An Introduction To Microbes - Bacteria, Archaea, Viruses & Eukaryotes Flashcards
(342 cards)
1
Q
What are the 2 types of microbial reproduction?
A
- Sexual reproduction (less common) requires fusion of 2 haploid nuclei (micronuclei)
- Asexual reproduction - cell division into 2 cells (same or different sizes)
2
Q
What are the 3 main examples of asexual reproduction?
A
- Multiple fission - large number of nuclei generated and each forms a daughter cell which then separate
- Budding - Daughter nuclei migrate into a protrusion (bud) that separates
- Binary fission - continual division of 1 cell into 2 identical cells
3
Q
Are bacteria haploid or diploid?
A
Bacteria are haploid with one set of genes (useful in genetic studies)
4
Q
Can cell division of microbes continue indefinitely?
A
Yes, cell division will continue indefinitely if the conditions are right.
5
Q
What type of gene transfer is involved in binary fission?
A
Vertical gene transfer - each daughter cell contains exactly the same chromosomal genetic material
(NOTION 2.1)
6
Q
What type of chromosome do prokaryotes have?
A
Circular chromosomes
7
Q
What are some characteristics of DNA replication in prokaryotes?
A
DNA synthesis:
- Starts at a single origin of replication
- Occurs bidirectionally
- New replication forks start before cell divides
8
Q
Bacteria often contain…
A
Bacteria often contain plasmids = small circles of DNA
These usually encode non essential functions, and replicate in the same way as the main chromosome.
9
Q
What are the 2 main types of gene transfer in bacteria?
A
- Vertical gene transfer - Asexual reproduction, transfer from mother to daughter cell (= growth by binary fission)
- Horizontal gene transfer - Exchange of genetic information from one cell to another that is not its offspring
10
Q
What are the 3 methods by which bacteria are able to horizontally transfer some DNA from cell to cell?
A
- Conjugation
- Transduction
- Transformation
11
Q
Provide more detail for the 3 main mechanisms of horizontal gene transfer.
A
Transduction = Bacteriophages
Transformation = Naked DNA Transposons (tn). Plasmids
Conjugation = Integrons. Conjugative Transposons. Mobile Plasmids.
12
Q
- What is involved in transformation?
- In order to take up DNA a cell must become?
- Give an example of bacteria which is capable of interspecies DNA uptake, and one which is not.
A
- Transformation = The uptake of free DNA by a bacterial cell; the DNA is then integrated into the bacterial chromosome.
- In order to uptake DNA a cell must become competent.
Not all bacteria can become competent. - Streptococcus is capable of interspecies DNA uptake, the bacterium Neisseria is not.
13
Q
- What can streptococcus pneumoniae cause?
- Is streptococcus pneumoniae gram +ve/-ve?
- What two forms can it exist in?
- Why is S pneumococci dangerous?
A
- It is a causative agent of some pneumonias.
- Gram positive Cocci.
- Exists in two forms:
- ‘S’ smooth colonies; virulent
- ‘R’ rough; avirulent - ‘S’ smooth pneumococci produce a polysaccharide capsule that helps defend bacterium from host immune responses -> bacterial infections & even death
14
Q
Who was Griffith, and what did he study?
What was involved in his bacterial transformation experiment?
What were the findings?
A
Griffith was a British microbiologist (1879-1941) who served in World War I as a medical officer.
He studied the virulence in Streptococcus pneumoniae.
In his bacterial transformation experiment, he found that mixing heat killed ‘S’ form streptococci with ‘R’ form streptococci, killed the mouse.
The substance released from the heat killed ‘S’ form had transformed the ‘R’ form streptococci into virulent, encapsulated ‘S’ form.
(NOTION 2.2)
15
Q
Who continued Griffith’s research into the virulence of streptococci pneumoniae?
What did his experiment involve?
And what were the findings?
A
Oswald Avery (American Doctor & microbiologist) [1877 - 1955] continued Griffith’s studies.
He carried out the same experiment four more times, but in each one he used a different enzyme:
I) Lipase
II) RNase
III) Protease
IV) DNase
He found that the mice still die in experiments I), II), III). And only DNase prevented the killing effect. The transferred substance must be DNA!
16
Q
What does conjugation involve?
What does it usually occur between?
A
The transfer of DNA from one bacterial cell to another via a special protein structure called a pilus = bacterial mating:
- Bacteria is physically intact
- DNA moves freely from one cell to another through conjugation ‘bridge’
- Plasmids and COPIED parts of the donor cell chromosome move
- Usually occurs between related bacterial species
(NOTION 2.3)
17
Q
What did Lederberg and Tatum’s experiment (in 1946) into gene transfer between two strains of E. coli involve?
A
They mixed two strains of E. coli (with different nutritional requirements):
- Strain 1 requires methionine & biotin for growth
- Strain 2 requires threonine & leucine for growth
Individually, both of these strains did not grow on minimal medium.
However, on minimal medium with no supplements, a few colonies of prototrophic bacteria (Met+, Bio+, Thr+, Leu+) grow.
Therefore it could be seen that: Any colonies growing on minimal media MUST result from gene transfer between the parent strains.
(NOTION 2.4)
18
Q
What is a capsule a determinant of?
Where are genes encoding capsule synthesis located?
A
A capsule is a pathogenicity determinant.
Genes encoding capsule synthesis are located on a plasmid and transferable to other strains.
19
Q
Where might antibiotic resistance genes be located?
A
In other bacterial species, antibiotic resistance genes are plasmid-borne.
20
Q
What are keys structures of a bacteriophage?
A
- Head Capsid
- Tail Sheath
- Baseplate
- Tail fibres
(NOTION 2.5)
21
Q
What is a bacteriophage?
A
Bacteriophage = A virus that infects a bacterium.
22
Q
What does transduction involve?
A
Transduction - Transfer of DNA from one bacterium to another with a virus (bacteriophage) acting as a gene carrier.
Transduction can involve movement of phage DNA and host cell DNA.
23
Q
Generalised vs Specialised Transduction
A
Generalised transduction (Random DNA encapsulation)
Specialised transduction (Lysogenic integrated phage, specific encapsulation)
24
Q
What is corynebacterium?
A
Corynebacterium is the casual agent of diphtheria.
25
What is the diphtheria toxin gene transmitted via?
The diphtheria toxin gene is transmitted via a bacteriophage.
26
Why is horizontal gene transfer important?
It is the key to rapid bacterial evolution, & essential for bacterial adaptation and survival.
27
What are the five main microorganisms?
Bacteria, archaea, fungi, algae, protozoa
(Viruses and prions are microscopic but are not able to grow and replicate outside their host, so may be classed as non-living).
28
Are all unicellular organisms microorganisms?
Yes, all unicellular organisms are microorganisms, but NOT all microorganisms are unicellular.
29
Multicell complexes vs multicellular organisms
Multicell complexes - Composed of a group of identical cells carrying out all the same identical functions.
In contrast a multicellular organism consists of different cells with different functions.
30
What were the first organisms on earth?
Microorganisms
31
What was the timing of life on earth?
NOTION 1.1
32
The human body supports ________ microbes, only ________ cells are human.
The human body supports about 4 x 10^13 microbes, only 3 x 10^13 cells are human.
33
How many microbial cells does a gram of soil contain?
A gram of soil can contain 1,000,000,000 microbial cells - as many microorganisms as there are people in China.
34
What % of earth’s biomass is estimated to be microbial?
10-20%
35
The tree of life (including microbes)
NOTION 1.2
36
What are the 3 domains on the Universal Phylogenetic tree?
- Bacteria
- Archaea
- Eukarya
37
What 6 kingdoms do microbes exist in?
Microbes exist in 6 kingdoms - Archaea, bacteria, Protista, Fungi, Plantae and Animalia
38
What are fungi?
In nature fungi are decomposers, breaking down matter to nutrients that plants and animals reuse.
39
What are some examples of species of fungi?
Over 100, 000 species, include mushrooms, yeast and moulds.
40
What is an example of a fungus?
An example is the yeast Saccharomyces cerevisiae (S. cerevisiae). A single celled microbe.
41
Are mushrooms microbes?
No mushrooms are fungi, but they are multicellular and are therefore not microbes.
42
What are algae?
A diverse group of photosynthetic microbes,
exist in a wide range of forms (microscopic to
macroscopic (phytoplankton)
43
How do algae capture light energy?
