Microbiology (Lecture 1-8) Flashcards
1
Q
Why is Whittaker’s five kingdom tree wrong?
A
- Oversimplifies the complexity of life on Earth
- Does not account for evolutionary relationships b/w organisms
2
Q
Why are there so many microorganisms?
A
- Rapid growth rate
- Many chances of speciation through random mutations
- Lateral gene transfer (exchange of genetic material)
- Every available niche is occupied by specifically adapted microbes
3
Q
“Every available niche is occupied by specifically adapted microbes” explain this statement
A
- Microbes hv been alive for so long thus
- They’ve been in many places n adapted
- Hence, can live in all these places
4
Q
What are prototrophs?
A
- Organism that synthesize all their own cellular components
- Amino acids, nucleotides, vitamins
5
Q
What are fastidious bacteria?
A
Bacteria that need organic components
6
Q
Asexual reproduction in bacteria
A
- Done by binary fission or budding
- Cells double in size then split into two
- Exponential growth bc 1 cell becomes 2 becomes 4
7
Q
How do microbes grow?
A
Assimilating nutrients n cell division
8
Q
Lag phase
A
Adapting to new conditions → takes some time for the organism to adjust
9
Q
Log/exponential phase
A
- Exponential growth
- Once adapted, cells begin to grow n reproduce rapidly
10
Q
Stationary phase
A
- Limitation by nutrients, buildup of waste product that inhibit growth
- Runs out of space n nutrients + waste build up → less growth n reproduction to occur
11
Q
Death
A
- Organisms start dying off n may lyse
- Eventually will feed off dying cells
12
Q
How can we measure bacterial growth?
A
- Cell number
- Optical density
- Fresh/dry weight
- Protein
- DNA
13
Q
What is the traditional way to identify microorganisms?
A
- Microscopy n staining
- Growth on selective/differential media
- Testing substrate spectrum supporting growth
- Characterization of cell chemical constituents
- Lipids, cell wall components, DNA base ratio, quinones
14
Q
Describe selective media
A
- Allows the growth of only some types of organism
- Used to culture/identify presumed pathogens from clinical specimens
15
Q
Describe differential media
A
- Allows the identification of organisms based on growth n appearance on that medium
- Often based on colour differences
- Used to determine whether a culture is potentially pathogenic
16
Q
Describe the ApiZym test system for microbial identification
A
- Culture of organism is grown
- Cells resuspended in a buffer
- Test wells are inoculated
- Resulting colour in test strip is compared against database
- Can help to distinguish between pathogenic n benign strains
17
Q
What is an S-layer?
A
- Regularly structured
- Paracrystalline outer layer composed of protein or glycoprotein
- External to cell wall in some bacterial n archaeal species
18
Q
Function of S-layer
A
- Protection against ion n pH fluctuations, osmotic stress n predators
- Protect against host defenses
- Sometimes act as a virulence factor
19
Q
How did cellular life first evolve?
A
- Surface origin hypothesis
- Subsurface origin hypothesis
20
Q
What is the surface origin hypothesis?
A
- Warm little pond
- Primordial soup (Evidence that organic molecules can form spontaneously)
- Hypothesis unlikely due to hostile conditions on surface
- High UV, meteor strikes, volcanic activity
21
Q
Subsurface origin hypothesis
A
- Hydrothermal vents at ocean floor
- More stable conditions
- Constant source of energy (reduced inorganic compounds)
22
Q
Key features of the origin of cellular life
A
- Self replicating RNA
- Enzymatic proteins
- DNA
- Evolution of biochemical pathways
- Divergence of lipid biosynthesis
- Divergence of cell walls
23
Q
Landmarks in biological evolution
A
- Early life probably dependent on H2 n CO2 (fixed by bacteria)
- Bacteria makes acetate
- Archaea makes methane
- Energy n carbon metabolisms diversif
- Phototrophy, using H2S as electron donor
- Evolved into an oxygenic photosystems using H2O
24
Q
Characteristics of molecular sequences in phylogenetic analysis
A
- Must be universal
- Ribosomes, DNA, RNA
- Contain variable n conserved regions
- Not subject to HGT
- Homologous (same purpose)
25
Why are ribosomal RNA genes a universal molecular marker?
Present in all forms of life
26
What are the potential chain of events in evolution of eukaryotes?
