BIOS 312 Exam 3 Review

Unit 3 - Covers all lectures and microbes

What are viruses?

• Genetic elements that cannot replicate independently of a living cell

• Obligate intracellular parasites

What are the extracellular forms of viruses?

Virus particles or virions

• Virions are the complete, infectious, and physical particle of a virus

• These exist outside of the host

• Facilitate transmission

• Only replicate within a host

T/F: Most viruses are larger than prokaryotic cells

False; they range from 0.02 to 0.03 um

Describe the structure and components of a virion

Includes

• Genome

• Capsid: a protective shell formed by protein subunits called capsomeres

• Nucleocapsid: combination of the nucleic acid and capsid

• Viral tegument: a cluster of proteins that lines the space between the envelope and nucleocapsid

• Envelope (optional): a lipid bilayer containing viral glycoproteins (spikes) for attachment

• Enzymes (optional): specific proteins required for replication, sometimes packaged inside the capsid

Compare naked viruses vs. enveloped viruses

• Naked viruses (most bacterial viruses) have no other layers

• Enveloped viruses (many animal viruses) have an outer layer consisting of a phospholipid bilayer (from host cell membrane) and viral proteins

Describe the size of a viral genome

Ranges from a 1.75 kilobase single strand within a circovirus

to a 1.25 megabase pair genome within a megavirus

Describe viral genomes

• Can be either DNA or RNA; so they can be either single or double-stranded

• Single-stranded genomes can be in either plus sense (same as mRNA) or minus sense (complimentary to mRNA)

• Generally the genomes are smaller than those of cells

Are viral genomes circular or linear?

Most are linear, but some can be circular

How are viruses classified?

They are classified on the basis of the hosts they infect and their genomes

• Bacterial viruses (bacteriophages; model systems)

• Archaeal viruses

• Animal viruses (extensively studied)

• Plant viruses (less well-studied)

• Other viruses

How does the capsid of a virus form?

It is made of individual proteins called capsomeres, that are arranged in precise, highly repetitive patterns around the nucleic acid

They can be assembled spontaneously (self-assembly) or with assistance from host-cell folding proteins

Helical virions are ___-shaped viruses

Rod-shaped (ex: tobacco mosaic virus)

• Length of virus determined by length of nucleic acid
  Width of virus determined by size and packaging of capsomeres

Icosahedral Virions

• Viruses that are roughly spherical

• Most efficient arrangement of subunits in a closed shell

• Requires fewest capsomeres

Describe enveloped viruses

• Have a lipoprotein membrane surrounding their nucleocapsid

• Can be either RNA or DNA viruses

• Can attach to and infect animal host cells

  • A few can infect plants/bacteria because of the cell walls surrounding the cell membrane

• During infection, the entire virion enters animal cell

• Can exit more easily

What kinds of enzymes can exist in a virion?

These are pre-formed enzymes

• Lysozymes: makes hole in cell well to allow nucleic acid entry; lyses bacterial cell to release new virions

• Neuraminidases: destroys glycoproteins and glycolipids; allows liberation of viruses from cell

• Nucleic acid polymerases

  • RNA replicases: RNA-dependent RNA polymerases

  • Reverse transcriptase: RNA-dependent DNA polymerase in retroviruses

What is the major difference between prokaryotic and eukaryotic viruses?

• Nucleic acid entry in prokaryotes

• Virion entry in eukaryotes

What are the phases of viral replication in a permissive (supportive) host?

1. Attachment (adsorption) of virion

2. Penetration (entry, injection) of the virion nucleic acid

3. Synthesis of viral nucleic acid and proteins by cell host metabolism as redirected by virus

4. Assembly of capsids and packing of viral genomes into new virions

5. Release (lysis) of mature virions from host cell

This is a prokaryotic virus

Describe the viral growth curve

• Typically characterized by a one-step growth curve

• Viral numbers increase once cell lyses

Phases of the curve

• Eclipse: genome replicated and proteins translated

• Maturation: packaging of nucleic acids in capsids

• Latent period: eclipse + maturation

• Release: cell lysis, budding, or excretion

  • Burst size: number of virions released

Time of life cycle

• Bacterial viruses: 20-60 minutes

• Animals: 8-40 hours

Compare virulent (lytic) viruses vs. lysogenic viruses

• Virulent viruses: infect their host, replicate in the host, and then destroy the host to escape

