Exam II- Microbiology Lecture

Differences between viruses and cells; review size comparisons of viruses, bacteria and eukaryotic cells (such as the yeast)

 -Viruses are much smaller than cellular microorganisms(yeast and bacteria)

• (yeast[7um], bacteria[E.coli= 2um], etc.)

            Ex.) Poxvirus (250nm), Yellow Fever Virus (22nm)

• viruses are non-living

• Viruses need a living host to reproduce

• Viruses are…

  • Obligate intracellular parasites

  • not cellular in nature; structure is very compact & economical

Viral Structure

Virions Definition

a complete virus particle that consists of an RNA or DNA core with a protein coat sometimes with external envelopes

Viral Structure

Capsids Definition

protective protein shell that surrounds viral nucleic acid

Viral Structure

Nucleocapsids Def

consists of capsid + nucleic acid

Viral Structure

Helical Capsids Definitions

a type of capsid symmetry- have rod-shaped capsomers that form hollow discs that resemble a bracelet

Viral Structure

Icosahedral Capsids Definitions

type of capsid symmetry= 20-sided 3-dimentional structure with 12 corners

What microscope is used to see viruses?

electron microscopes

A naked virus does not have ___________

an envelope

What kind of macromolecules make up the capsid?

proteins

What is the viral envelope made of? How does a virus acquire its envelope during replication? (e.g. at which step of the replication cycle does the virus acquire the envelope?)

Viral Envelope:  composed of phospholipid bilayer and envelope proteins

How do virus acquire its envelope: viruses acquire the envelope during the release step (at the end of replication) when a part of the host membrane is taken to the envelope the new virion

-       Viruses enter the cell through membrane fusion

What are (envelope) spike proteins? Function of spike proteins?

Envelope spike proteins: viral glycoproteins on the envelope surface

Function: essential for attachment to the host(animal) cell

Why are viral genetic materials said to be diverse compared to cellular organisms?

• viruses contan either DNA or RNA, but NOT both -

• in inactive phage

  • DNA viruses: most have a double stranded DNA (dsDNA) molececule

  • RNA viruses: most have ssRNA molecules

  • genomes can be one or several segments

• Smaller genome size compared to cellular organisms

  • only contain the genes to invade/manipulate host cells

  • no metabolic genes

Steps (sequence of events) in animal virus multiplication (replication cycle)

1. Adsorption- involvement of spikes, which determine the host range

2. Penetration- virus enters host cell through endocytosis or fusion

3.Uncoating- release of viral nucleic acids from viral coat

4.Synthesis- viral nucleic acid and capsid protein synthesis using host machinery

5.Assmebly- formation of nucleocapsids and viral particles

6. Release- Newly synthesized viral particles leaving the host cell through budding or exocytosis

Examples of cytopathic effects (abnormal host cell morphology resulting from viral infections) - What are syncytia? What is an oncovirus?

-Inclusion body formation in host cell: viral mass in nucleus or ctoplasm

-Syncytia: fusion of host cells, formation of giant cell w/ multiple nuclei (cells growing together)- it’s a sign of viral infection; it allows virus to escape host antibodies/detection

-Oncovirus: a virus that can cause cancer

What are phages or bacteriophages? Major differences between bacteriophages and animal viruses during multiplication

Phages or Bacteriophages: are viruses that infects bacteria specifically

Differences

-Bacteriophages

• inject their DNA through a trail structure directly into the bacterial cytoplasm

• infect bacteria (prokaryotic cells)

• 
  

-Animal Viruses

• fuse with the host cell membrane or enter by endocytosis

• infect eukaryotic cells(animals, plants, fungi)

Lytic Phage Def

lyse the host cells and release phage progeny at the end of infection cycle

Note:

• Lyse: the breakdown of a cell caused by damage to its plasma (outer) membrane

Temperate Phage Def

don’t always lyse the host cell(Temperate, “mild”, as opposed to “virulent”); they can persist within the host cll or lyse the host cell

Note:

• Lyse: the breakdown of a cell caused by damage to its plasma (outer) membrane

Temperate phage

a virus that can infect bacteria in two ways: lysogeny and lysis

What is a prophage?

A prophage is a bacteriophage genome that is integrated into the circular bacterial chromosome

What happens when a prophage is activated (induction)

“Induction”: the lysogenic state induced to enter the lytic state

What is lysogenic conversion? How does that lead to emergence of new pathogenic bacteria?