- Endosymbiotic theory: acquisition of mitochondria
- May hv led to the divergence of eukaryotes n archaea
- Nucleus forms n separates itself in the cell → complex organelles form
- Acquisition of cyanobacteria
- Becomes a chloroplast
- Produces lineage of plants capable of photosynthesis
27
Describe aquifex
- Isolated from a hot spring
- Hyper thermophilic
- Chemolithoautotrophic
- Oxides H2 to water using O2 as e- acceptor
- Autotrophic
- C-fixation
28
Describe deinococcus
- Radiation resistant
- Isolated from canned meat sterilized by gamma irradiation
- V rapidly reassembles radiation damaged
29
Describe cyanobacteria and plastids
- Blue green algae
- Plastids were originally cyanobacteria
- Morphologically diverse
- Widely distributed
- Oxygenic phototrophs
30
Describe chlamydia
- Obligate intracellular parasites
- Distinct life cycle
- Important human pathogens
31
Describe spirochaetes
- Helically shaped, motile, gram negative
- Flagellum inside the cell
- Heterotrophs
- Contains free living, symbiotic n parasitic species
32
How are species defined in higher organisms?
Groups of individuals that can interbreed n produce viable offspring
33
Why is the definition of species in higher organisms problematic in microbiology?
Due to:
- Asexual reproduction
- Lateral gene transfer
- Phenotypic n genotypic plasticity of microorganisms
34
Current definition of species in microbiology
Group of strains that show a high degree of overall similarity and differ considerably from related strain groups with respect to many independent characteristics
35
Describe the gram staining procedure
1. Spread culture in thin film over a slide
2. Dry the culture in air
3. Pass slide through flame to fix culture to slide
4. Staining process:
1. Culture is flooded with crystal violet for 1 minute
1. Cells turn purple
2. Add iodine solution for 3 minutes (to wash out crystal violet)
1. Cells remain purple
3. Decolorize with alcohol briefly
1. Gram-positive cells are purple
2. Gram-negative cells are colorless
4. Counterstain with safranin for 1 - 2 minutes
1. Gram positive cells are purple
2. Gram negative cells are pink to red
5. Drop of oil is placed on the slide
1. Examined under microscope
36
Why do gram-positive cells stain purple?
- Hv thick cell wall w LPS layer
- Crystal violet can bind to thick LPS layer
37
Why do gram-negative cells stain purple?
- Thin walls
- When washed w alcohol, thin cells hv their violet washed out [weak binding]
38
Biochemical test — decomposition of simple carbohydrates
- Observing how microorganisms react to different types of carbohydrates under anaerobic or aerobic conditions
- Glucose decomposed → acid produced → changes pH n induces a colour change in medium
39
Biochemical test — fermentation test
- Identify the ability of microorganisms to produce acids (lactate n acetate) by fermenting sugars
- Acid production results in a change in pH n can induce a color change in the medium
- Gas production is collected in Durham tube n observed
40
How can the metabolic capabilities of microorganisms aid in their identification?
- By identifying the metabolic pathways that are used by microorganisms, we can classify bacteria and determine their potential pathogenicity.
- Different tests can be used to identify the presence of specific enzymes or the ability to decompose different substrates, providing important information about the microorganisms.
41
Psychrophiles
- Inhabit permanently cold environments
- Arctic, antarctic deep sea, glaceris
42
How do psychrophiles adapt to low temperature environments?
Modifying protein structure n membrane fluidity
43
What are hyper-thermophiles and where are they generally found?
Microorganisms that grow at temperatures above 80°C and are generally found in hot springs and thermal vents.
44
What is FAME analysis and how is it used in molecular analyses?
- Determination of fatty acid profiles of membrane lipids
- Used in molecular analyses by comparing the chromatograms of the extracted lipids and chemically modified methyl esters to a database for the best match.
45
What is AFLP and how is it used to distinguish closely related strains of bacteria?
- Technique used to distinguish closely related strains of bacteria by targeting repetitive elements in the bacterial genome
- It determines the number of repeats in a genome and compares the electrophoretic patterns of the results.
46
From outside to inside, what are the components of bacterial cells?
- Capsule, S-layer
- Then:
- Gram positive bacteria
- Cell wall
- Gram negative bacteria
- Outer membrane
- Periplasmic space
- With peptidoglycan in Gram –ves and some Gram +ves
- Cell (plasma) membrane
- Universal to all cells
47
Describe a capsule
- Glycocalyx (sugar coat)
- Polysaccharide components outside the cell wall
- Loose network of polymer fibres extending outward from wall
48
Describe the capsule’s slime layer
- Diffuse, unorganized, easily removed
- Does not exclude small particles
- Can be difficult to visualize
49
What are the roles of capsules?
- Not required for growth/reproduction
- Carbon store
- Protection against desiccation
- May be involved in the capture of nutrients
- Acquisition of ions from the environment
- Confer advantages in vivo
- Attachment to surfaces
- Biofilms, holdfast to eukaryotic cells
- Exclude phage, antimicrobials and disinfectants
- Pathogens often capsular and resist phagocytosis
50
What is the difference b/w a capsule n a slime layer?
- Capsule: organized, tight matrix that is not easily removed, excludes small particles (e.g. India ink)
- Slime layer: diffuse, unorganized layer that is easily removed n does not exclude small particles
51
What are bacterial adhesins?