They use chlorophyll to capture light energy, found in aquatic environments
44
What is an example of algae?
Chlorella
45
What do some algae form?
Some algae form symbiotic relationships with other organisms
46
What are lichens?
Lichens are an example of symbiosis between algae and fungi
47
As well as existing as single cells, how else can algae exist?
- Single cells
- Filaments
- Colonies
- Diatoms
48
After the Mount Saint Helens eruption in 1980, what were the first life forms colonising sterile ground?
Lichens & mosses
49
What are Protozoa?
- Eukaryotes
- Single celled predators and parasites,
50
How large are Protozoa?
They are 1000 times larger than bacteria
51
What is an example of Protozoa?
An example is Amoeba. Amoeba constantly change form as the internal contents flow when they move.
52
What are rumen protozoa?
Rumen protozoa :
• diverse,
• highly specialised,
• colonise the animal gut in large numbers,
• move rapidly,
• engulf and degrade plant material
53
What are some examples of rumen Protozoa?
Examples - Entodinium, Epidinium, Diplodinium
54
Most rumen Protozoa are ______
Ciliated Protozoa (motile)
55
What are archaea?
- prokaryotes – single celled organisms with some eukaryote-like biochemical processes;
- often inhabit extreme environments such as salty or high temperature environments – extremophiles
- Reproduce asexually
56
Do archaea contain peptidoglycan in their cell wall?
No
57
How many RNA polymerases do archaea contain?
3 (and they contain different rRNA to bacteria)
58
What are some metabolic activities that archaea exhibit?
Archaea exhibit some unique and diverse metabolic activities;
- As methanogens they make methane in guts of ruminants and humans
- Inhabitants of ‘black smokers’ at deep-sea vents;
- Inhabitants of extremely saline environments: Dead Sea
-Extremely temperature-resistant
59
Pyrococcus furiosus survive at what temperature?
100 degrees
60
What are bacteria?
How long have they existed for?
How many species are there?
Bacteria (singular: bacterium)
- Single-celled prokaryotes,
- Have existed on Earth for > 3.5 billion years.
- More than 5,000 species,
- New ones constantly being discovered.
- Grouped as Gram-positive or Gramnegative based on cell wall ultrastructure
61
How many RNA polymerases do bacteria contain?
Single RNA polymerase, multiple copies of
16S rRNA gene
62
What is a widely known bacterium?
Escherichia Coli
63
What are viruses?
Viruses (singular: virus)
• smallest and simplest microbes.
• reproduce by injecting their genes into a host cell to produce thousands of new viruses.
• Thus they need a living host to survive
64
How numerous are viruses in the environment?
Incredibly numerous in the environment; 50 million viruses per milliliter in seawater.
65
What are some examples of viruses?
Poliovirus and Coronavirus
66
What did a survey of bacterial microbiota on the forearm involve?
• Six people tested
•182 bacterial species identified
• 98 isolates potentially new to science (in 2007)
• Only 4 of the 182 species common to all tested
67
What does biological diversity examine aspects of?
Biological diversity examines aspects such as variation in:
- Structure
- Physiology
- Biochemistry
- Genetics
68
What classifications tools are used to detect diversity in microbial life?
Classification tools
– Cell ultra-structure ; eukaryote/prokaryote
– Overall cell morphology (size, shape);
– Motility
– Physiological diversity; growth characteristics
– DNA sequence diversity
69
Prokaryotic vs eukaryotic microbes
Prokaryotic microbes do NOT contain a nucleus or other internal membranes.
Eukaryotic microbes do contain a nucleus, and may also contain organelles such as mitochondria/ chloroplasts.
70
Out of the main microbes, which ones are eukaryotes/prokaryotes?
Eukaryotes:
- Fungi
- Algae
- Protozoa
Prokaryotes:
- Bacteria
- Archaea
Acellular:
- Viruses
71
What is the typical size of the bacterium Escherichia coli?
1-3 micrometers
72
What is the typical diameter of the yeast Saccharomyces cerevisiae?
10 micrometers
73
Smallest vs largest prokaryotes
Nanoarchaeum equitans = 0.5 micrometers (marine archaea)
Thiomargarita Namibiensis = 750 micrometers (marine bacterium)
74
What are the 5 morphologies of bacteria and archaea?
- Coccus
- Rods; bacillus
- Square
- Comma shape vibrio
- Spiral
75
Are bacteria mainly monomorphic?
Yes, Bacteria are either mainly monomorphic (consistent
shape/size) but some are pleomorphic/pleiomorphic, changing shape under specific environmental/growth conditions
76
Gram +ve vs Gram -ve bacteria
Gram +ve
- Stains blue/purple
- Thick peptidoglycan layer, no outer lipid membrane
Gram -ve
- Stains red/ pink
- Thin peptidoglycan and outer lipid membrane
(NOTION 1.3)
77
Fungal morphology
Filamentous branching structure:
- Spores/ Conidia
- Hyphal growth
- Yeast budding
78
Many bacteria are heterotrophic, what does this mean?
Requires an organic ‘food source’
79
Some bacteria are autotrophic, what does this mean?
Require an inorganic source of carbon i.e CO2
80
What are some unusual food sources of bacteria?
(Acidithiobacillus ferrooxidans) extract energy from the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+).
81
What can the use of DNA sequencing to classify diversity highlight?
It can highlight serious flaws in relying on gross morphology or even biochemistry to distinguish species.
82
All bacteria have the 16S rRNA gene essential for bacterial growth, how many copies are there per bacterial cell?
How many nucleotides make up the gene?
What regions are conserved, what are some variable regions?
There are 4-16 copies of the gene per bacterial cell.
Additionally, the gene is made up of around 1500 nucleotides.
Eight regions conserved (bold)
- Used for primer design
Nine regions variable
- facilitates detection of differences
This can therefore be used in bacterial classification.
(NOTION 1.4)
83
Coenocytic hypha vs septate hypha
NOTION 1.5
84
Can generic bacterial primers detect archaea?
Generic bacterial primers often fail to detect archaea - different sequences in conserved regions within 16S rRNA gene.
85
What is the process involved in binary fission?
1. DNA Replication
2. Cell elongation
3. Septum formation
4. Completion of septum & division into 2 daughter cells
NOTION 3.1
86
What is the doubling time of a bacteria?
Doubling Time = The time taken for two daughter cells to be produced from one original bacterial cell (each with identical DNA).
87
How long can the doubling time of a bacterial cell usually take?
Bacterial cells exhibit doubling times that vary from 10 min to several days.
88
What bacterial species is one of the fastest dividing? What is its doubling time?
Escherichia coli is one of the fastest dividing every 20min in the lab.
89
What bacterial species is one of the slowest dividing? What is its doubling time?
Mycobacterium species are some of the slowest taking
days or even weeks (M. leprae can take more than 20
days to divide)
90
What environmental factors impact on a bacterial cells doubling time?
Environment:
- Nutritional requirements (simplicity vs complexity of
nutrients)
- Physical environment (Temp, pH, osmolarity)
91
What is the time taken to replicate DNA in E Coli?
The time taken to replicate the DNA in E Coli = 40 minutes
92
Is the time taken for DNA to replicate constant or variable?
Is the time taken for one complete cell division constant or variable?
The time taken to replicate DNA is constant.
The time for one complete cell division is variable -
depending on nutritional and genetic factors.
93
How can the time taken to replicate DNA in E Coli be longer than its doubling time?
DNA synthesis starts at single point on chromosome (origin of replication).
Fast growing bacteria have multiple cycles of DNA
replication in progress when growing rapidly.
Multiple replication forks.
94
What is Growth?
What is Growth Rate?
Growth is an increase in the number of cells in the population;
Growth rate is the change in cell number per unit time.
95
What is the interval required for the formation of two cells from one cell called?
This is called a generation. And the time required is known as the generation time.
96
What is the generation time?
Generation time = time required for cell number to double (= doubling time).
97
What are the 5 phases of growth cycle of prokaryote populations?
1. Lag phase
2. Exponential/ logarithmic
3. Deceleration
4. Stationary
5. Death
NOTION 3.2
98
What does the Lag phase involve?
Lag - adjustment of cell metabolism to new conditions
99
What does the exponential stage involve?
