MB 206 : Module 1 - D

1. Extra-chromosomal Elements MB 206 : Module 1 - D

2. Bacteriophages • bacterial viruses or phages • Extrachromosomal Elements • Can survive outside host cell • Infect bacteria: • Replcate  lysis of cell – lytic • Intergrate without cell death = lysogenic

3. Infection of E. coli by Phage l • Virulent phage replicate and kill their host by lysing or breaking it open • l phage can infect cells but don’t necessarily kill • Two paths of reproduction • Lytic mode: infection progresses as in a virulent phage • Lysogenic mode: phage DNA is integrated into the host genome

4. Bacteriophages • Infectious agents, replicate as obligate intracellular parasites in bacteria • Morphologically different - polyhedral, filamentous, and complex (polyhedral heads with tails attached) • consist of a protective shell (capsid) surrounding the tightly packaged nucleic acid genome • genomes vary in size (~ 2 to 200 kb); either dsDNA, ssDNA, or RNA • genes encodes proteins - for replication & phage assembly

5. Lysis plaques of l phage on E. coli bacteria lawn

6. Plasmid Plasmid – small, circular, extrachromosomal DNA which replicates independently of host chromosomal DNA • First plasmid described was discovered in Japan in Shigella species during an outbreak of dysentery in the early 1940‘s • 3 main components: • Origin of replication • Selectable marker • Restriction enzyme site(s) • Enzymes that cut at specific sequence on DNA

7. Plasmids Content • Replication factors • Genes

8. Ori Region • Ori, actual site of replication • Proteins that assist in replication (varies) • Recognition sequences for control factors • The ori determines the Range

9. The Large Virulence Plasmid of Shigella flexneri

10. Plasmids • Discrete, extrachromosomal genetic elements in bacteria • Usually much smaller than bacterial chromosome • Size varies from < 5kb to > 100 kbp • Mostly supercoiled, circular, ds DNA molecules • Replicate independently of the chromosome • Exist in multiple copies in bacterial (the average number of plasmid per bacterial is called copy number). • Usually encode traits that are non-essential for bacterial viability.

11. Plasmid Genes

12. F plasmids • codes for sex factor of bacteria • also called conjugative plasmids • function: - genes promote transfer of plasmid - donor to recipient - genes code for proteins required for their replication • usually large plasmids (>40 Kbp), small copy number (1 to several per chromosome) • partition themselves among daughter cells during cell division similar to bacterial chromosome

13. R plasmids: • medically important, eg, resistant to Penicillin (carries genes of the Bla operon) • In early 1940's, Penicillin was introduced for general use 1946 - 14% of Staphylococcus aureus were penicillin resistant 1947 - 38% PenR 1969 - 59% PenR 1970's - almost 100% PenR • resistance to one or several antibiotics (R factor)

14. Col plasmids: • produce colicins, a type of bacteriocin that affect sensitive cells (Col-) & inhibit growth • may or may not be self-transmissible • ColE1 is mobilizable but non-conjugative • size : <7.5 Kbp • high copy numbers (typically 10-20 per chromosome) • rely on their bacterial host to provide some functions required for replication • are distributed randomly between daughter cells at division.

15. Function of plasmids • Many plasmids control medically important properties of pathogenic bacteria, contain genes that code for : a) resistance to one or several antibiotics b) production of toxins eg. heat-labile & heat-stable enterotoxins of E. coli, Shiga toxins of Shigella exfoliative toxin of S. aureus tetanus toxin of C. tetani c) synthesis of cell surface structures required for adherence or colonization • Some plasmids are cryptic = no recognizable effects on the bacterial host • Comparing plasmid profiles = for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies

16. Plasmid DNA replication • Plasmid replication by - Theta model (either uni- or bidirectional) or - Rolling circle • Replicon - DNA molecules that can replicate autonomously (plasmids, chromosomes, phage) • Replicon must have on origin of replication, called ori • Functions of the ori region: Host range - narrow or broad host ranges • Broad-host-range plasmids = encode all of their own proteins required for replication initiation • Regulation of copy number Stringent - low copy number (F factor) Relaxed - high copy number (pBR322 =16 copies; pUC =30 to 50) Requires host proteins for replication

17. Theta Model Replication fork Ori Rep Replication fork Ori Rep Replication bubble

18. Rolling circle replication • Enable rapid synthesis of multiple copies of circular DNA or RNA (plasmid or phage genomes). • A striking feature: = one strand is replicated first (which protrudes after being displaced) and the second strand is replicated after completion of the first one.

19. Mechanism of Rolling circle DNA replication • An initiator protein encoded by the plasmid DNA nicks one strand of the ds plasmid at the ori site. • The initiator protein binds to the 5' PO4 end of the nicked strand • The free 3' OH end serve as a primer for DNA synthesis by DNA polymerase III, using the un-nicked strand as a template. • The 5' PO4 ssDNA strand is displaced by helicase PcrA in the presence of the initiation protein. • Continued DNA synthesis can produce multiple ss linear copies of the original DNA in a continuous head-to-tail series called a concatemer. • These linear copies are converted to ds circular plasmid by: the initiator protein makes another nick to terminate synthesis of the first (leading) strand. DNA polymerase III replicate the ss ori to make complementary strand, RNA primer removed, DNA ligase joins the ends to make ds circular plasmid.