- Allow cells to stick to n attack other cells
- Includes capsular polysaccharides, EC slime, fimbriae n lectins
52
Describe an S-layer
- Generally not associated w pathogens
- Paracrystalline outer wall layer composed of protein/glycoprotein
- Regularly structured layer external to cell wall
- May protect against ion and pH fluctuations, osmotic stress, predators such as Bdellovibrio
- May protect against host defenses
- Sometimes a virulence factor
53
What is the composition of peptidoglycan in both Gram-positive and Gram-negative bacteria?
Alternating residues of NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid)
54
How are the NAG and NAM residues arranged in peptidoglycan?
Cross-linked by amino acid side chains creating amide bonds
55
What is the function of peptidoglycan in Gram-positive bacteria?
Forms a mesh-like polymer that retains Gram stain
56
What are the non-protein amino acids found in peptidoglycan?
- D-glutamic acid
- D-alanine
- DAPA
57
How do D-amino acids protect peptidoglycan from degradation by proteases?
Proteases typically break down proteins that use L-amino acids
58
Why does the capsule use D-amino acids?
D-amino acids cannot be broken down by proteases
59
Peptidoglycan structure
- Chains of linked peptidoglycan subunits
- Joined by cross-links between the peptides
- Cross-links often occur between carboxyl group of terminal D-alanine and amino group of diamino pimelic acid (DAPA)
60
What is the function of peptidoglycan?
- Provides structural support and shape to bacterial cell walls
- Porous, elastic, and stretchable
- Strong enough to retain shape when isolated
61
What are the cross-links in peptidoglycan synthesis for E. coli and Staphylococcus aureus?
- E. coli: Cross-links between DAPA and D-alanine
- Staphylococcus aureus: Cross-links between L-lysine and glycine
62
How are peptidoglycan chains cross-linked to form a solid structure?
- Peptidoglycan chains form sheets
- Sheets are cross-linked with each other to form a solid-like structure
63
Lysozyme
Antibacterial enzyme that degrades β, 1-4 glycosidic bonds in peptidoglycan backbone
64
What does the loss of PG lead to?
Makes bacterial cells sensitive to changes in osmotic pressure → cells more likely to burst
65
Lysozyme function
- Host defense against bacteria
- Present in saliva, tears and secreted in airways
66
How does penicillin halt cell wall synthesis?
- Inhibits peptidoglycan synthesis
- Targets transpeptidation step (pencilin sensitive)
- Transpeptidation: involves cleaving 1 of the 2 D-Ala residues in the linker peptide n linking it to DAPA (-) or L-Lys (+)
- Penicillin prevents this → cells become osmotically sensitive n lyse
67
Why is penicillin more effective for gram positive bacteria?
Gram negative doesn’t depend on peptidoglycan as much as the gram positive
68
How do archaeal cell walls differ from bacterial cell walls?
- Archaeal cell walls lack:
- Peptidoglycan/murein
- Archaeal cell walls contain:
- Pseudomurein
- Other polysaccharides or glycoproteins
- S-layers
69
What is pseudomurein and how is it different from peptidoglycan/murein?
- Structural component found in some methanogenic archaea
- Similar to peptidoglyan/murein
- Composed of alternating NAG n NAT subunits
- Linked by β 1,3 instead of β 1,4 glycosidic bonds
- Lacks D-amino acids in linker
70
Why are archaeal cell walls not susceptible to degradation by lysozyme or inhibition by penicillin?
Archaeal cell walls lack the specific structural features (peptidoglycan/murein, D-amino acids in the linker) that are targeted by lysozyme and penicillin in bacterial cell walls, making them resistant to these agents.
71
Role of sterols/hopanoids in membranes
- Stabilize membrane structure
- Make membrane more rigid
72
Difference between hopanoids n sterols
- Hopanoids
- Bacteria
- Rigid, planar molecules
- Sterols
- Eukaryotes
- Flexible
73
Are hopanoids found in archaea?
No, instead hv isoprene structures
74
How do hopanoids and sterols respond to a rise in temperature?
Stabilize membranes
75
Why is the outer membrane of Gram-negative bacteria asymmetric?
Insertion of LPS into external layer of outer membrane
76
What is Braun's lipoprotein?
Protein that is covalently linked to peptidoglycan n embedded in OM of gram negative bacteria by hydrophobic end
77
What is the function of Braun’s lipoprotein in the outer membrane of Gram-negative bacteria?
- Attaches peptidoglycan layer to OM n serves as an adhesion site for 2 membranes
- Allows transport of substances b/w membranes
78
How can adhesion sites between the inner and outer membranes be visualized?
- Induce plasmolysis → increase space b/w membranes
- Transmission electron microscopy (TEM)
- Immunogold staining → used to track viral particles entering cell thru adhesion sites
79
What is the significance of adhesion sites in Gram-negative bacteria?