Exponential - cell growth at max. rate possible in the medium chosen
100
What does the deceleration phase involve?
Deceleration - Limiting supply of one nutrient or accumulation of toxic metabolites
101
What does the stationary phase involve?
Stationary - no net growth, at least one nutrient depleted - possible growth at expense of cell death and lysis
102
What does the death phase involve?
Death - Decline of viable cells due to exhaustion of energy or accumulation of toxic byproducts
103
How can cell growth be plotted on a graph?
It is usually best to plot on a semi log scale!
NOTION 3.3
104
How can growth rate be calculated?
Growth rate (u) = In (cells2 - cells1)/ (t2 - t1)
NOTION 3.4
105
What do aseptic techniques involve?
- Exclusion of contaminants
- Culture media sterilised to eliminate contaminants
- All material contacting sterile media must be kept sterile
- Essential to maintain pure cultures of bacteria
106
What are the 6 main types of bacterial culture?
1. Streak Plate
2. Broth culture
3. Stab culture
4. Slope culture
5. Spread plate
6. Pour Plate
NOTION 3.5
107
What are 3 ways by which the growth of bacteria can be measured?
Cell number:
- Cell counts (microscope)
- Viable counts (agar plates)
Cell molecules:
- Total dry mass
- Protein
- DNA
- Diaminopimelic acid (component of peptidoglycan)
Light scattering:
- Optical density measurements (OD)
108
What is involved in using viable counts to measure cell growth?
It involves serial dilutions of bacterial sample, which then decreases the number of bacterial cells, to a point where you can feasibly count them.
One viable cell forms one colony = 1 colony forming unit (CFU). Therefore the number of colonies indicates no. of cells in starting culture.
NOTION 3.6
109
What is involved in using the microscope to count the number of bacterial cells?
Each grid square has a precise volume; counting
the number of bacteria per grid and then multiplying
by a factor determined by the volume of the grid gives cells/ml.
Cells are often killed with dilute formalin to prevent them moving!
110
What is involved in the light scattering method?
Light scattering - simplest and commonest method for measuring growth. Gives measurement of optical density.
111
How can you choose which method to use to measure bacterial growth?
Direct counts: Dead/Live? Small cells? Accuracy? Needs high cell densities
Viable counts: Accuracy depends on medium used - damaged cells/ small colonies. Clumps? [CFU]
Light scattering: good between 0.05 - 0.7 ( around 5 x 10^7 to 8 x 10^8 ) - use dilutions to extend range
112
What methods to measure cell growth help provide:
- Simplicity
- Accuracy
- The ability to deal with Low Cell numbers
Simplicity:
- Light scattering (OD)
- Useful for measuring growth rates
Accuracy:
- Biochemical assay - protein, DNA
- Fluorescent in situ hybridisation (FISH)
Low cell numbers ( < 10^7/ ml)
- Viable cell counts
113
FISH involves a large degree of replication. What does this involve?
Count 30 views per hybridisation.
Count at least 2 slides per probe target = 20-100 hybridising cells per view.
Total count = average of minimum of 60 individual counts
114
What calculation is used following FISH?
Calculate - Number of hybridising cells per gram faeces
Calculate - % hybridising cells as proportion of total eubacteria count
115
What is involve in light scattering?
NOTION 3.7
116
Batch vs Continuous Culture
Closed system = Nutrients run out (Growth stops)
Open system (Chemostat) = Nutrients replenished (Growth continues)
NOTION 3.8
117
What are the +ve’s & -ve’s of batch vs continuous culture?
Batch growth – no addition after inoculation; growth rate varies, medium composition varies; difficult to change growth rate.
Chemostat growth – continuous addition of fresh medium, constant volume, constant environment, can vary growth rate.
118
Can pH affect growth rate?
What pH do acidophilic bacteria survive at?
What pH do neutrophilic bacteria survive at?
What pH do alkaliphilic bacteria survive at?
Yes, growth rate is also affected by the pH of the environment!
NOTION 3.9
119
Can growth rate be affected by temperature?
What temperature do psychrophile bacteria survive at?
What temperature do mesophile bacteria survive at?
What temperature do thermophile bacteria survive at?
What temperature do hyperthermophile bacteria survive at?
Yes, growth rate is affected by temperature!
NOTION 3.10
120
What are black smokers?
What causes them?
Water temp as high as 400 degrees.
Black smokers caused by precipitated iron sulphide.
121
Can growth rate be affected by osmolarity?
At what osmolarity do Nonhalophiles survive at?
What is an example of a Nonhalophile?
At what osmolarity do Halotolerants survive at?
What is an example of a Halotolerant?
At what osmolarity do Halophiles survive at?
What is an example of a Halophile?
At what osmolarity do Extreme Halophiles survive at?
What is an example of a Extreme Halophile?
Yes, growth rate is affected by osmolarity!
NOTION 3.11
122
What can weak organic acids act as?
They can act as preservatives (this is because the growth rate of bacteria is affected by pH).
123
What are the 2 factors that affect bacterial growth?
Bacterial growth depends on bacterial species and environmental conditions.
124
Why is it important that we understand the nutrition of microbes?
1. Environmental cycling of the major elements (N, S, C) is absolutely dependent upon activity of micro organisms
2. Ability of microorganisms to derive energy from chemical oxidation has important industrial applications; uranium, copper, gold recovery from mine spoil
3. Ability of microorganisms to grow on different media has important biotechnological application; oil spill remediation
4. Ability of microorganisms to grow on different food sources has implications in food processing and manufacture
5. Understanding nutritional requirements of specific members of gut microbiota enables us to selectively boost growth for health
125
What are the 2 main functions of nutrients?
Nutrients serve two functions:
1. They are used as building blocks for the construction of cell components
2. They supply the energy required to drive cellular processes
126
What are the 5 main macromolecules that cells are constructed from?
Cells are constructed from macromolecules: proteins,
polysaccharides, RNA, DNA and phospholipids.
These are assembled from amino acids, sugars, nucleotides and fatty acids respectively.
127
What do metal ions serve as within a cell?
Metal ions serve as enzyme cofactors, as osmoregulators and help stabilise structures e.g ribosomes
128
Polymerisation involves significant loss of ________
Entropy
129
What is anabolism?
Anabolism - the biochemical processes involved in the synthesis of cell constituents from simpler molecules (Generally requires energy)
130
What is catabolism?
Catabolism - biochemical reactions leading to the production of energy by a cell
131
Why is ATP a high energy storage molecule?
ATP contains two phosphate-phosphate high energy bonds (repulsion of negative charges).
132
What are 2 examples of bacteria which have simple nutritional requirements? What is an example of one bacteria which has a more complex nutritional requirement?
Cyanobacteria and E.coli have simple nutritional
requirements while lactic acid bacteria have more
complex ones.
133
What are the 8 basic components of a medium for growing microorganisms?
1. Water
2. Carbon source
3. Nitrogen source
4. Phosphorus source
5. Sulphur source
6. Inorganic salts in macro-amounts Na+, K+, Mg2+
7. Inorganic salts in trace amounts Cu2+, Mn2+, Co2+, Zn2+, Ni2+
8. Source of energy e.g light, oxidation of organic compounds
134
What are the 2 main carbon sources?
Carbon source:
1. Can be CO2 = Autotroph
2. Can be organic molecules = Heterotroph
135
Pseudomonas strains can grow on what types of carbon sources?
Pseudomonas strains can grow on hydrocarbons, sugars, fats etc.
136
Typical bacterium is ____ nitrogen by dry weight
Typical bacterium is 12% nitrogen by dry weight (present in proteins, nucleic acids, cell wall).
137
What are the 2 main sources of nitrogen?
Most bacteria obtain N as either ammonium ions (NH4-) or nitrate (NO3-).
138
How do Cyanobacteria obtain their nitrogen source?
Some bacteria (some cyanobacteria) can fix atmospheric nitrogen (N2) to form ammonia (NH3).
139
What is the main source of phosphorus?
Generally available as phosphate ions (PO4 3-).
Required for synthesis of nucleic acids and phospholipids.
140
What is a key structural role of sulphur?
What is the main source of sulphur?
Structural role in 2 amino acids and for some vitamins.
Available as sulphate (SO4 2-) or sulphide (S2-).
141
What are some key functions of Mg2+ in a cell?
