CH. 18: THE GENETICS OF VIRUSES & BACTERIA

Well-researched: Pneumococcus, Escherichia coli, bacteriophages, TMV

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Bacteria

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Bacterial cell shapes

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Gram stainingClassification of bacteria according to what color stain they retain. A sample of bacteria (on a slide) is first stained with a violet dye. The slide is then rinsed with ethanol.If the violet stain washes off, a red dye is added (“counterstaining”).Depending on the structure of the cell walls, some types of bacteria (such as staphylococcus and streptococcus) retain the violet stain and are called Gram-positive. Other types (such as pseudomonas and salmonella) retain the red, but not the violet, stain and are called Gram-negative. This technique is named after the Danish bacteriologist Hans C. J. Gram (1853-1938) who invented it in 1884.

Gram + (top), Gram – (bottom)

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• Bacterial chromosome is GENERALLY a single, continuous (circular) thread of double- stranded DNA.

• Approx. 1 mm long when fully extended (only 2mm in diameter); contains about 4.7 million base pairs.

• More recently, it has been found that bacteria may actually have different kinds of chromosomal material 5

The E.coli Chromosome:

• Prokaryotic Cells Replicate their DNA in a bidirectional fashion (replication)

• Replication begins at a specific base sequence.diagram:

6“theta” replication

Transcription and Its Regulation

• Transcription begins when RNA polymerase (enzyme) begins formation of an mRNA strand along DNA strand, beginning at promoter site.

• A segment of DNA that codes for one specific protein is known as a structural gene

• There may be several "start" and "stop" codons along the mRNA strand, marking the beginning and end of each structural gene. – "leader" sequence (of nucleotides) at 5’ end– "trailer" sequence (of nucleotides) at 3’ end

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The Need for Regulation

This is regulated by several means: • 1) can be induced by presence of a material

(ex. Lactose presence induced E. coli to synthesize beta-galactosidase enzyme)

• 2) can be inhibited: presence of substance prevents formation of an enzyme "repressible" (ex: E. coli; tryptophan inhibits tryptophan forming enzymes)

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The Lac Operon

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The Operon Model (1965 Nobel Prize) François Jacob, André Lwoff, and Jacques Monod "for their discoveries concerning genetic control of enzyme and virus synthesis"- arose from study of mutant cells

studies done on E. coli cells making the enzyme: beta-galactosidase ; found it was "blocked" by a repressor binding to operator.

Operator – gene that activates transcription of a structural gene

Regulator - This gene codes for a repressor protein

Repressor: protein that can bind to the operator gene, thus obstructing* the promotor (blocks the RNA polymerase from moving along ("reading") the molecule no mRNA transcription can occur.

*when the repressor is removed, mRNA transcription begins

animations of lac and tryp operons

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CAP-cAMP Complex

• The catabolite gene activator protein (CAP) is a sequence-specific DNA binding protein that starts transcription of some gene systems and represses others, when bound to cyclic adenosine monophosphate (cAMP)

• this CAP-cAMP complex binds to the promotor and maximizes transcription.

• It increases the binding of RNA polymerase, among other things

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Bacterial plasmids are often used in genetic engineering, as a “gene of interest” can be inserted

easily into them, then cloned into hundreds of copies when the bacteria multiply.

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Recombinant DNA: involves modifying/combining DNA from a variety of different sources and inserting these altered

molecules into other cells, in which the "new "genes are expressed.

Bacterial Reproduction

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Binary fission

• Bacteria cell goal: to grow and multiply • many can double number every 20 minutes!

Plasmids and Conjugation

• Although bacterial chromosomes carry all the genes necessary for growth and reproduction of the cell, they also carry additional DNA molecules called Plasmids

• Plasmids usually carry only between 2 to 30 genes; generally small)

• 2 important types: – "sex factor" plasmids = F (fertility) – "drug resistance" plasmids = R (resistance)

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The F Plasmid• We now know that the fertility

factor or F factor is a very large (94,500 bp) circular dsDNA plasmid; it is generally independent of the host chromosome. http://www.mun.ca/biochem/courses/3107/Lectures/Topics/conjugation.html.

• They make a cell capable of conjugation.

• F+ (male) "donor" cells: make pili (protein "bridges" that form to connect 2 cells for transfer of genetic material)

• F- cells lack the F plasmid and can’t form pili (female); these are "recipient" cells

15“rolling-circle replication”

Hfr Cell: Sometimes the F factor gene can be incorporated within the main bacterial chromosome. Because such strains transfer chromosomal genes very efficiently, they are called Hfr (high frequency of recombination).

This can then transfer a portion of bacterial chromosome to a F- cell:

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R plasmids = resistance plasmids • Plasmids carrying genes responsible for antibiotic (or antibacterial

drug) resistance among bacteria may pass them to other species of bacteria as well! ex: E. coli -------> Shigella (dysentery)

• "Horizontal gene transfer“ also called Lateral gene transfer (LGT), is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism.

• By contrast, vertical transfer occurs when an organism receives genetic material from its ancestor, e.g. its parent or a species from which it evolved.

• It is most often thought of as a sexual process that requires a mechanism for the mobilization of chromosomal DNA among bacterial cells.