- Structural integrity of Gram-negative bacteria by attaching peptidoglycan layer to OM
- Allows transport of substances b/w membranes
80
Describe the structure of lipopolysaccharide (LPS)
- Lipid A
- Core polysaccharide
- O side chain
81
What can LPS induce when it interacts with the innate immune system?
Massive immune response (septic shock)
82
Describe lipid A
- 2 glucosamine residues linked to fatty acids and phosphate (occasionally pyrophosphate)
- Lipid A is integrated into outer membrane
- Immunogenic
- Remaining LPS projects from cell surface
83
Describe core polysaccharide
- AKA R-antigen or R-polysaccharide
- Side chain of NAG, phosphate and ethanolamine
84
Describe O side chain
- Variable region → responsible for antigenic make-up of bacteria
- Flexible n bent
- Highly variable composition
- R/S variants depend on side chain length
85
Functions of LPS
- Lipid A stabilizes outer membrane structure
- Core polysaccharide charged
- Contributes to negative charge on surface
- Charged, hydrophilic external layer → reduces permeability of hydrophobic substances
- Prevents entry of bile salts, antibiotics
- Protects against host defences
- Rough variants are more susceptible to phagocytosis
- Loss of O-antigen in *E.coli and Salmonella →* reduced virulence
- O-antigen v variable → key diagnostic tool
86
What is endotoxin?
- Molecule that can be released during the growth or lysis of bacterial cells
- Composed of LPS
87
When is endotoxin released?
During cell division or by lysis of bacterial cells
88
What happens when LPS is in the blood?
Causes septic shock syndrome
89
How to test for endotoxin?
- Rabbit pyrogen test
- LAL assay
90
Function of endotoxin
Prime to immune system against a pathogen, preparing it to act again by becoming aware of the antigen
91
Describe Limulus Amaebocyte Lysate (LAL) test
- Amaebocytes: blood cells of Limulus polymephus
- Contains a clotting factor that’s released if comes in contact w bacterial endotoxin
- Natural immune mechanism that contains bacterial infection
92
Properties of endotoxins
- Heat stable
- Toxic in nanogram amounts
- Interacts w innate immune system cells
- Can trigger cytokines release in a cascade
- Activates transcription factors (e.g. interferon-beta n tumour necrosis factor)
93
Endotoxin can result in
- Inflammation
- Fever
- Vasodilation
- Thrombosis
94
What are porins?
Protein channels that permit passage of small molecules up to around 600 Da
95
How do larger molecules cross the outer membrane?
- By attaching to carriers n passing through active transport systems
- NOT accomplished through porins
96
What is the porin superfamily?
- Homotrimeric, transmembrane proteins that form water-filled channels in the OM
- Highly conserved structure
- Similar amino acids involved in binding n transferring molecules across the membrane
97
What is the selectivity of most porins?
- Slightly cation selective → prefer to move cations
- Non-specific channels that allow the passage of small (<600 Da) hydrophilic molecules
98
Are all porins non-selective?
- Some porins are selective
- E.coli LamB is a maltose n maltodextrin selective porin that was first identified as the receptor for bacteriophage Lambda
99
Porin structure
- Common β strand structure
- 16 stranded anti parallel β-barrel
- Unusual structure is exceptionally stable → produces essentially closed structure
100
Why are porins important for the survival of Gram-negative bacteria in harsh environments?
- High thermal stability
- Resistant to protease n detergent degradation
101
How are porins stabilized?
Formation of a salt bridge b/w N- n C- termini → provides extra stability to the structure
102
What is the oligomeric state of porins?
Trimers
103
What is the shape of a porin monomer?
- Hourglass shape w a central constriction
- Has a hydrophobic of 25A that sits in the membrane
104
What dictates the size of the solute that can traverse the channel in porins?
- Charges inside the pore of porins
- Also dictate what species can go thru the porin
105
What is the periplasmic space?
Space b/w the OM n the cytoplasmic membrane in gram-negative bacteria
106
What is the gel-like consistency of the periplasmic space due to?
Abundance of proteins
107
What is the function of hydrolytic enzymes such as alkaline phosphatase in the periplasmic space?
- E.g. alkaline phosphatase
- In periplasmic space
- Strip phosphate from any molecule it sees → providing phosphate for IC usage
108
How do some enzymes in the periplasmic space contribute to energy conservation in bacteria?
- E.g. electron transport
- Transport them thru inner membrane
109
What is the function of peptidoglycan synthesis enzymes in the periplasmic space?
- Located in the periplasmic space
- Responsible for synthesizing peptidoglycan in that area
110
What is the role of periplasmic binding proteins in bacterial cells?
- Periplasmic binding proteins deliver specific compounds to ABC transporters in the cytoplasmic membrane
- Facilitates the uptake of nutrients by bacterial cells
111
What is the function of chemoreceptors located in the periplasmic space?