• Lysogenic viruses: viral DNA integrates into the host cell’s DNA, which alters the host as the viral DNA becomes part of its genome

Describe bacterial viruses (bacteriophages)

• Very diverse group

• The best studied are those that infect enteric bacteria (E. coli, Salmonella enterica)

• Usually naked, although some can have lipid envelopes

• Structurally complex and contain heads, tails, and other components

• Most phages have dsDNA genomes

T/F: Attachment of a virion to a host is highly specific

True

• Attachment requires complementary receptors on the surface of a susceptible host and its infecting virus

• Receptors on host cell carry out normal functions for cell

• Receptors include: proteins, carbohydrates, glycoproteins, lipids, lipoproteins, or complexes

T/F: The attachment of a virus to its host can result in changes to both virus and cell surface that facilitate penetration

True

What is a permissive cell?

A host cell that allows the complete replication cycle of a virus to occur

Describe Bacteriophage T4

• A virus of E. coli

• One of the most complex penetration mechanisms known

1. Virions attach to the cell via tail fibers that interact specifically with polysaccharides on the E. coli cell envelope

2. The tail fibers retract, and the tail core makes contact with the E. coli cell wall

3. Lysozyme-like enzymes forms a small pore in the peptidoglycan

4. The tail sheath contracts and viral DNA passes into the cytoplasm

Describe the synthesis step of the viral life cycle

• The virus hijacks the host cell’s metabolism to synthesize proteins (including sigma factors) and replicate nucleic acids

• RNA polymerase is also modified to recognize virus promoters

Describe the assembly and packaging step of the viral life cycle

Structural proteins for virion self-assembly and packaging of the nucleic acid in the capsid

Describe the lytic cycle

Lytic infection cycle of virulent phages

1. A phage virion attaches and injects its phage DNA into a host cell; degrades host DNA

2. New viral DNA and proteins are synthesized followed by assembly into virions

3. Viral enzymes (lysozymes) induce the release of progeny phages

T/F: All viruses are lytic

False

T/F: Lysogenic viruses are ecologically significant

True; many environmental bacteria are infected with lysogens

T/F: The virulence of many human pathogens is controlled by lytic bacteriophages

False; it is controlled by lysogenic bacteriophages

What is the Bacteriophage Lambda?

• A temperate phage that infects E. coli used for studies

• Has a linear, dsDNA genome

• Complementary, single-stranded regions 12 nucleotides long at the 5’-terminus of each strand

• Upon penetration, DNA ends base-pair forming the cos site; DNA ligates and forms double-stranded cricle

• When lysogenic, integrates into E. coli chromosomes at the lambda attachment site

Temperate bacteriophages carry out two types of life cycles. What are they?

The lytic and lysogenic cycle

• The lytic cycle for temperate bacteriophages is similar to the lytic cycle for virulent phages

Compare the lytic vs lysogenic cycle for viruses

• Lytic cycle: immediate, rapid replication of viruses, and the destruction (lysis) of the host cell.

• Lysogenic cycle: allows virus to remain dormant, integrating its genome into the host's DNA to be replicated passively during cell division without destroying the host

What determines if a virus becomes lytic or not?

Gene promoters and regulatory proteins

Describe what happens when Bacteriophage Lambda enters the lytic cycle

• It synthesizes long, linear concatemers of DNA by rolling circle replication

• Genome-sized lengths cut as cos sites; genomes packaged into phage heads

• After the tails are added, lysis occurs

• Transduction: packaging of host chromosomal genes and transfer to a new host can also occur

• When it is lysogenic, its DNA integrates into E. coli chromosome at the lambda attachment site using lambda integrase

What are the two key repressor proteins involved in temperate phage replication?

• Cl protein (lambda repressor): causes repression of lambda lytic events

• Cro repressor: represses lambda cII and cIII proteins required for lysogeny, thus controls activation of lytic events

What are the outcomes of lysogeny?

1. Via binary fission, host cell and the provirus DNA are both replicated and passed on to the progeny

2. Upon induction of some event (ex: stress), the provirus DNA excises out of the host chromosomal DNA and initiates the lytic cycle

3. Genetic information in the provirus can provide new characteristics for the host cell

What components do animal viruses share with other viruses?