Lysogenic Conversion: conversion of phenotype

How does that lead to emergence of new pathogenic bacteria?

a bacteriophage (a virus that infects bacteria) introduces new virulence factors or traits that make the bacterium more harmful to the host

What is lysogenic conversion? How does that lead to emergence of new pathogenic bacteria?

lysogenic conversion: conversion of phenotype

Differentiate “lysogenic conversion” and “induction”

• Examples of Lysogenic conversation

Difference: lysogenic conversion alters the bacterium's traits, …

… while induction activates the bacteriophage to begin producing new virus particles, causing cell lysis.

- Ex.) many nonpathogenic bacteria can become pathogenic after acquiring
a temperate phage which may carry toxin genes

• Corynebacterium diphtheriae: the diphtheria toxin is encoded by the
  prophage b. When the nontoxigenic bacterium C7 is infected by b, àit
  becomes a toxin-producing pathogen C7(b) and can cause diphtheria, a
  respiratory infection that can lead to death

• Cholera toxin (Vibrio cholerae) and botulism toxin (Clostridium botulinum)
  genes are all phage-encoded

Viral cultivation methods;

why can we culture bacteria on agar plates or in broth, but can’t grow viruses directly on agar plates?

Viruses cannot be cultured directly on agar plates because they are not living cells and require a host cell to reproduce.

What methods are used for viral cultivation

In vitro method

• cell or tissue culture

In vivo method(for animal viruses)

• lab animals

• embryonic bird tissue (chicken egg, turkey, duck)

What is a plaque? (in context of viral nfection)

plaque: is the area of host lysis

• plaque can be used to enumerate viruses

What are prions and viroids? (Which types of macromolecules are they made of?)

Prions: are infectious protein particles from diseased animals (no nucleic acid, no envelope, no capsid)

• Made of: Proteins

Viroids: contain RNA only, plant pathogens (tomatoes, potatoes, cucumbers), short RNA pieces (1/10^th size of normal viruses), naked strands of RNA, no capsid proteins

 Diseases caused by prions and viroids in animals and plants, difference between a satellite virus and a regular virus

Disease caused by… prions and viroids

-Prions: Mad cow disease (CGAD in humans) & sheep scrapies= FATAL

-Viroids: primarily effect plants and we don’t know much on them

Satellite viruses: defective viruses dependent on other viruses for replication (steal spike protein form HPV)

Ex.) delta agent (hepatitis D virus), can only be multiple in the presence of Hepatitis B virus

Regular viruses: Regular viruses are fully independent, capable of replicating on their own, while satellite viruses depend on another virus (the helper virus) for replication and often exhibit smaller and simpler genomes.

Anabolism (biosynthesis) and catabolism: definitions; relationships between the two

Anabolism: building of complex molecules from numerous simple ones.

Catabolism: breakdown of complex molecules into numerous simple ones

Relationship: Catabolism breaks down molecules to release energy, while anabolism uses that energy to build and repair essential cellular components.

• essential for life and are closely regulated to ensure energy homeostasis in the cell.

ATP molecule structure: how many high energy bonds does it have?

2 high energy bonds

Define phosphorylation and dephosphorylation

phosphorylation: the process of adding a phosphate group to a molecule or ion

dephosphorylation: is when ATP+ becomes (ADP-) ---> energy is released, to be used in endergonic run

ATP

Adenosine triphosphate is the energy carrier in the cell

-       a nucleotide that stores energy in the form of high-energy bonds

-       2 high energy bonds will release energy when broken

ADP- adenosine diphosphate

ATP—> ADP +Pi: is this reaction dephosphorylation or phosphorylation? Dephosphorylation

Electron carrier pairs: NADH/NAD⁺, FADH₂/FAD: Which one is the reduced form and oxidized form in the pair? Which one in the pair can donate electrons? Which one can accept electrons?

NAD+ accepts 2 e- and becomes NADH

NADH can donate 2 e- to the respitory chain and become NAD+ again

Consider this overall reaction that depicts glucose catabolism in aerobic respiration:

Glucose + O₂ + ADP  6 CO₂ + 6 H₂O + ATP

Pathways of aerobic respiration

Stage 1) Glycolysis, Stage 2) TCA cycle, Stage 3) Electron transport (ETC)

At which stage(s) is ATP produced?

Gylcolysis, TCA

At which stage is oxygen (O₂) directly involved?