20. Plasmid amplification and curing • Plasmid amplification by chloramphenicol treatment - inhibits protein synthesis - inhibit chromosomal but not plasmid replication. - Chromosomal replication requires new protein synthesis but plasmid replication uses only stable bacterial replication proteins. - Plasmids replicated to high copy number because no repressor protein to control copy number • Plasmid curing - with acridine orange - inhibits plasmid but not chromosomal replication - unknown how this occurs

22. Plasmid is an ideal structure for genetic engineering because • Simple in structure • Easy to extract & isolate in the lab • Easy for genetic manipulation & transformed back into bacteria • Contains genetic information which can be used by the bacteria • Most plasmid present in high copy number • Plasmid codes for antibiotic resistant gene eg. Ampicillin, Apr or Tetracyclin Tcr - selection of bacteria with transformed plasmid. • Non-essential for bacteria’s growth, thus possible to manipulate plasmid DNA without affecting bacteria growth.

23. Exchange of Genetic Information in bacteria • Medically important - rapid emergence and dissemination of antibiotic resistance plasmids - flagellar phase variation (eg. Salmonella) - antigenic variation of surface antigens (eg. Neisseria & Borrelia) • Sexual processes in bacteria involve transfer of genetic information from a donor to a recipient, results in: - substitution of donor alleles for recipient alleles - addition of donor genetic elements to the recipient genome. • 3 major types of genetic transfer found in bacteria: a) Transformation b) Transduction c) Conjugation In all three cases, recombination between donor and recipient DNA result in formation of stable recombinant genomes        

24. Types of transfers: • Non-transmissible = cannot initiate contact with recipient or transfer DNAConjugative = can initiate contact with recipient bacteriumMobilizable = can prepare its DNA for transferSelf-transmissible = is both conjugative & mobilizable • 4 stages of plasmid transfer: a) Effective contact b) Mobilization - preparation for DNA transfer c) DNA transfer d) Formation of F in recipient • Donation - a conjugative plasmid (F) can provide conjugative function to a mobilizable plasmid (eg. ColE1) such that both plasmids can be transferred. • Plasmid conduction - a self-transmissible plasmid (F) can recombine with a non-mobilizable plasmid and transfer the co-integrate.

25. Fin genes & plasmid transfer • fin genes - fertility inhibition - codes for repressor that prevents transcription of genes required for transfer. • F plasmid has 1 fin gene so transfer system is always ‘ON’ • R plasmid has 2 fin genes so cannot always transfer. - in new recipients (repressor is absent) so transfer can occur soon after receiving the R plasmid but after time (when repressor is made) transfer can't occur

27. (a) Bacterial Transformation • introducing DNA from donor to recipient • result in uptake and integration of fragments of donor DNA into recipient genome. • produce stable hybrid progeny. • is most likely to occur when the donor and recipient bacteria the same or closely related species.

28. Bacteria transformation in the lab "Transformation" is simply the process where bacteria manage to "uptake" a piece of external DNA.   Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell.

29. Bacteria Transduction • Bacteriophage infect donor bacterium • form rare abnormal bacteriophage particles contain DNA from donor bacteria. • abnormal bacteriophageinfect recipient bacteria & inject DNA into recipient • donor DNA integrated / recombined into recipient DNA resulting in transduced bacterium.

30. Bacterial Conjugation • Transfer of DNA between 2 bacteria in contact with each other • Contact between donor and recipient (initiated by sex pili) • DNA transfer through a conjugation bridge • Mediated by a plasmid • Called an F-factor (fertility factor) or conjugative plasmid

33. Several important properties of F   • F is a self-replicating plasmid and is maintained in a dividing cellular population. • Cells carrying F produce pili, minute proteinaceous tubules that allow the F+ cells to attach to other cells maintaining contact. • F+ cells can transfer its F plasmid to a F- cell , turning the recipient cell into an F+ cell. F+ cells are usually inhibited from making contact with each other. • Occasionally, F can integrate into the host bacterial chromosome and transfer the host chromosomal markers to the recipient cell.

34. Restriction-modification (RM) systems • consist of methylases = methylate the adenine or cytosine residues at specific sequences in their own DNA • corresponding restriction endonucleases cleave foreign DNA which are not methylated at the same target sequences. • RM systems = protect bacteria against invasion by phages or plasmids. • Barrier to genetic exchanges between different bacterial strains or species. • Recent evidence suggests that plasmid-borne RM systems = strategy to ensure plasmid maintenance in a host strain since cells that lose these plasmid (and the corresponding protective methylase gene) are killed by restriction enzyme, which attacks the newly replicated & unmodified chromosomal DNA.

36. Thank U!!!