- Detect changes in the chemical environment
- Transmit signals to the cytoplasmic membrane to direct bacterial movement
112
Where do chemoreceptors sit in Gram-negative bacteria?
Surface of the inner membrane in gram-negative bacteria
113
What is the Sec pathway?
- Nascent polypeptides are transported from the inside of the inner membrane to periplasmic membrane space in bacteria
- Ribosomes on inside of inner membrane synthesize polypeptides n the Sec protein exports them thru the cytoplasmic membrane using a translocase
114
How does protein folding occur in the periplasmic space?
After the nascent polypeptide has been transported there thru the Sec pathway
115
What is the TAT pathway in bacterial protein export?
- Fully folded enzymes are exported across the cytoplasmic membrane in bacteria
- Polypeptides hv a twin-arginine in the N-terminal region
- Acts as a signal to go thru the TAT pathway
116
How does the TAT pathway differ from the Sec pathway in bacterial protein export?
- TAT: exports fully folded enzymes
- Sec pathway: exports nascent polypeptides that fold after translocation into the periplasmic space
- TAT pathway also requires a different single peptide than the Sec pathway
117
What is the role of the translocase in the Sec pathway of bacterial protein export?
Transporting nascent polypeptides across the cytoplasmic membrane n into the periplasmic space
118
What are the 3 different transport types across the CM?
- Simple transport
- Group translocation
- ABC system
119
Describe simple transport
- H+ transported down the concentration gradient
- Energy used to transport another substance
120
Describe group translocation
- Higher concentration of substance outside of the cell
- Substance transported down gradient
- Addition of phosphate to transported substance
- Prevents change in concentration gradient
- Molecules can still diffuse down concentration gradient
121
Describe ABC system
- Takes component to transporter
- Transports components
- ATPase → uses hydrolysis energy to transport
122
What are flagella?
- Long, thin EC helical structures
- Made of protein subunits that aid in motility
- Connected to a motor that spins them clock or anti-clockwise allowing bacteria to swim and change direction
123
Describe the structure of a flagellum
- Long, thin, EC helical structure made of protein subunits that aid in motility
- Connected to a motor that spins them clock or anticlockwise
- Allows bacteria to swim n change direction
124
What is the motor of the flagellum?
Driven due to proton transfer thru ring structure (from periplasm to cytoplasm)
125
What happens when the flagella fall off?
- If the flagella falls off, motility is lost
- Rings n hook of flagellum are rigid n attached to the membrane n cell wall/OM
- Flagellum shaft is easy removed by vigorous shaking
126
Describe flagella synthesis
- Flagella synthesis starts with the MS and C rings in the cytoplasmic membrane
- Form the motor proteins for the flagellum.
- The P and L rings, hook, and cap are assembled next in the periplasm.
- Flow of flagellin monomers through the channel in the hook leads to growth of the filament at the tip.
- Once assembled, the flagellum is anchored to the cell wall and outer membrane through complex ring structures.
- The flagellum constantly grows to repair shear damage caused by movement, and can be removed by vigorous shaking.
127
How does the motor work?
- Rotary motion provided by basal structures
- This mechanism is not completely understood
- Powered by proton motive force
- Protons move across the cytoplasmic membrane thru the MOT complex
128
What is the mot complex?
- Collection of proteins in the bacterial cytoplasmic membrane that forms the basal structure of the flagellum motor
- MotA n MotB (2 integral membrane) interact to create a channel for protons to flow across the membrane
- Flow of protons thru the channel generates a torque that drives flagellum rotation
129
What is the role of Asp residue in MotB in the motor?
- Mutational analysis suggests dependence on an Asp residue in MotB
- Hypothesis: Changes in charge lead to conformational change of MotA, causing movement relative to MotB
- Second conformational change upon loss of charge may provide another step of the motor
130
Describe bacterial movement - run
- Motor rotates anti-clockwise
- Flagellar filaments (w LH helices) form bundle n propels cell
131
Describe bacterial movement - tumble
- Quick reversal of motor to clockwise rotation
- Produces twisting force that transforms flagella into a right-handed helix
- Causes bundle to fall apart rapidly
132
How do separated filaments act?
In an uncoordinated way to generate forces that change cell orientation
133
Aerotaxis
Towards oxygen
134
Chemotaxis
Towards nutrients, away from toxins
135
Magnetotaxis
Movement along the lines of magnetism
136
Phototaxis
Towards light
137
How does bacteria sense a change in nutrient concentration in the environment?
- Transmembrane proteins
- MCP: detect n measure changes in the environment
138
How do MCPs control movement?
- Interact w cytoplasmic membrane, which interact w the rings of the motor that regulate the direction in which it turns
- Dictates run or tumble by a switch from anti-clockwise to clockwise rotation
139
What are transducer proteins?
Another name for MCPs (act as receptors that detect certain molecules)
140
What happens when an attractant binds to an MCP?