• Capsid

• RNA/DNA genome

  • Many pathogenic viruses have RNA genomes

What differentiates animal viruses from other viruses?

• The entire virion enters the animal cell

• Eukaryotic cells contain a nucleus, which is the site of replication for many animal viruses

Describe the viral infection of animal cells

• Animal viruses bind specifically to host cell receptors, which are typically used for cell-cell contract or immune function

• Different tissues and organs express different cell surface proteins

  • Viruses often only infect certain tissues

• Entry usually occurs by fusion with the cytoplasmic membrane or endocytosis

Describe the process of animal virus infection

• Uncoating occurs in the cytoplasm

• DNA genomes enter the nucleus; RNA genomes are converted into DNA within the capsid

What are the outcomes of animal viral infection?

• If the virus is virulent: host cell is lysed (most common outcome!)

• If latent infection: viral DNA exists in host cell chromosomes and virions are not produced; cell is unharmed

• If persistent infection: virions are released by budding and the cell does not lyse

• The cell can also undergo transformation and become cancerous

What are retroviruses?

RNA viruses

• Enveloped virions that contain two copies of the RNA genome

• Virion contains several enzymes, which include reverse transcriptase to make DNA copies of the genome

• Genes included: gag, pol, env

  • Some have a fourth gene downstream of env that is active in cellular transformation

What are hepadnaviruses?

DNA viruses

How does retroviral replication work?

1. Virus enters the cell; it fuses to the cytoplasmic membrane at receptor sites

2. Envelope is removed. Genome and enzymes remain in the core

3. Reverse transcription leads to the creation of a linear double-stranded DNA that enters the nucleus

4. Retroviral DNA integrates into the host genome similar to a temperate bacteriophage

5. Transcription leads to the formation of mRNAs and viral RNA genome

6. Genomic RNA and nucleocapsids assemble

7. Enveloped virions are released from the cell by budding

Enumeration of viruses

• Viruses replicate only in certain types of cells or in whole organisms

• Bacterial viruses are easiest to grow = model systems

• Animal viruses (and some plant viruses) can be cultivated in tissue or cell cultures

• Plant viruses are typically most difficult because study often requires growth of the whole plant

Titer

The number of infectious agents per volume of fluid

Plaque Assay

One way to measure virus infectivity

• Analogous to the bacterial colony

• Plaques are clear zones that develop on lawns of host cells

• Lawn can be bacterial or tissue culture

• Each plaque results from infection by a single virus particle

Satellite Virus

A subviral agent composed of nucleic acid

• The success of the virus depends on coinfection with another virus for their replication

Virioid

Infectious RNA that lacks a protein coat

(Smallest known pathogens (246-399 nucleotides) - only known to infect plants)

T/F: The death rate of bacteria is constant

True

What factors influence how well a disinfectant works?

1. The number of microbes (more microbes = more time needed to kill them)

2. Environmental factors (temperature; presence of organic material, etc)

3. Time of exposure: antimicrobials often require an extended time to affect resistant microbes or endospores

4. Microbial characteristics can also affect choice of chemical/physical growth control methods

What kind of environment does a disinfectant work better in?

In warm solutions or in the presence of organic matter (blood, vomit, feces)

Can also inhibit the action of chemical microbials

Why are bacteria encased in a biofilm more difficult to treat with disinfectant?

Because the microbes are suspended in media that is rich in proteins and fats

Many disinfecting agents target what of a microbe?

• The cell membrane because damage to it can cause cellular contents to leak out of the cell and interfere with cell growth

• Can also alter shape of enzymes (through heat or pH) leave cells susceptible to death

• Alterations to DNA or RNA (ex: UV treatment) leaves cell unable to divide

Sterilization

The destruction of all living microorganisms

• Most common method: heating

Commercial Sterilization

Destroying all pathogenic and spoilage microorganisms as well as heat-resistant spores, especially from C. botulinum, from shelf-stable foods

This means that a very small number of resistant bacterial spores may remain, but will not be dangerous

Complete sterilization is not required for many areas (like food or surgical wounds)

Disinfection

Microbial control aimed at destroying harmful bacteria

• Eliminates only vegetative bacteria (not spores)

• Methods: UV light, chemical disinfectants

Antisepsis: when used on living tissue; thus the agent becomes an antiseptic

Degerming

Mechanically eliminating germs instead of killing them directly

Ex: getting skin swabbed with alcohol for a flu shot

Sanitization

Reducing the number of microbes to prevent disease

What is the suffix for treatments that eliminate/kill microorganisms?