Mg2+ functions to stabilise ribosomes, cell membranes and is required for the activity of many enzymes.
142
What are some key functions of Ca2+ in a cell?
Ca2+ - stabilises the bacterial cell wall and key role in the heat stability of endospores.
143
What are some key trace elements?
Trace elements: Co, Zn, Mo, Cu, Mn, Ni usually required by
a small number of enzymes.
144
What types of low molecular weight organic compounds are required by some microbes?
Low molecular weight organic compounds required by some
microbes: vitamins, amino acids, purines and pyrimidines.
145
Liquid vs Solid media
Liquid media:
- Easiest to prepare and use.
- Good for growing large quantities of microbes needed for analysis or experiments
- Not useful for separating microbes.
Solid media:
- Made by adding agar, a seaweed extract, to appropriate liquid.
- 1.5% w/v agar is standard for plates.
- Agar is solid at 40oC.
- Few microbes can degrade agar, as it is normally not a source of C.
146
Complex vs defined media
Complex media: composition of media not completely known. Made from inexpensive organic materials (slaughterhouse wastes/ soybeans/ yeast wastes from brewing/ animal blood/ rumen fluid, etc)
Some examples of complex media = Nutrient agar; contains peptone and beef extract
Complex media are often used to culture organisms from clinical samples.
Defined Media: Composed of known quantities of chemically pure organic and/ or inorganic compounds.
147
Defined culture medium for Cyanobacteria
NOTION 4.1
148
Defined culture medium for Escherichia Coli
NOTION 4.2
149
Defined medium for Leuconostoc mesenteroides, a lactic acid bacterium with complex nutrient requirements
NOTION 4.3
150
Complex culture medium for gut anaerobes
NOTION 4.4
151
What are autotrophs?
What are heterotrophs?
What are phototrophs?
What are chemotrophs?
Organisms can be classified by their source of carbon:
- Autotrophs derive the bulk of their carbon from CO2
- Heterotrophs derive the bulk of their carbon from organic sources e.g. sugar.
Organisms can also be classified according to their source of energy:
- Phototrophs use light energy.
- Chemotrophs use the energy released by redox reactions
152
What are the 4 nutritional categories for microbes?
There are 4 categories:
1. Photoautotrophs
2. Photoheterotrophs
3. Chemoautotrophs
4. Chemoheterotrophs
153
What is an example of a photoautotrophs?
Cyanobacteria are examples of photoautotrophs:
- Simple nutritional requirements; salts & sunlight
- Cyanobacteria are found in damp soil, lakes and springs
154
What is an example of a photoheterotroph?
Purple non sulphur bacteria (These bacteria grow in anaerobic conditions, gaining their energy from light reactions but using organic acids as their carbon source for cellular synthesis).
155
What is an example of a chemoautotroph?
How does it derive it’s energy?
What is the energy then used for?
Where can it be found?
Nitrosomonas europaea are examples of chemoautotrophs:
- Derives its energy from the oxidation of ammonia to nitrite.
- Consumes oxygen
- Energy generated used to convert carbon dioxide into cellular material
- Found in soil and sewage
156
Reaction for the oxidation of ammonia to nitrite
NOTION 4.5
157
What is a 2nd example of a chemoautotroph?
How does it derive its energy?
What are the electrons then used for?
What is its optimum temperature?
What is its optimum pH?
Acidithiobacillus ferooxidans:
- Derives its energy from the oxidation of Fe2+ (ferrous) to Fe3+ (ferric)
Acidithiobacillus thiooxidans:
- Derives it’s energy by transforming Sulphur compounds; sulphide to sulphate
- S2- (-2 oxidation state) to SO4 2- (+6 oxid. state)
- Sulphuric acid is therefore generated
Electrons fed into electron transport chain
- Oxygen is the terminal electron acceptor
- Proton gradient generated ; used to make ATP via ATP synthase
Acidithiobacillus thiooxidans is a Mesophile (temperature optimum 28oC)
And is also an Acidophile (pH optimum <4)
158
What does bioleaching involve, and what are the effects?
The sulphide mineral pyrite (FeS2), often present in mine waste materials: contains valuable copper, gold, uranium
4CuFeS 2 + 11O 2 + 6H2O → 4CuSO 4 + 4Fe(OH) 3 + 4S (1)
2S + 3O 2 + 2H2O → 2H2SO 4 (2)
The equations above demonstrate the recovery/release of copper during bioleaching. However the generation of sulphuric acid + iron III (rust) is an unwanted side effect.
159
What are some examples of chemoheterotrophs?
Most bacteria, all fungi and Protozoa = Chemoheterotrophs
160
What does the Winogradsky column involve?
What are the findings?
This involves:
- Taking pond water
- Adding carbon (sawdust, sodium bicarbonate, shredded newspaper)
- Adding sulphur (ground eggshell and egg yolk)
- Shake the mixture
- Leave for 3-5 months
What is found is that the microbial organism in the pond water, separate throughout the column. This can be seen in the following diagram: NOTION 4.6
161
What is the transit time along the GIT?
Mouth = 1 min
Oesophagus = 4-8 seconds
Stomach = 2-4 hours
Small intestine = 3-5 hours
Colon/ large intestine = 10 hours to several days
162
Going down the GIT what happens to the:
- Presence of oxygen
- Density of bacteria
- Dominance of obligate anaerobes
What are the densities of microbes in the:
- Stomach
- Small intestine
- Colon/ large intestine
Going down the GIT:
- Increasingly anaerobic conditions
- Increasing bacterial density
- Increasing dominance of obligate anaerobes
Stomach = 10^3 - 10^4 / ml
Small intestine = 10^8 / ml
Colon/ large intestine = 10^10 - 10^11 / ml
163
What types of energy is absorbed in the stomach/small intestine?
What about in the large intestine?
NOTION 4.7
164
What are the 2 subtypes of chemotrophs?
1. Chemolithotrophs
2. Chemoorganotrophs
165
What are chemolithotrophs?
- They use inorganic compounds for source of energy
- H2S, NH3, H2
- Usually also autotrophs
166
What are chemoorganotrophs?
- They use organic compounds for energy source
- Glucose, ethanol etc
- Usually heterotrophs (Same energy & carbon source)
167
Who first observed motile microorganisms, and when?
How did he do this?
When were bacteria later discovered to rotate their flagella?
Motile microorganisms first observed 1673; Dutch tailor and amateur scientist Antonie van Leeuwenhoek.
Leeuwenhoek used a single lens microscope for his observations and sent detailed descriptive sketches of
microorganisms to the Royal Society of London.
300 years later bacteria was discovered to rotate their flagella
168
How do prokaryotes move?
- Flagella are used by bacteria/ archaea for locomotion
- Composed of the protein flagellin
- Rotation is achieved by a complex protein motor embedded in the plasma membrane
169
What are the 4 different prokaryotic flagella arrangements?
Different flagella arrangements:
- monotrichous - polar
- lophotrichous – ‘tuft’
- Amphitrichous
- peritrichous – ‘around’
NOTION 5.1
170
What is the general size of flagella?
Length = 5 - 20 micrometers
Width = 0.01 - 0.02 micrometers
171
What 3 structures does a flagellum consist of?
A flagellum consists of three different sections:
• A long filament (composed of a protein called flagellin)
• A hook
• A basal structure; essentially like a mechanical wheel bearing
NOTION 5.2
172
What is the function of the basal structure of a flagellum?
Basal structure converts chemical energy into rotary motion (rotates clockwise/anticlockwise)
173
What is the shape of the flagellum?
Flagellum is helical [corkscrew]
174
How many genes are required for flagellar synthesis?
Over 40 genes required for flagellar synthesis – big energy investment
175
Differences to archaeal flagella
Archaeal flagella (archaella) have a similar
rotational movement, but different structure
176
What is chemotaxis?
How is this achieved?
Bacterial motility allows bacteria to respond to chemicals in their environment (chemotaxis).
They swim towards attractants and away from repellents.
Achieved via chemoreceptors located within the cell membrane.
Bacteria with polar flagella change direction by reversing the flagellar rotation direction
177
Movement in peritrichous bacteria is made up of two types of action. What are these 2 types?
Movement in peritrichous bacteria is made up of two types of action – runs and tumbles:
➢ Anticlockwise flagellar rotation produces a forward
movement.