- Attractant binds to MCP
- Decrease in activity of sensor kinase CheA
- RESULT: less phosphorylation n kinase activation
141
What happens when a repellent binds to an MCP?
- Repellent binds
- Increase in activity of CheA
- RESULT: more phosphorylation and activation of the kinase
142
What is the role of CheA in bacterial chemotaxis?
- Sensor kinase that can autophosrylate
- Activated
- Initiates a signaling cascade that results in the regulation of the direction of flagellar rotation in the bacterium
143
How do MCPs interact with CheA in bacterial chemotaxis?
- Binding of attractants or repellents to MCPs modulates the activity of CheA
- Leads to changes in the direction of flagellar rotation n dictates the bacterium’s movement
144
How do MCPs detect signals?
- When MCP binds, they undergo a conformational change
- This change is transmitted to the cytoplasmic domain of the protein
- Domain interacts w signaling pathway that regulates the flagellar motor’s rotation
- Interaction b/w MCP n signaling pathway is modulated by CheA
- Two results: phosphorylated or dephosphorylated
- Allows the bacteria to regulate their movement in response to changes in the environment
145
What is the role of CheY in bacterial chemotaxis?
- Cytoplasmic protein that is phosphorylated by CheA-℗
- Response to signals detected by MCPs
- CheY-℗ (not CheY) can bind to flagellar motor n change the rotation from CCW to CW → tumbles
146
What is the role of CheZ in bacterial chemotaxis?
- Phosphatase that dephosphorylates CheY-℗
- Allows the CCW motion to resume n bacterium to continue running
147
How do attractants and repellents affect bacterial chemotaxis?
- Attractants → decrease CheY-℗ → less switching n longer runs
- Repellents → increase CheY-℗ → more tumbling
148
What is the adaptation in chemotaxis?
- Bacteria integrate signals from attractants or repellents over time via methylation of MCP
- CheR constantly methylates MCP
- Allows the cell to become less sensitive to a signal that is present for a long time
- Fully methylated MCP is insensitive to attractant
- If attractant binds to fully methylated MCP → nothing happens
149
How is MCP methylation regulated in bacterial chemotaxis?
- Regulated by CheR n CheB
- CheR: constantly methylates MCP
- CheB: methylesterase that is phosphorylated by CheA-℗ → increases methylesterase activity → demethylates MCP
- Low conc of attractant → high conc of CheA-℗ n CheB-℗ → demethylation of MCP → increasing sensitivity to the attractant → longer runs
- High conc of attractant → low CheA-℗ n CheB-℗ → high methylation lvl of MCP → decreasing sensitivity to the attractant + increasing autophosphorylation of CheA → making it more likely to tumble
150
What is the role of methylation of MCP in chemotaxis?
Methylation of MCP desensitises the cell to a specific concentration of attractant or repellent and serves as a bacterial "memory" of previous exposures.
151
How does methylation of MCP affect bacterial behavior in high concentrations of attractant?
In high concentrations of attractant, MCP is methylated (more cheA-℗), resulting in shorter runs and tumbling to stay in a "good" environment.
152
How does methylation of MCP affect bacterial behavior in high concentration of repellent?
In high concentration of repellent, MCP is methylated (less cheA-℗), resulting in longer runs and less tumbling to leave a "bad" environment.
153
What is the significance of the bacterial "memory" in chemotaxis?
The bacterial "memory" allows the cell to alter its behavior to changing environments based on previous exposures, which increases its chances of survival and efficient use of resources.
154
Gliding motility in Flavobacterium johnsoniae
- Without flagella
- Bacterium secretes slime n glides across the surface
155
What is the mechanism of gliding motility in F. johnsoniae?
- Adhesive molecules move laterally along the cell surface during gliding
- Suggests a tank wheel like mechanism
156
What are the Gld proteins involved in gliding motility?
- 8 Gld proteins in total
- 3 of them are components of an ABC transporter
- 5 are lipoproteins located in the cytoplasmic / OM
157
What happens when gld genes are disrupted?
- Loss of motility
- Increased resistance to bacteriophages that infect wild type cells
- Loss of the ability to digest the insoluble polysaccharide chitin
158
What is the mechanism of twitching motility via type IV pilus?
- Based on the pili first extending from the cell surface then being retracted, dragging the cell along the surface
- Process is powered by ATP hydrolysis
- Throws out pilsu n use it to pull itself along
- Like a grappling hook
159
Why do protein vesicles contain gas?
- Confers buoyancy to the cell
- Allows cell to float up to oxygenated water/towards the light
- Involved in vertical migration in aquatic systems
160
Is the ability to produce ß-galactosidase and indole from tryptophan necessary for a bacteria to be classified as E. coli?