-cide

What is the suffix for treatments that inhibit the growth of microorganisims?

-stasis

What are the physical methods used to control microbial growth?

1. Heat

2. Filtration

3. Low temperatures

4. High pressure

5. Desiccation

6. Osmotic Pressure

7. Radiation

How does heat affect microbial growth?

• Heat denatures enzymes

• Thermal Death Point (TDP): lowest temperature at which all cells in a liquid culture are killed in 10 minutes

• Thermal Death Time (TDT): minimal time for all bacteria in a liquid culture to be killed at a particular temperature

• Decimal Reduction Time (DRT): Minutes to kill 90% of a population at a given temperature

Autoclave

Provides steam under pressure at 121C at 15 psi for 15 minutes

• Kills all organisms and endospores

• Steam must contact the item’s surface

• Large containers require longer sterilization times

• Test strips used to indicate sterility

Pasteurization

Uses heat to reduce spoilage organisms and pathogens

Equivalent treatments

• 63C for 30 min

• High-temperature short-time (HTST): 72C for 15 sec

• Ultra-high-temperature (UHT): 140C for 4 sec

Thermoduric organisms survive

Dry Heat Sterilization

Kills by oxidation

• Flaming

• Incineration

• Hot-air sterilization

Filtration

Passage of substance through screen-like material

• Used for heat-sensitive materials

• High-efficiency particulate air (HEPA) filters remove microbes > 0.3um

• Membrane filters remove microbes >0.22 um

T/F: Low temperature has a bacteriostatic effect

True; slows the growth

• Refrigeration

• Deep-freezing

• Lyophilization (freeze drying)

Desiccation

Absence of water prevents metabolism

Osmotic pressure uses salts and sugars to create a hypertonic environment, causing plasmolysis

What are the different types of radiation that can be applied to controlling microbial growth?

• Ionizing radiation (x-rays, gamma, electron beams)

  • ionizes water to create reactive hydroxyl radicals

  • damages DNA causing lethal mutations

• Nonionizing radiation (UV, 260nm)

  • damages DNA by creating thymine dimers

• Microwaves

  • kill by heat; not especially antimicrobial

What should be known about chemical methods for controlling microbial growth?

• Can be used to control growth of microbes on living/non-living tissue

• Few agents achieve sterility - most reduce microorganism numbers to safe levels

• Not all agents work on all microorganisms

What is the Use-Dilution Test?

• To measure the effectiveness of liquid disinfectants on hard, non-porous surfaces

• A metal/glass cylinder is dipped into a culture and then submerged into a disinfectant

  • The cylinders are placed into a sterile broth medium to check for growth - turbidity determines effectiveness

Disk Diffusion Test

• Filter paper disks are impregnated with a disinfectant and placed onto a lawn of bacteria

• Zone of clearing around disk = the agent is effective at disinfecting the bacteria

What are phenols?

• A type of disinfectant

• Not frequently used on living tissue (is an irritant, does not smell good)

• Significant antimicrobial effects at 1% solution

• Phenolics (derivatives of phenols) reduce the irritant qualities of phenol

• Works well with soap and detergents

• Persist for a long time after application

What are bisphenols?

• A type of disinfectant derived from phenols

• Part of the prescribed lotion pHisoHex

• Triclosan (another version) is found in antimicrobial soups and in cutting boards/plastic knife handles to prevent foodborne illness

  • Prevents synthesis of fatty acids

What are biguanides?

• Broad spectrum disinfectants

• Attacks the bacterial membrane

• Works well against gram-positive bacteria

  • Can work on gram-negative, except for pseudomonads

• Generally not sporacidal

What are halogens?

• Iodine and chlorine → effective alone and as constituents of inorganic/organic compounds

• Iodine: one of the oldest disinfectants and is available in tinctures

• A liquid form of compressed chlorine gas can be used to disinfect municipal water supplies and swimming pools

What do alcohols do?