➢ Clockwise flagellar rotation results in the cell tumbling
NOTION 5.3
178
Runs and tumbles in chemotaxis
NOTION 5.4
179
Why do bacteria move?
Reasons for bacterial movement:
1. Facilitates host colonization and survival
2. Motile bacteria that can swim toward food particles – chemotaxis - have a better chance of obtaining nutrition and surviving in an ecosystem
3. Motile bacteria that can swim toward mucosal surfaces –chemotaxis - may have a better chance to make contact with the mucous membranes of the bladder and intestine, attach, and colonize.
180
Give an example of the importance of bacterial movement
Eg.non-motile mutants of Vibrio cholerae are less virulent than the motile wild types.
Motility contributes to bacterial survival and pathogenesis.
181
What has the bacterial flagellin protein in E Coli been used for?
The bacterial flagellin protein has been widely used to serotype bacterial strains, including E.coli.
To help identify different E. coli strains, cell surface structures that are capable of eliciting an immune responses are used.
182
What has the well profiled pathogenic E Coli O157:H7 been found to have?
The well-profiled pathogenic E. coli O157:H7 has distinct lipopolysaccharide antigens (O) and flagella antigens (H).
183
What is a direct method by which you can detect bacterial motility?
Direct – looking for movement
1. Use of a phase contrast microscope – helps to reveal fine detail not available via bright field microscopy.
2. Use motility medium, (diagnostic in clinical microbiology)
184
Why are direct methods for detecting bacterial motility useful?
Pneumonia is caused by Gram -ve bacilli including:
- Klebsiella pneumoniae (non-motile),
- Pseudomonas aeruginosa (motile).
Rapid, easy diagnosis, enables specific treatment options.
185
What do indirect methods to detect bacterial motility involve?
Indirect – looking for the presence of flagella
1. Flagellar stains add extra width to the structure and
make them visible under the light microscope.
2. Electron microscope – the high magnification makes flagella easily visible
186
What type of microbes can contain flagella & cilia?
Eukaryotic microbes
187
What type of eukaryotic microbe contains cilia?
Cilia are restricted to one group of protozoa, the ciliates.
188
Eukaryotic vs prokaryotic flagella
Eukaryotic flagella used for cell movement are different in structure from those of prokaryotes.
Structure of a eukaryotic flagellum described as a 9 + 2 arrangement - nine peripheral pairs of microtubules plus one central pair.
189
Structure of eukaryotic flagellum
Structure of eukaryotic flagellum:
- Microtubules composed of protein, tubulin.
- Flagella dimensions: 0.15 - 0.3 micrometers x 40 micrometers
- Cilia and eukaryotic flagella are identical in cross section;
they only differ in length.
Eukaryotic flagella do NOT rotate but have whip-like motion.
190
Cilia vs flagella
NOTION 5.5
191
What are the 4 main categories of Protozoa?
1. Flagellates
2. Ciliates
3. Amoeba
4. Sporozoans (generally non motile)
NOTION 5.6
192
What are amoeba?
Amoeba are propelled by the flow of cytoplasm (cytoplasmic streaming).
Cytoplasmic streaming assists in phagocytosis: recognition and ingestion of (food) particles - chemotaxis
193
Structure of Protozoa flagellates
NOTION 5.7
194
What are 3 other examples of directed motility
Other examples of directed motility:
1. Aerotaxis
2. Phototaxis
3. Magnetotaxis
195
What is aerotaxis?
Aerotaxis – movement towards or away from oxygen.
196
What is phototaxis?
Phototaxis - movement towards light
197
What is a magnetotaxis?
Magnetotaxis – orientation and movement along lines of magenetic force.
Certain bacteria swim in the direction of the Earth’s magnetic field because of the presence of magnetosomes.
198
How do bacteria communicate?
Bacteria converse with one another and with plants and animals by emitting and reacting to chemical signals.
Cell-to-cell communication is achieved via chemical signal molecules called ‘autoinducers’.
199
What is Quorum Sensing?
What is the uses of Quorum Sensing?
(QS) production of signalling molecules by each cell.
If sufficient cell population present, the autoinducer
molecules accumulate
• Each cell can sense population density / cell concentration
• Populations of bacteria can co-ordinate their activity
200
What was the first organism in which quorum sensing was observed?
What is the auto inducer used by this organism?
Vibrio fischeri, a luminescent marine bacterium, was the first
organism in which quorum sensing was observed; QS triggers light production.
The autoinducer in Vibrio fischeri is homoserine lactone.
201
What is bioluminescence?
Bioluminescence - visible light emission in living organisms that accompanies the oxidation of organic compounds (luciferins) mediated by an enzyme catalyst (luciferase).
202
What is the point in bioluminescence?
To attract prey or to camouflage
203
What is the concentration of V. fischeri freely living in
the seawater?
What happens in high cell densities?
The concentration of V. fischeri freely living in the seawater is less than 10^2 cells ml-1. The autoinducer diffuses out of the bacteria but there is no observable luminescence.
At the high cell densities (>10^10 cells ml^-1), found in light organs the autoinducer accumulates and reaches a critical concentration required for activation of the luciferase enzyme → luminescence.
204
What is a biofilm?
What happens as cells form a biofilm?
A biofilm is an encased community of microbial cells attached to a surface by a gelantinous adhesive.
As cells form a biofilm (e.g. dental plaque), the concentration of signalling molecules increase.
QS triggers the synthesis of matrix polysaccharide.
205
Bacterial communication in Salmonella
Toxin producing bacteria e.g. Salmonella wait until the cell density is sufficient before releasing a toxin to sicken their host. Otherwise the immune system is more likely to destroy the pathogen.
Salmonella release a quorum sensing protein that accumulates and once a certain concentration reached, indicating a certain cell concentration, toxin production switched on.
206
What is an example of an amoeba?
Pseudopodia
207
What is an example of a flagellate?
Euglena
208
What are 2 examples of ciliates?
Paramecium & vorticella
209
What are magnetosomes generally made from?
Magnetosomes - Intracellular crystals of magnetite (Fe3O4)
210
How many base pairs makes up the E Coli Genome?
E Coli Genome is circular and 4.64 million bp long
NOTION 6.1
211
1. How many genes makes up the E Coli genome?
How many potential proteins are there?
2. In a growing cell, how many proteins actually exist?
3. How many copies of each protein are there usually?
4. What are the differences in protein abundance down to?
1. E. coli has ~4500 genes with a potential ~4400 proteins.
2. In a growing cell approx 1000-2000 proteins are made.
3. Protein abundance varies from 10-20 copies/cell (e.g. a repressor) to 200,000 copies/cell (e.g. ribosomal protein)
4. Differences are due to regulation of transcription/translation
212
Stages of Gene Expression
DNA - (Transcription) - mRNA - (Translation) - Protein
213
What does base pairing ensure?
Base pairing ensures accuracy of information transfer - for both vertical transmission (Replication) & gene expression.
214
Some genes are synthesised continuously. What is this called?
“Constitutive” expression
215
Levels of regulation of gene expression
NOTION 6.2
216
How many possible codons are there?
What is the start codon?
What are the stop codons?
What do the rest of the codons code for?
4 x 4 x 4 = 64 possible codons
1 start codon = AUG = formylmethionine & methionine
3 termination codons = UGA, UAA, UAG
Rest code for 19 remaining amino acids (redundancy).
217
Structure of a tRNA molecule
NOTION 6.3
218
How do tRNA’s become charged?
Charging of tRNA’s take place by Amino Acyl tRNA synthetases.
NOTION 6.4
219
The start determines the ‘Reading Frame” and hence the amino acid sequence. What happens when the frame of the sequence is shifted?
NOTION 6.5
220
Starting stages of Translation
1. Start codon AUG base paired with f-Met tRNA
2. f-Met tRNA occupies the acceptor site
NOTION 6.6
221
How does the ribosome distinguish the start from other AUG codons?
Initiation of translation needs Shine Dalgarno sequence & AUG. The Shine Dalgarno sequence = ribosome binding site.
222
Single mRNA can be coated with many ribosomes. What is this called?
Single mRNA can be coated with many ribosomes - a “Polysome”.
223
Can transcription & translation take place at the same time, in prokaryotic cells?
Yes, there can be coupled transcription & translation in prokaryotic cells.
224
What is involved in protein folding & secretion?