By definition, E. coli produces the enzyme ß-galactosidase and produces indole from tryptophan at 44°C. Knocking out these abilities through single mutations could mean that the bacteria is no longer considered E. coli. This is an important consideration for bacterial classification.
161
Role of fimbria/pili
- Loose association
- Adhesion, role for pili/fimbriae
- Invasion into or thru submucosal epithelial cells
162
Explain how fimbria/pili assisted adhesion
- Pili establish contact w host cell using its adhesive tip protein
- host cell surface and bacterial surface are (-)vely charged
- pili sticking out of bacterial surface to avoid electrostatic repulsion
- resist flushing by moving contents of intestine/urine
- pilus' adhesive tip protein binds to a receptor that's usually a glycolipid or glycoprotein
- pili depolymerise
- pulls bacterium close to host cell
- additional adhesins bind to host cell
- colonisation can begin
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What is the Type 1 fimbriae and where is it found?
- Thin surface polymer
- Well-characterized n widespread in Enterobacteriaceae, including most E. coli
- Important virulence factor in a range of pathogens
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Describe the structure of the Type 1 fimbriae
- FimH: tip adhesin
- Binds to structures containing D-mannose
- FimF n FimG link FimH adhesion onto fimbriae
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Function of FimD usher protein in Type 1 fimbriae assembly
- FimD → usher protein
- Catalyzes Fim A polymerization at base of pili
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Which pathogens use the chaperone-usher pathway to assemble their pili/fimbriae?
- E.coli
- Salmonella spp
- Vibrio cholerae
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What is the role of FimC in the assembly of Type 1 fimbriae?
- Chaperone protein
- Plays a critical role in the assembly of Type 1 fimbriae
- Helps in the proper folding n stabilization of FimA subunits, which are the building blocks of Type 1 fimbriae
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What happens without FimC in terms of the assembly of Type 1 fimbriae?
Subunits may misfold n become unstable, resulting in the assembly of non-functional or defective fimbriae
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What are P-pili and what is their function?
- Critical virulence factors of UPEC
- Causes cystitis n pyelonephritis
- Involved in adhesion to glycolipids on uroepithelial cells
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What is pyelonephritis and why is it dangerous?
- Urinary tract infection that reaches the kidneys
- Can be dangerous n fatal if not treated properly
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How are P-pili structured and assembled?
- Structure n assembly similar to that of type I fimbriae
- Consist of a helical cylinder composed of many protein subunits
- PapG as tip adhesin
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What is the function of PapG in P-pili?
- Tip adhesin of P-pili
- Binds to surface globoside receptors (glycolipids) in uroepithelial cells → allowing the bacteria to attach n invade the host cells
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What are the variants of PapG and what do they bind to?
- 3 variants of PapG: PapG1, PapG2, PapG3
- Bind to surface globoside receptors on eukaryotic cells, allowing the bacteria to adhere to n infect the host
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What are Type IV pili and where are they found?
- Thin n flexible fibres
- Found in some Gram-positive bacteria
- Clostridia
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How are Type IV pili different from fimbriae?
- Typically longer than fimbriae
- Only present in a few numbers per cell
- Typically found at both cell poles n are often not hollow like flagella
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What are the roles of Type IV pili?
- Host cell adhesion
- Biofilm formation
- Twitching motility
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What is twitching motility?
- Form of movement in bacteria that is powered by ATP hydrolysis n controlled by retraction proteins
- Important for bacterial colonization n pathogenesis
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What is the relationship between bundling and virulence in Type IV pili?
- Can aggregate laterally to form bundles
- Bundling mutants, which lack the ability to form these bundles, hv reduced virulence
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What is the specificity of E.coli pathogens determined by?
- LPS (O antigen) and fimbriae (K antigen)
- CFA (colonising factor antigen) fimbriae are present on E. coli pathogenic to humans
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What is the specific interaction between Enterotoxigenic E. coli (ETEC) strains and mucosal epithelium?
Interacts w mucosal epithelium thru a surface antigen called CFA (colonizing factor antigen)
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Role of CFA in E.coli
- Factor pathogenic to humans
- Allows ETEC strains to adhere to the mucosal epithelium, facilitating their colonization n infection
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Where do pathogenic strains of E. coli adhere?
- Adhere to tissues other than the colon where they express their pathogenicity by producing toxins
- E.g. small intestine n urinary tract
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Role of F plasmid in gene transfer
Enables the transfer of genetic material (e.g. plasmids) b/w bacterial cells
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How does the F pilus facilitate adhesion and information transfer?
- Acts similar to Type 4 pili in adhesion
- Attaches to another cell via its tip
- Retracts to bring the cells together
- Once attached, initiates the transfer of genetic information b/w the cells
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What is the process of pilus-mediated gene transfer between an F+ donor and an F- recipient?
