 in E. coli chromosome • 20 different Hfr strains possible – Other Insertion Element sites permit integration as well • IS3 • Hfr strain DNA transfer • Expression of tra genes – Cell-cell contact – Relaxase expression • Chromosome nicked at oriT • Plasmid DNA strand transferred into recipient – with replication in donor Hfr strain DNA transfer • Plasmid DNA transfer followed by chromosomal DNA – Entire chromosome transferred in 100 minutes at 37C – Usually only fragment of chromosome transferred – Fragment can be incorporated into recipient chromosome by recombination • otherwise lost: not all of plasmid transferred Hfr strain DNA transfer • Hfr and recipient of different genotypes – Transfer of alleles can result in recombinant transconjugant • Genes close to oriT transferred at higher frequency than genes further away – gradient of transfer Hfr gene mapping • Genetic markers – Alleles, mutations – Transposons – Phenotypic consequences allow position of marker to be mapped • in recipient (transconjugant) cell – Select for recombinant with one marker • test for other markers Hfr gene mapping • Transfer is linear and unidirectional – So order of markers on chromosome with respect to oriT can be determined from frequency of transfer – By selecting for one marker, then scoring frequency of recombinants for other markers, gene positions can be deduced • Most matings interrupted before entire chromosome transferred Hfr gene mapping • To map rif marker, cross donor with F- at 42 minutes – donor is proC and rif-8 (sensitive to rifampicin) Hfr gene mapping F in this strain E. coli chromosome genetic map Hfr gene mapping • Recipient is hisG1, argH5, and trpA3 – and rif (resistant to rifampicin) • Donor & recipient allowed to mate • Plated for selection – No proline or (histidine, arginine, or tryptophan) Hfr gene mapping • Recombinants will be recipients that have received wildtype hisG, argH, or trpA from donor • Replica-plate to tester medium for unselected markers – e.g. His+ recombinant: plate to –Arg, -Trp, and Rif plates Hfr gene mapping • Plot frequency of marker recombinants versus E. coli genetic map position – In this case, relative to hisG • Frequency of unmapped marker indicates its location on chromosome Prime Factors • Plasmids that contain chromosomal genes – F plasmid with chromosomal genes: F’ – R plasmid with chromosomal genes: R’ • Products of recombination or transposition – e.g. from Hfr – Recombinational excision can carry chromosomal fragment with it Prime Factors • Complementation testing – Allelism: are 2 mutations in same or different gene? – Dominance/recessivity – Molecular nature of mutation (point, indel…) – Function acting in cis or in trans (cis – Prime Factors create partial diploids (merodiploids) Prime Factors • Selection – Early transfer of distal markers – Replicons Prime Factors • Selection – Early transfer of distal markers • Markers that were far from oriT in chromosome of Hfr will be close in F’, and transferred early • Recipient is merodiploid and transconjugant, not recombinant • Capable of conjugation Prime Factors • Selection – Replicons • Capable of replication independent of chromosome • If recipient is defective in recombination, prime factor transconjugants can acquire and transmit selected marker but no recombinants from Hfr individuals form De scriptive Statistics N points Valid N (lis twis e) 17 17 Minimum 62.50 Maximum 148.13 Mean 99.6691 5 Count 4 3 2 1 80.00 100.00 points 120.00 140.00 St d. Deviation 20.83829 De scriptive Statistics N points Valid N (lis twis e) 16 16 Minimum 62.50 Maximum 118.13 Mean 96.6406 5 Count 4 3 2 1 70.00 80.00 90.00 points 100.00 110.00 St d. Deviation 17.22987