NOTION 6.7
225
How many types of RNA polymerase can be found in bacteria/ archaea?
What subunits make up the RNA polymerase found in bacteria/ archaea?
Bacteria and archaea have a single RNA polymerase.
The core enzyme (required for transcription) is made up of:
- 2 alpha subunits
- 2 beta subunits
The holoenzyme (required for promoter recognition & initiation) is made up of:
- 2 alpha subunits
- 2 beta subunits
- 1 sigma factor
226
Steps involved in RNA synthesis
NOTION 6.8
227
What 2 key sequences makes up the promoter region?
The promoter sequence consists of two main sequences:
- The -10 box (aka TATA box) TATAAT
- The -35 sequence (aka consensus sequence) TTGACA
NOTION 6.9
228
What is the importance of the sigma factor of RNA polymerase?
What is the general promoter sequence of a “housekeeping gene”? What sigma factors recognise this sequence?
What is the general promoter sequence of a “Heat Shock gene”? What sigma factors recognise this sequence?
What sigma factor recognises the sporulation genes in Bacillus Subtilis?
RNA polymerase can complex with different sigma factors that recognise different promoter sequences.
Most housekeeping genes:
- Promoter: TTGACA 17bp TATAAT
- Sigma factor 70
Heat Shock Genes:
- Promoter: CCCTTGAA 17bp CCCCATXTA
- Sigma factor 32
Sporulation genes:
- In Bacillus subtilis
- Sigma Factor F
229
Genes of the same metabolic pathway are clustered into ________.
Genes of the same metabolic pathway are clustered into OPERONS and transcribed as a polycistronic mRNA.
NOTION 6.10
230
What stable genes are clustered together in E Coli?
NOTION 6.11
231
What are stem loops?
Stem loops are signals for transcription termination
NOTION 6.12
232
Bacteria have 2 types of terminators, what do these include?
1. Rho-independent terminators are located after the end of the open reading frame - Most genes have these (NOTION 6.13)
2. Rho-dependent termination - most frequently occurs in genes that are not translated (NOTION 6.14)
233
What prevents transcription termination by Rho?
Coupled transcription and translation prevents transcription termination by Rho as the mRNA is covered with ribosomes.
234
When might Rho be useful?
What is the end result of this?
If the mRNA is not translated due to a premature stop codon introduced by mutation, Rho can terminate transcription.
Net result: Both the mutated gene and the downstream gene are not transcribed. This is called ‘polarity’.
235
What is anti-termination?
rRNA and tRNA are not translated - need anti termination systems to prevent premature termination by Rho.
Anti-terminator proteins bind to RNAP and allow read through of terminators.
236
What are the 2 main categories of regulatory proteins?
Regulatory proteins - Repressors & activators
237
What type of proteins increase the DNA binding specificity & affinity?
Dimeric proteins increase the DNA binding specificity & affinity - NOTION 7.1
238
What are the 3 main DNA binding motifs which allow interaction between regulatory proteins & DNA?
- Helix-turn-helix
- Leucine Zipper
- Zinc finger
NOTION 7.2
239
What happens when you add arginine to cells growing in arginine free medium? What is this known as?
When you add arginine to cells growing in arginine free medium, this switches off the biosynthetic enzymes (which where originally producing arginine). This is an example of repression.
NOTION 7.3
240
How does the arginine repressor operate?
Arginine repressor binds to its operator in the presence of its co-repressor (arginine). Only when the co-repressor is bound can the repressor bind the operator - this is allostery.
NOTION 7.4
241
What happens when you add lactose to cells growing in lactose free medium? What is this known as?
When you add lactose to cells growing in lactose free medium, this switches on the catabolic enzymes. This is known as induction.
NOTION 7.5
242
What happens to the lactose repressor, in the presence of lactose?
Lactose repressor does not bind the operator in the presence of its inducer (lactose).
NOTION 7.6
243
What is an example of gene activation (Hint: Maltose)?
RNA Polymerase cannot bind to the promoter region unless the activator is bound. Activator protein only binds when maltose is present.
NOTION 7.7
244
What is an example of catabolite repression (“Diauxic Growth”)?
Bacteria grow on glucose (preferred carbon source) until it is exhausted before they induce lactose catabolism genes. Glucose is repressing the lactose catabolism genes - catabolite repression.
NOTION 7.8
245
The lactose operon is controlled by two different regulators. What do these include?
The lactose operon is controlled by two different regulators:
- Catabolite activator protein (CAP)
- Lactose Repressor
246
When is CAP (Catabolite Activator Protein) active?
CAP only activates lac genes when cAMP is bound. cAMP levels are low when glucose is present and high when glucose is absent.
When glucose runs out, cAMP binds CAP, activator binds, RNA polymerase binds & transcription begins.
247
What is attenuation?
Attenuation = Controls expression of genes for amino acid and nucleoside synthesis
248
What are leader peptides?
The leader peptide ‘senses’ the level of amino acid in the cell. Leader peptides are rich in the amino acid being synthesised by the gene cluster downstream.
NOTION 7.9
249
What is downstream of the leader peptide?
Downstream of the leader peptide is a region in the mRNA that can form different secondary structures. This region contains 4 important sequences:
- Sequences 3 and 4 can form a stem loop terminator
- Sequences 2 and 3 can form a stem loop - antiterminator
NOTION 7.10
250
Effect of excess tryptophan on transcription of tryptophan structural genes
NOTION 7.11
251
Effect of limiting tryptophan on transcription of tryptophan structural genes
NOTION 7.12
252
How can small RNAs regulate gene expression?
Antisense RNA binds to mRNA to block translation.
NOTION 8.1
253
What is an example of gene expression regulated by small RNAs (Antisense RNAs)?
EXAMPLE: Small non-coding RNAs regulate virulence
of Staphylococcus aureus (biofilm, iron metabolism,
exotoxin production, hydrolytic enzymes)
254
What are riboswitches?
mRNA encodes a biosynthetic enzyme and the small metabolite that binds the RNA is product of that reaction. The binding of this metabolite blocks the progression of translation.
NOTION 8.2
255
What is an example of a Riboswitch?
Example: Thiamine Riboswitch
256
What are 2 other important cell regulation systems?
Other important cell regulation systems:
1. Cell signalling by “Quorum Sensing”
2. Signal Transduction by ‘Two - component sensor regulator systems’
257
What signal molecule is released, during quorum sensing, by:
- Gram negative bacteria
- Gram positive bacteria
Some, possibly all, bacteria secrete a signal molecule at a constant rate
– Acyl homoserine lactone (Gram negative)
– Peptides (Gram positive)
258
What is the ultimate effect of quorum sensing?
- Concentration of signal increases with cell density
- When threshold concentration reached, responsive genes are expressed
259
Diagram displaying production of AHL
NOTION 8.3
260
What are 8 examples of group behaviour in bacteria?
Group behaviours in bacteria:
- Symbiosis
- Virulence
- Competence
- Conjugation
- Antibiotic production
- Motility
- Sporulation
- Biofilm formation
261
Where can Vibrio fischeri be found?
What is their effect?
Vibrio fischeri live in special organs in ‘flashlight fish’. The bacteria luminesce when there are sufficient numbers present.
262
What is a common bacteria found in cystic fibrosis patients?
Why is this bacteria dangerous?
Psuedomonas aeruginosa infection of cystic fibrosis patients.
QS induces large number virulence genes.
Generates biofilm – resistance to antibiotics.
263
Diagram of Toxin Production in E Coli
NOTION 8.4
264
What makes up the Two Component System, involved in allowing bacteria to sense changes in environment & respond?
What is this system known as?
Composed of:
(1) Histidine kinase (often membrane localised)
(2) Cytoplasmic response regulator
Also known as a Phospho-relay system, where there is a phosphoryl transfer from histidine kinase to the response regulator.
265
Diagram of the “Two Component System”
NOTION 8.5
266
Examples of Two Component Regulatory Systems that regulate transcription in Escherichia Coli
NOTION 8.6
267
What are some niche-specific environmental conditions within the human body?
- pH/ Temperature
- Oxygen Levels
- Micronutrients
- Host Defence Mechanisms
268
How do pathogenic strains of E Coli acquire virulence genes?
Pathogenic strains of E. coli acquire virulence genes and regulators of virulence genes by horizontal gene transfer.