• http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/genetic-exchange/exchange/exchange.html

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Because they are unable to reproduce sexually, bacterial species have acquired several mechanisms by which to

exchange genetic materials

• Transformation - the uptake of naked DNA is a common mode of horizontal gene transfer that can mediate the exchange of any part of a chromosome; this process is most common in bacteria that are naturally transformable; typically only short DNA fragments are exchanged.

• Conjugation - the transfer of DNA mediated by conjugal plasmids or conjugal transposons; requires cell to cell contact but can occur between distantly related bacteria or even bacteria and eukaryotic cells; can transfer long fragments of DNA.

• Transduction - the transfer of DNA by phage requires that the donor and recipient share cell surface receptors for phage binding and thus is usually limited to closely related bacteria; the length of DNA transferred is limited by the size of the phage head.

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VIRUSES

diagram: bacteriophages TMV

• consists of a molecule of nucleic acid encased in a protein coat (capsid) contain no other "cell machinery", but can move from cell to cell and utilize the host cell’s "machinery" to replicate the viral genes "obligate intracellular parasites"

• Viral nucleic acids vary: may be either DNA or RNA; double or single-stranded, circular or linear.

Viral classifications – Viruses are generally classified by size (nm), shape/capsid, and type of nucleic acid they contain.

CHARACTERISTICS:1. Contain a varying, but small amount of DNA (or RNA) surrounded by protein

T7 bacteriophage has DNA and ~100 genes; The poliovirus has 7,600 RNA nucleotides; the vaccinia (cowpox) virus has 240,000 DNA nucleotide pairs

2. Small viruses that don’t have room for a lot of DNA use overlapping genes3. Retroviruses (ex: HIV) are RNA viruses that use an enzyme called Reverse Transcriptase to make DNA to

replicate itself during infection stage) 19

Viroids and Prions• Viroids:

(mostly cause plant diseases)• are tiny strands of RNA, usually only a few

hundred nucleotides long. • Viroids can interfere with a plant's

metabolism. • Generally speaking, where viroids come

from and how they can disrupt the host cell are not known.

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Prions• Prions are infectious agent, a misshapen protein, which have been defined as "small

proteinaceous infectious particles which resist inactivation by procedures that modify nucleic acids".

• The discovery that proteins alone can transmit an infectious disease has come as a considerable surprise to the scientific community.

• Prion diseases are often called spongiform encephalopathies because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum.

• Probably most mammalian species develop these diseases. Specific examples include: – Scrapie: sheep – TME (transmissible mink encephalopathy): mink – CWD (chronic wasting disease): mule deer, elk – BSE (bovine spongiform encephalopathy): cows

• Humans are also susceptible to several prion diseases:– CJD: Creutzfeld-Jacob Disease – GSS: Gerstmann-Straussler-Scheinker syndrome – FFI: Fatal familial Insomnia – Kuru– Alpers Syndrome

• http://www-micro.msb.le.ac.uk/3035/prions.html 21

Viral Cycles• Lytic cycle - causes destruction of the host cell quickly

– Attachment- phage attaches to cell surface – Entry- phage injects DNA into host cell – Replication- phage DNA "tells" host to make more phage DNA and protein coats – Assembly- new viruses are assembled; host cell becomes a "virus factory" – Release- cell lyses (breaks open), releasing viruses

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Lysogenic Cycle• some viruses incorporate their DNA into a cell’s

chromosome. • The cell may then cause a sudden eruption of viral activity

(can remain latent for many generations)

• Temperate bacteriophages- viruses that can integrate their DNA into bacterial chromosome at specific sites

• Prophage- an integrated bacteriophage

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Viral Transduction

• the transfer of cellular DNA from one host cell to another by means of viruses = recombinant DNA

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Transduction1. General TransductionTransduction is bacterial

chromosome DNA transfer mediated by a virus. This is a one way nonreciprocal virus mediated transfer from one bacterial cell to another.

2. Restricted (specialized) Transduction

The situation in which a particular phage will transduce only specific regions of the bacterial chromosome.

25* can you think of the implications to genetic engineering?  gene therapy?

Introducing Lambda• Lambda is the best studied of the temperate bacteriophages

– Viral form: linear, double-stranded (2 free ends) – When inserted into a bacterial cell, it becomes circular

• integrase: enzyme produced by a retrovirus that enables its genetic material to be integrated into the DNA of the infected cell.

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Lambda plaques on an E.coli culture

Transposons • discovered by Barbara McClintock• Are segments of DNA that can move around to different positions in the

genome of a single cell. • These mobile segments of DNA are sometimes called "jumping genes". • In the process, they may cause mutations increase (or decrease) the

amount of DNA in the genome. • Also contain a gene for enzyme "transposase"

At each end, they contain a sequence of repeats• direct repeats -ATTCAG-ATTCAG- • often used to I.D. insertion points • indirect repeats -ATTCAG-GACTTA-

• Can carry genes for mutations, protein synthesis, drug resistance, etc...from one place in the genome to another

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Many transposons move by a "cut and paste" process: the transposon is cut out of its location (like command/control-X on your computer) and inserted into a new location (command/control-V).

This process requires an enzyme — a transposase — that is encoded within some of these transposons.

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