extra-chromosomal elements. bacteriophages  bacterial viruses or phages  extrachromosomal...

Download Extra-chromosomal Elements. Bacteriophages  bacterial viruses or phages  Extrachromosomal Elements  Can survive outside host cell  Infect bacteria:

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  • Extra-chromosomal Elements

  • Bacteriophagesbacterial viruses or phages Extrachromosomal ElementsCan survive outside host cellInfect bacteria:Replcate lysis of cell lyticIntergrate without cell death = lysogenic

  • Infection of E. coli by Phage lVirulent phage replicate and kill their host by lysing or breaking it openl phage can infect cells but dont necessarily kill

    Two paths of reproductionLytic mode: infection progresses as in a virulent phageLysogenic mode: phage DNA is integrated into the host genome

  • BacteriophagesInfectious 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

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

  • PlasmidFirst plasmid described was discovered in Japan in Shigella species during an outbreak of dysentery in the early 1940s3 main components:

    Origin of replication Selectable marker Restriction enzyme site(s) Enzymes that cut at specific sequence on DNA

  • Plasmids ContentReplication factors Genes

  • Ori Region Ori, actual site of replicationProteins that assist in replication (varies)Recognition sequences for control factorsThe ori determines the Range

  • The Large Virulence Plasmid of Shigella flexneri

  • Plasmids

    Discrete, extrachromosomal genetic elements in bacteriaUsually much smaller than bacterial chromosomeSize varies from < 5kb to > 100 kbpMostly supercoiled, circular, ds DNA moleculesReplicate 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.

  • Plasmid Genes

  • F plasmids

    codes for sex factor of bacteriaalso called conjugative plasmidsfunction: - genes promote transfer of plasmid - donor to recipient - genes code for proteins required for their replicationusually large plasmids (>40 Kbp), small copy number (1 to several per chromosome)partition themselves among daughter cells during cell division similar to bacterial chromosome

  • 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)

  • Col plasmids:produce colicins, a type of bacteriocin that affect sensitive cells (Col-) & inhibit growthmay or may not be self-transmissibleColE1 is mobilizable but non-conjugativesize :
  • 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 studiesFunction of plasmids

  • Plasmid DNA replicationPlasmid 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 oriFunctions 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

  • Theta ModelReplication forkOri Rep Replication forkOri Rep Replication bubble

  • 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.

  • Mechanism of Rolling circle DNA replicationAn 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 strandThe 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.

  • Plasmid amplification and curingPlasmid 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

  • Plasmid is an ideal structure for genetic engineering becauseSimple 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 bacterias growth, thus possible to manipulate plasmid DNA without affecting bacteria growth.

  • Exchange of Genetic Information in bacteriaMedically 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

  • Types of transfers:Non-transmissible = cannot initiate contact with recipient or transfer DNA Conjugative = can initiate contact with recipient bacterium Mobilizable = can prepare its DNA for transfer Self-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.

  • Fin genes & plasmid transferfin 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

  • 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.

    (a) Bacterial Transformation

  • "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.

    Bacteria transformation in the lab

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

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