NOTION 8.7
269
Pathogenicity Islands in E Coli Genome
NOTION 8.8
270
Summary of Gene Expression & Pathogenesis
1. Repressors and activators regulate transcription by binding to operators in response to metabolite concentrations
2. mRNA leaders can also control transcription by sensing the rate of translation (attenuation) or can be targets for small RNAs or may contain a riboswitch (binding site for the metabolite)
3. Quorum sensing ensures that certain genes are only switched on when the cell density is high
4. Two component sensor regulators systems transmit environmental signals from outside the cell so that the cell can respond
271
What are viruses?
- Very small acellular infectious agents
- Obligate intracellular parasites
- Widely distributed in nature, infecting animals, plants, and microorganisms
- Numerically most successful biological entities
272
No of viral particles on Earth
10^31 viral particles on Earth
273
Why study viruses (Diseases)?
- SARS-Cov-2
- Influenza virus
- Smallpox (eradicated in 1980)
- Polio virus (aims to be eradicated)
- HIV/ AIDS
- Zika Virus
- Ebola Virus
- Aphthovirus
- Tobacco mosaic virus
- Tomato bushy stunt virus
NOTION 9.1
274
Why study viruses (Technology)?
- Virus like particles
- CRISPR
- Vaccines
- siRNA
- Detection of viruses
- Novel Therapies
- mRNA
275
No of viruses per litre of seawater
There are approximately 100,000,000,000 (10^11) viruses
per litre of seawater.
276
What organisms can viruses infect?
Viruses can infect animals, plants, fungi, protozoa,
archaea, and bacteria
… And viruses.
277
History of Viruses
- 1400 BC = First written record of virus infections in heiroglyphics found in Memphis, Egypt
- 100 BC (China) = Protection from smallpox using variolation
- 1796 = Edward Jenner vaccinated a boy with cowpox - infected material
- 1880 (Germ Theory) = Robert Koch & Louis Pasteur. Pasteur identified rabies to be caused by an agent he termed “virus” from the Latin for “Poison”
- 1892 = Dmitri iwanowksi showed that ceramic filtered extracts from diseased plants could transmit disease to other plants
- 1898 = Martinus Beijerinick work on TMV. Friedrich Loeffler & Paul Frosch work on foot-and-mouth disease in cattle
- 1909 = Landsteiner & Popper. Poliomyelitis (Yellow Fever) was the first human disease to be recognised as a viral disease
- 1915 - 1917 (Bacteriopahges) = Frederick Twort & Felix d’Herelle discovered viruses which infect bacteria
- 1950 - 2003 = Discoveries of Ebola, West Nile, HIV, SARS
278
Size of viruses in comparison to eukaryotic/ prokaryotic cells
NOTION 9.2
279
Size of varying viruses
NOTION 9.3
280
How were giant viruses detected?
Systematic sampling of a variety of aquatic environments.
Scientists have recently revived several large viruses that had been buried in the frozen Siberian permafrost for tens of thousands of years
281
When was the oldest Pandoravirus identified?
Around 48,500 years old.
Still infectious after a good thaw.
282
What is the largest virus?
- Dimensions
- Size of genome
- Number of proteins encoded
- What does it infect?
Pithovirus sibericum:
- 1.5 micrometers in length, 500nm in diameter
- The genome of Pithovirus is a circular, double-stranded DNA (dsDNA) molecule with a size of 610,000 bp
- It encodes 467 putative proteins
- Infects amoeba species
283
What is the smallest virus?
- Size of genome
- Number of proteins encoded
- What does it infect?
Porcine circovirus 1&2
- Smallest known animal virus
- The genome of PCV 1 is a circular single-stranded DNA molecule with a size of 1,800 – 3,800 bp
- It encodes 3 proteins - a capsid and two replicases
- PCV 2 causes Post weaning multisystemic wasting syndrome in pigs
284
What are key components which make up the structure of viruses?
Viruses are made up of :
• Genetic material - Either DNA or RNA
• Capsid - Protein coat surrounding the centre of the virion
• Nucleocapsid – Combination of the nucleic acid and the capsid
• Membrane Envelope- Surrounds the nucleocapsid
• Protein spikes – Help viruses attach themselves to the host
285
Structure of the following viruses:
- Human Immunodeficiency Virus
- Bacteriophage
- Influenza virus
- Adenovirus
- Tobacco mosaic virus
- Ebola virus
NOTION 9.4
286
What types of genomes can viruses have?
Viral Genomes:
• Either DNA or RNA genomes
• Some circular, but most linear
287
Classification of DNA viruses
NOTION 9.5
288
Classification of RNA viruses
NOTION 9.6
289
What are the 3 viral capsid symmetries?
- Filamentous
- Spherical (Icosahedral)
- Complex
290
Give an example of a filamentous virus
Tobacco mosaic virus
291
What shape are filamentous viruses?
How are the nucleic acids/ proteins arranged?
What is the length of the virus determined by?
What is the width determined by?
Helical symmetry: rodshaped viruses (e.g.,TMV)
– Linear array of nucleic acid and the protein subunits
– Length of virus determined by length of nucleic acid
– Width of virus determined by size and packaging of
protein subunits
292
What is an example of a spherical virus?
Human papilloma virus
293
Why does a spherical virus form an efficient shape?
Most efficient arrangement of subunits in a closed shell, require lesser amounts of energy, evolutionarily favoured
294
What is an Icosahedron?
Icosahedron:
- 20 faces – each an equilateral triangle
- 30 edges – each an interface between 2 faces
- 12 vertices – each a point where 5 faces meet
NOTION 9.7
295
Shape of bacteriophages
NOTION 9.8
296
What are 4 viruses which have weird structures/ shapes?
- Bicauduviridae
- Pandoravirus salinus
- Guttaviridae
- Lipothrixviridae
NOTION 9.9
297
Some virions contain enzymes critical to infection. Give some examples of these enzymes
Some virions contain enzymes critical to infection:
– Lysozyme
- Makes hole in cell wall
- Lyses bacterial cell
– Nucleic acid polymerases
– Proteins that counter host defence
– Neuraminidases
- Enzymes that cleave glycosidic bonds
- Allows liberation of viruses from cell
298
What is a susceptible cell?
A susceptible cell – functional receptor for a particular virus – cell may or may not support viral replication.
299
What is a resistant cell?
A resistant cell has no receptor
300
What is a permissive cell?
A permissive cell has the capacity to replicate virus – it may or may not be susceptible.
301
What is a susceptible & permissive cell?
A susceptible and permissive cell is the only cell that can take up a virus particle and replicate it.
302
What are the 2 traditional methods by which viruses were detected?
Traditional methods:
- Electron microscopy
- Viral cultures
303
What are 3 more modern methods by which we can detect viruses?
Modern Methods:
- Immunoassays to detect antibodies
- Polymerase Chain Reaction (PCR)
- Next generation sequencing
304
What is a titre?
Titre: number of infectious units per volume of fluid
305
What is the most efficient way to measure a titre?
Plaque assay: analogous to the bacterial colony; one way to measure virus infectivity.
NOTION 9.10
306
What is a virome?
What is involved in the study of virome composition?
Virome is the total collection of viruses in a particular habitat.
Study of virome composition has benefited from new non-culture-based metagenomic methods.
307
What are some examples of methods used to examine the composition of a virome?
(A) Flow cytometric enumeration of phage particles, obtained from faecal filtrate.
(B) Agar overlay showing plaques of different morphologies on E. coli from enriched sample.
(C) Relative abundance of metagenomic contigs obtained by shotgun DNA sequencing of human faecal VLP fractions
(D) SDS-PAGE gel electrophoresis displaying predominant polypeptide fractions
(E) TEM of phage particles purified human faecal VLP fraction
NOTION 9.11
308
What did the Hershey Chase experiment involve?
Used T2 bacteriophages:
- Labelled Proteins with S35
- Labelled DNA with P32
They then mixed the bacteriophages with bacteria.
They found that, when centrifuged:
- Labelled protein was found in the supernatant with phages
- Labelled DNA in the pellet with bacteria
This is evidence that DNA carries our genetic code!
309
Virus replication is typically characterised by a ______-step growth curve. What does this involve?
Virus replication is typically characterised by a one-step growth curve. This involves a latent period: eclipse + maturation.
NOTION 10.1
310
What is “Burst size”?