(Does glycolysis need oxygen?)

ETC

• Glycolysis DOESN”T need oxygen

At which stage(s) are electrons released from organic molecules to produce either NADH or FADH2?

Glycolysis, TCA

At which stages does substrate-level phosphorylation occur?

glycolysis

At which stage(s) does oxidative phosphorylation occur?

ETC

Which process (stage) is responsible for the majority of the cellular ATP produced?

ETC

Does substrate-level or oxidative phosphorylation produce more ATP?

oxidative pophoylation produces more ATP

Cellular locations of the TCA cycle and the electron transport chain (ETC) in bacteria and in eukaryotic cells

Bacteria: TCA and ETC (cytoplasm, specifically plasma membrane)

- Eucaryotic Cells: TCA (mitochondrial matrix), ETC (inner mitochondrial membrane)

Anaerobic respiration vs. aerobic respiration:

• What is the terminal electron acceptor for aerobic respiration?  

oxygen

What is the terminal electron acceptor for anaerobic respiration using nitrate?

Nitrate (NO3-)

In the absence of oxygen: what type(s) of microbial metabolism can take place?

• aerobic respiration AND fermentation

Fermentation reactions in alcoholic and lactic acid fermentation

• What are the main fermentation products in the yeast (Brewer’s or Baker’s yeast)?

ethanol and gas

In general, what is the fermentation product for most bacteria?

usually produce acids and sometimes gas

How about in E. coli? (These two fermentation products are important for the diagnosis and identification of E. coli)

produces large amounts of acids AND gas is a mixture of CO2 + H2); positive for the Methyl Red test (MR test, positive if acid is produced)

What fermentation products does the MR-VP test detect?

MR-VP test for acid production (MR) and ethanol (VP)

Based on its metabolism in the presence and absence of oxygen (aerobic and anaerobic respiration, fermentation

• what is a facultative anaerobe?

If O2 is present they produce lots of energy, if O2 is absent they have to use fermentation and have enzymes to protect them

What is a (strict anaerobe)?

doesn’t have membrane chains & don’t use anaerobic/aerobic respiration, but ferment and must live in O2 free environments; don’t have enzyme to neutralize

What is a (strict) aerobe?

Must use oxygen and have enzyme to protect them

Which enzymes are required for the metabolism of polysaccharides such as lactose and starch?

1. Enzymes for Starch: Amylase and maltase

   • Ultimately broken down into glucose, which can enter glycolysis or be stored as glycogen (in animals) or starch (in plants).

2. Enzymes for Lactose: galactose, lactase

   • Converted to glucose and galactose, which can enter glycolysis for energy production.

Compare genome vs. chromosome; parts of the eukaryotic cell where genetic materials can be found

Genome: all genetic material from an organism

-        Composed of double stranded DNA(dsDNA)

Chromosome: carry DNA into nucleus

Eukaryotic cell: genetic material is found in the Nucleus

Review DNA structure

• what are the 3 parts of a nucleotide?

-        Phosphate group, sugar(pentose), nitrogen

base pairing rules in DNA and RNA

  

Nucleotide in DNA	Matching nucleotide
	In DNA	In RNA
A	T	U
G	C	G
T	A	U
C	G	C

67 Is the following pentose (5-carbon sugar) a ribose or deoxyribose? Label the 1’, 2’ 3’, 4’ and 5’ carbon on the sugar ring; where does the pentose connect to the nitrogen base? Where does it connect to the phosphate?

-        Deoxyribose 5’,4’, 3’, 2’, 1’

where does the pentose connect to the nitrogen base?

• carbon 1’

Where does it connect to the phosphate?

• carbon 5’

What is the antiparallel structure of a DNA molecule

-        Two DNA strands run in opposite directions (one in 5’3’ and the other in 3’ 5’) – antiparallel

Which of the following is an accepted way for DNA sequence?

a.     5’ AMP GMP AMP AMP TMP GMP GMP 3’

b. 5’ dAMP dGMP dAMP dAMP dTMP dGMP dGMP 3’

c.    5’ AGAATGG 3’

d.      5’AGAAUGG 3’

c. 5’ AGAATGG 3’

DNA replication:

What is an origin? What is a DNA polymerase? What is the semiconservative nature of DNA replication

-        Origin: the specific location on a DNA molecule where the replication begins, acting as a starting point for the process

-        DNA polymerase: is the enzyme that plays a crucial role in the synthesis of new DNA strands during replication. Its primary function is to add nucleotides (the building blocks of DNA) to the growing DNA strand by reading the template strand.