• Kill bacteria and fungi, not endospores and non-enveloped viruses

• Generally denature proteins and dissolve lipids (disrupts membranes)

• Evaporate rapidly and leave no residue

• Common forms: ethanol and isopropanol

• Can be used to enhance the activity of other disinfectants

What do heavy metals and their compounds do?

• Several metals are biocidal and antiseptic (mercury, copper)

• Silver and copper can prevent growth (oligodynamic action)

What are surfactants?

• Surface active agents

• Can decrease surface tension and dislodge bacteria and other microorganisms from surfaces

What do soap and detergent do?

• Allows for the mechanical removal of microbes when added to scrubbing

• Soap is an emulsifier and breaks things up into small pieces

• Not antiseptic

What are acid-anionic sanitizers?

• Important for cleaning food-processing facilities

• Combinations of phosphoric acid and surface-active agents

• Have negatively charged particles that disrupt plasma membranes

What are quaternary ammonium compounds?

• The most widely used surfactants

• Positively-charged

• Bactericidal against gram-positive bacteria

• Fungicidal

What is used as chemical food preservatives?

• Sulfur dioxide = prevents food spoilage

• Organic acids = inhibit metabolism

  • Sorbic acid, benzoic acid, calcium propionate prevent molds in acidic foods

• Nitrates and nitrates prevent endospore germination

What are bacteriocins?

Proteins produced by one bacterium that inhibits another

What type of antibiotic prevents the spoilage of cheese

Nisin and natamycin

What do aldehydes do?

• Inactivate proteins by cross-linking with functional groups

  • -NH2, -OH, -COOH, -SH

• Used for preserving specimens and in medical equipment

  • Formaldehyde and ortho-phthalaldehyde

  • Glutaraldehyde: one of the few liquid chemical sterilizing agents

Describe chemical sterilization

• Gaseous sterilants cause alkylation (replacing hydrogen atoms of a chemical group with a free radical)

• Cross-links nucleic acids and proteins

• Used for heat-sensitive material

  • Ethylene oxide

What is plasma?

• Fourth state of matter, consisting of electrically excited gas

• Free radicals destroy microbes

• Used for tubular instruments

What are supercritical fluids?

• CO₂ with gaseous and liquid properties

• Used for medical implants

What are peroxygens and other forms of oxygen used for?

• Oxidizing agents

• Used for contaminated surfaces and food packaging

  • O₃, H₂O₂, and peracetic acid

What did Paul Ehrlich do?

• Introduced concept of a “magic bullet” that selectively destroys pathogens and leaves the surrounding tissue unaffected

• Basis for chemotherapy and selective toxicity

What did Alexander Fleming do?

• Discovered the first true antibiotic penicillin

• Accidentally inoculated a plate of S. aureus with a mold from the air and observed clearance around the mold

What is an antibiotic?

A substance produced by a microorganism that inhibits the growth of other microorganisms

• Easy to discover

• Very few of them useful clinically

• More than half of our antibiotics are produced from Streptomyces spp

  • Filamentous bacteria that lives in the soil

• A few are produced by endospore-producing bacteria like Bacillus

• Others produced by mold

• Either bactericidal: kills cells

• or bacteriostatic: prevents microbes from gorowing

T/F: Synthetic drugs like sulfa are antibiotics

False, they are antimicrobial drugs

Antibiotics naturally occur from microorganisms

How were many of the antibiotics used today discovered?

By screening colonies of antibiotic-producing organisms or by screening soil samples

What is selective toxicity?

• Generated through attacking structures unique to prokaryotes (ex: lipopolysaccharides, porins)

• To pass through the channels made by porins, drugs must be small and hydrophilic

• Lipophilic drugs do not pass through Gram-negative cell walls

Compare broad-spectrum and narrow-spectrum agents

• Narrow-spectrum antibiotics: act against a limited group of bacteria

• Broad-spectrum antibiotics: act against a larger group of bacteria

T/F: During treatment, narrow-spectrum antibiotics are used first, then broad spectrum antibiotics

False

Giving broad-spectrum antibiotics first saves time, but it can also act on normal microbiota

The harvesting of energy is mostly done through what kind of reactions?

Redox reactions

• The most electronegative element is the electron acceptor