Extra-chromosomal Elements MB 206 : Module 1 - D

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

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

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

Lysis plaques of l phage on E. coli bacteria lawn

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

Plasmids Content Replication factors Genes

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

The Large Virulence Plasmid of Shigella flexneri

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.

Plasmid Genes F factor (fertility factor) Carries genes for six pili and transfer of the plasmid partition themselves among daughter cells during cell division similar to bacterial chromosome R plasmids (antibiotic resistance) medically important, eg, resistant to Penicillin (carries genes of the Bla operon) resistance to one or several antibiotics (R factor) Col (colicinogenic) Encode enzymes for catabolism of unusual compounds produce colicins, a type of bacteriocin that affect sensitive cells (Col-) & inhibit growth

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

R plasmids: resistance to one or several antibiotics (R factor) 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 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.

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

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

Theta Model Replication fork Ori Rep Replication fork Ori 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 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.

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 Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. It is cell-permeable, and interacts with DNA and RNA by intercalation or electrostatic attractions. When bound to DNA, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). The dye is often used in epifluorescence microscopy. Acridine orange is prepared from coal tar and creosote oil. Acridine orange can be used in conjunction with ethidium bromide to differentiate between live and apoptotic cells

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.

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        

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 transfer - 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

(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.

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. Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. If the foreign DNA has an origin of replication recognized by the host cell DNA polymerases, the bacteria will replicate the foreign DNA along with their own DNA. When transformation is coupled with antibiotic selection techniques, bacteria can be induced to uptake certain DNA molecules, and those bacteria can be selected for that incorporation. Bacteria which are able to uptake DNA are called "competent" and are made so by treatment with calcium chloride in the early log phase of growth. The bacterial cell membrane is permeable to chloride ions, but is non-permeable to calcium ions. As the chloride ions enter the cell, water molecules accompany the charged particle. This influx of water causes the cells to swell and is necessary for the uptake of DNA. The exact mechanism of this uptake is unknown. It is known, however, that the calcium chloride treatment be followed by heat. When E. coli are subjected to 42degC heat, a set of genes are expressed which aid the bacteria in surviving at such temperatures. This set of genes are called the heat shock genes. The heat shock step is necessary for the uptake of DNA. At temperatures above 42degC, the bacteria's ability to uptake DNA becomes reduced, and at extreme temperatures the bacteria will die.

Bacteria Transduction Bacteriophage infect donor bacterium form 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.

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

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.

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.

Thank U!!!