Burst size: Number of virions released from one host cell.
311
What are the 5 phases of viral replication?
Phases of Viral Replication
• Attachment (adsorption) of the virus to a susceptible host cell
• Entry (penetration) of the virion or its nucleic acid
• Synthesis of virus nucleic acid and protein by cell metabolism as redirected by virus
• Assembly of capsids and packaging of viral genomes into new virions (maturation)
• Release of mature virions from host cell
312
What is involved in the attachment phase of viral replication?
What is required for this phase?
What can the receptors include?
What is the effect of this phase on the cell surface & virus itself?
Attachment of virion to host cell is highly specific
- Requires complementary receptors on the surface of a susceptible host and its infecting virus
- Receptors on host cell carry out normal functions for cell (e.g., uptake proteins, cell to cell interaction)
- Receptors include proteins, carbohydrates, glycoproteins, lipids, lipoproteins, or complexes
The attachment of a virus to its host cell results in changes to both virus and cell surface that facilitate penetration.
313
What virus displays one of the most complex penetration mechanisms?
What is involved in the penetration of this virus?
Bacteriophage T4: virus of E Coli:
- Virions attach to cells via tail fibers that interact with polysaccharides on E. coli cell envelope
- Tail fibers retract and tail core makes contact with E. coli cell wall
- Lysozyme-like enzyme forms small pore in peptidoglycan
- Tail sheath contracts and viral DNA passes into cytoplasm
NOTION 10.2
314
What steps are involved in the production of viral nucleic acid & protein, once the virus has entered the host cell?
1. Once a host has been infected, new copies of the viral genome must be made and virus-specific proteins synthesized in order for the virus to replicate
2. Generation of messenger RNA (mRNA) occurs first
3. Viral genome serves as template for viral mRNA
4. In some RNA viruses, viral RNA itself is the mRNA
5. In some cases essential transcriptional enzymes are contained in the virion
315
What are the 5 rules of viral infection?
The rules:
• Attachment and entry
• Translation
• Genome replication
• Assembly
• Release of virus
316
Diagram displaying replication of a bacteriophage
NOTION 10.3
317
Diagram displaying replication of the Poliovirus
NOTION 10.4
318
Diagram displaying replication of Human HIV
NOTION 10.5
319
What is lysogeny?
Lysogeny = The ability of some viruses to integrate into the host genome.
320
What are temperate viruses?
Temperate viruses can infect cells using either lytic or lysogenic cycles.
321
What is involved in the lytic cycle?
Lytic cycle ⇢ Infection ⇢ extrachromosomal replication and
production of viral proteins ⇢ lysis and reinfection.
322
What is involved in the lysogenic cycle?
Lysogenic cycle ⇢ Infection ⇢ integration into host DNA ⇢ virus in this form is called a “prophage” ⇢ replication of viral DNA becomes part of natural growth and cell division of the host cell ⇢ eventually cells enter the lytic cycle.
323
Diagram of lytic vs lysogenic cycle
NOTION 10.6
324
Taxonomic classification of viruses
NOTION 10.7
325
What are the 3 main criteria for classifications of viruses?
Main criteria for classification:
- Nucleic acid
- Type of nucleic acid - DNA or RNA
- Single or double stranded
- Linear, circular, single molecule or segmented (Influenza)
- If single stranded: negative or positive (polarity)
- Capsid symmetry
- Icosahedra, Helical or complex
- Presence or absence of lipid envelope
326
What is Plus (+) sense RNA vs Negative (-) sense RNA?
Plus (+) sense RNA can be directly translated into proteins.
Whereas a Negative (-) sense RNA needs to flipped into Plus (+) sense RNA, before being translated into proteins.
327
What are the 7 classifications of viruses in the Baltimore classifications system?
7 viral genome types:
1. dsDNA
2. ssDNA (+ve strand)
3. Gapped dsDNA
4. dsRNA
5. ssRNA (+ve)
6. ssRNA (-ve)
7. ssRNA -> ssDNA (-)
NOTION 10.8
328
How common are dsDNA viruses? Give an example
How are these genomes prepared for transcription?
dsDNA viruses = The vast majority of discovered viruses, but not found in plants (yet).
Example = Herpes Simplex Virus.
Their genome can be transcribed directly.
329
How common are ssDNA viruses? Give an example
How are these genomes prepared for transcription?
Not very many examples but wide range of host cells:
- Prokaryotes
- Archaea
- Eukaryotes
Example = Parvovirus B19 (which causes “slapped cheek syndrome”)
Host cell needs to convert ssDNA to dsDNA.
330
How common are + ssRNA viruses? Give an example
How are these genomes prepared for transcription?
Can be found in:
- Prokaryotes
- Eukaryotes
- NOT ARCHAEA
Example = Zika virus or Norovirus
+ ssRNA strand is used directly as the mRNA for translation (i.e no transcription required).
RNA dependent replicase is used to replicate the + ssRNA strand.
331
How common are dsRNA viruses? Give an example
How are these genomes prepared for transcription?
Can be found in:
- Prokaryotes
- Eukaryotes
- NOT ARCHAEA
Examples = Cystovirus
Requires RNA dependent replicase to convert to + strand ssRNA, which is then used as mRNA.
332
How common are - ssRNA viruses? Give an example
How are these genomes prepared for transcription?
Only found in eukaryotes, not prokaryotes or archaea.
Can be segmented or non segmented!
Example = Influenza virus.
Requires a functional RNA replicase within the virion, to convert to + sense RNA (which can then be used as mRNA).
333
How common are + ssRNA retroviruses? Give an example
How are these genomes prepared for transcription?
Only found in eukaryotes, not prokaryotes or archaea.
Example = HIV
Involves reverse transcription to - ssDNA strand, which is then converted to dsDNA strand.
334
How common are disrupted dsDNA viruses? Give an example.
How are these genomes prepared for transcription?
Only found in animals and humans!
Requires reverse transcription.
Example = Hepatitis B.
The viral dsDNA is released into nucleus. Transcribed by host RNA polymerase. Genomic RNA is retrotranscribed in the cytoplasm into new dsDNA genomes.
335
1. What are viroids?
2. How are they transmitted?
3. What is their mode of action?
4. What is their genome like?
1. Viroids = An infectious entity comprising solely nucleic acid with no outer coat protein. They are primarily plant pathogens.
2. Cell to cell transport or direct injection e.g by aphids feeding from the plant.
3. Don’t generally make proteins but simply replicate their nucleic acids.
4. So far all of the viroids use circular RNA
336
1. What is a prion?
2. What are some diseases in which prions are responsible?
3. Why are fungal prions interesting?
1. Prion = A replicating inheritable entity that does not rely on DNA or RNA.
2. Responsible for a number of deadly diseases including Creutzfeldt-Jakob disease (CJD), scrapie and bovine spongiform encephalopathy (BSE).
3. Fungal prions are very interesting because they can confer a selective advantage to the host and protect against non-compatible strains.
337
1. Eukaryotic mechanisms to diminish viral infections
2. Prokaryotic mechanisms to diminish viral infections
1. Many eukaryotes possess mechanisms to diminish viral infections. For example, immune defense mechanisms, RNA interference.
2. Prokaryotes possess similar mechanisms. CRISPR - Similar to RNA interference.
338
What are 3 restriction modification systems, used to defend against viruses?
Restriction modification systems
• DNA destruction system; only effective against double-stranded DNA viruses
• Restriction enzymes (restriction endonucleases) cleave DNA at specific sequences
• Modification of host’s own DNA at restriction enzyme recognition sites prevents cleavage of own DNA (only dsDNA – ssDNA and RNA viruses unaffected)
339
Graph of Innate & Adaptive Immunity
NOTION 10.9
340
What mechanisms are involved in:
- The protection against infection via innate immunity
- The protection against infection via adaptive immunity
- The eradication of established infection via innate immunity
- The eradication of established infection via adaptive immunity
NOTION 10.10
341
Give some examples of viruses, which we currently have vaccines against
Vaccines:
- Smallpox
- Poliovirus
- Influenza A virus
- SARS-CoV-2
342
What are 3 examples of current treatments for viral infections?
Treatment of viral infections:
- Pre-formed antibodies (HBV immunoglobulin)
- Immunomodulation (Interferon - HBV, HCV)
- Antivirals