-        Semiconservative nature of DNA replication: refers to the way the two strands of the original DNA molecule separate (each used as a template for the synthesis of new strands)

• Each new DNA molecule consists of the original strand (from the parent DNA) and one newly synthesized strand.

Practice of DNA, RNA and protein sequences:

How should each be label (3’-5’)

protein sequence should have N-_______ C- terminus

Which strand of the DNA is the coding strand?

has the same sequence as the mRNA (except for T → U).

Which one is the template strand

the strand that RNA polymerase reads to make the mRNA.

  The terms: replication, transcription, and translation: complete the following table (choose from the following: DNA, RNA, promoter, DNA polymerase, RNA polymerase, ribosome, codon, anticodon, start codon, stop codon, etc.)

	Template type (DNA, RNA or protein)	Product type (DNA, RNA or protein)	Where does it start (names of the initiation sites)	Main enzyme or organelle required
replication	DNA	DNA	Origin	DNA polymerase
transcription	DNA	RNA	Promoter	RNA polymerase
translation	RNA	Protein	Start codon	ribosome

  If the coding region on a DNA strand is 1200 bases long (including the start codon by not including the stop codon), what is the size of the protein product (the number of amino acids in this peptide)? Hint: use the 3:1 ratio

o   The "3:1 ratio" mentioned refers to the fact that every 3 nucleotides in the DNA sequence code for 1 amino acid in the protein

-        If the coding region is 1200 bases long, and each codon (a group of 3 bases) codes for one amino acid, then the protein product will consist of 1200 bases / 3 bases per codon = 400 amino acids

1.     Cellular locations (cytoplasm or nucleus) for transcription and translation in eucaryotes

1.      

Genetic processes	Cellular location: cytoplasm or nucleus?
	Procaryotes	Eucaryotes
Replication	?	?
Transcription (RNA splicing occur?)	?	?
Translation (70s or 80s ribosome?)	?	?

      

Genetic processes	Cellular location: cytoplasm or nucleus?
	Procaryotes	Eucaryotes
Replication	Cytoplasm	Nucleus
Transcription (RNA splicing occur?)	Cytoplasm	Nucleus (RNA splicing occurs)
Translation (70s or 80s ribosome?)	Cytoplasm(70S)	Cytoplasm(80S)

What does the Ames test detect? What is the relationship between mutagens and cancers?

Detects: substance can cause mutations, helping identify potential mutagens.

The relationship between mutagens and cancer lies in the fact that mutations, especially in genes that regulate cell growth and division, can lead to uncontrolled cell growth—a hallmark of cancer

Point Mutation Types

Def of point mutations: change of a single nucleotide(letter), or nitrogen base

• Nonsense Mutation

• Silence Mutation

• Missense Mutation

• Frameshift Mutation

Missense Mutation

DEF: causes change in a single amino acid residue in the protein and can change the protein function dramatically

EX.) GGU (Gly) ——> GCU (Ala)

Nonsense Mutation

DEF: mutation on DNA changes a normal codon on mRNA to a stop codon

EX.) UAC (tyr) —> UAA (stop)

Silent Mutation

DEF: alters a base but does not change the amino acid

Ex.) GGC—> GGU

• base(letter) changes but both codons are both Gly doesn’t change

Frameshift Mutation

DEF: insertion or deletion of a nucleotide; adding an extra “letter”

very serious mutation 

What kind of mutation are these? Which will most likely cause a defective protein product?

i)      codon UGG is mutated to codon UGA

ii)     codon GGG is mutated to codon AGG

iii)   codon ACU is mutated to ACC

iv)    codon AAA is mutated to AAGA

i)      codon UGG is mutated to codon UGA= nonsense mutation

ii)     codon GGG is mutated to codon AGG= missense mutation

iii)   codon ACU is mutated to ACC= silent mutation

iv)    codon AAA is mutated to AAGA= frameshift mutation

•   Which of the above will most likely cause a defective protein product? Why is a frameshift mutation so serious to the cell?

  • Nonsense mutation (i) and Frameshift mutation (iv).

  • Frameshift mutations are particularly dangerous because they alter the entire downstream sequence of amino acids, often leading to a nonfunctional protein.