- F+ donor (carries F plasmid) forms a pilus
- Pilus of F+ donor attaches to surface of the F-recipient cell
- F+ pilus can only attach to an F- recipient
- Once attached
- Pilus contracts
- 2 cells pulled together
- 2 cell membranes fuse
- Form bridge that connects 2 cells
- Plasmid is replicated to the F-recipient cell
- Plasmid is replicated to F-recipient cell
- Sometimes other genes are transferred in the plasmid
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Purpose of bacterial endospores
- Dormant stage in bacteria life cycle
- Providing a survival mechanism for bacteria under unfavorable conditions
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Properties of bacterial endospores
- Highly resistant to
- Heat
- Desiccation
- Radiation
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How do bacterial endospores form?
- Vegetative cell undergoes sporulation in response to stress or unfavorable conditions
- Vegetative cell transforms into a format endospore (highly resistant)
- Endospore remains inside the bacterial cell until the cell dies → releases the spore into the environment
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Which types of bacteria commonly produce endospores?
- Soil bacteria
- Bacillus
- Clostridium
- Sporosarcina
- Only gram-positive bacteria hv the ability to produce endospores
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How do endospores contribute to bacterial survival?
- Highly resistant
- Enable bacteria to survive in harsh conditions that would be lethal to vegetative cells
- Dormant state of endospores
- Allows bacteria to withstand extreme conditions without undergoing metabolic activity or replication.
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Describe the sporulation process
1. DNA organised along cell axis
2. The genome is replicated
1. A genome copy is move to 1 pole and then enclosed in forespore septum
2. Forespore produced by membrane invagination
3. Cell membrane engulfs forespore in a second membrane
4. Cortex between the 2 spore membranes accumulates calcium and dipicolinic acid
1. Ca and the acid becomes dehydrated to <10% → no water = cell can’t overheat
2. Becoming resistance to chemicals and heat
5. Complex exosporium layers are produced
6. Spore matures with complete cortical layers
7. Release of spore
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Structural components of bacterial endospores
- Exosporium (outermost layer)
- Thin layer of proteins
- Not essential for survival, maybe for dispersal
- Spore coats (made up of spore-specific proteins)
- Peptidoglycan cortex: removes water, contributing to the spore’s heat resistance
- Core
- Contains spore’s genome, cytoplasm n ribosomes
- Metabolically inactive
- Protected from denaturation thru dehydration
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What makes bacterial endospores highly resistant to environmental stresses?
- Dehydration of the spore → prevents denaturation
- Presence of small acid-soluble proteins → maintains the integrity of the spore’s genome by preventing the formation of pyrimidine dimers in DNA
- Overall structure of endospore → protective barrier against detrimental factors
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What is the metabolic state of endospores?
- Metabolically inactive structures
- Inside the spore, no discernable metabolic activity
- The core contains the genome, cytoplasm, and ribosomes, but they are in a dormant state.
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How does germination occur in bacterial endospores?
- Germination is the process by which a dormant endospore returns to its vegetative state.
- It starts with the uptake of water and amino acids, which act as triggers for germination.
- This process is rapid, usually complete within 30 minutes.
- As germination progresses, the spore loses its refractile (shiny) appearance due to rehydration and the loss of resistance.
- The spore's protective layers are broken down, and the core becomes metabolically active.
- The released cell then begins to grow and divide in a normal manner.
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How does the cell wall composition change during germination?
- After germination, cell produced from endospore is usually -
- But peptidoglycan layer is rapidly built up
- Results in +
- Transition occurs as the vegetative cell regains its ability to synthesize peptidoglycan
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Virulence is determined by factors that aid in
- Adhesion to n entry into cells
- Antiphagocytic activity, immune system evasion
- Production of toxins
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Conventional virulence factors
- Bacterial toxins
- Adhesins
- LPS
- Secreted hydrolytic enzymes
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Meningitis
Inflammation of the protective membranes that cover brain n spinal cord
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What causes meningitis?
- Viruses
- Bacteria
- Fungi
- Protozoa
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What does C.tetani do?
Over-activity of motor neurons causing muscle spasms
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Describe Tetanus toxin’s mechanism of action
- Released by cell lysis, spreads through blood n lymph
- Targets CNS
- Binds to peripheral nerve terminals, transported along axons
- Halts release of glycine and GABA neurotransmitters
- They normally check nervous impulses
- Absence of these checks result in muscular spasms
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Describe Haemophilus influenzae
- Gram-negative coccobacillus
- Non-encapsulated strains carried asymptomatically in urinrary tract
- Cause of bacterial meningitis
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Hib vaccines
Vaccines that protect against Haemophilus influenzae type b (Hib) bacteria, a leading cause of severe bacterial infections in young children.
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Conjugated vaccines
Vaccines that use a peptide carrier to enhance the immune response to polysaccharide antigens, which are normally weakly immunogenic.
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PRP-T
One of the three conjugated Hib vaccines introduced in 1992. It uses a tetanus toxoid carrier
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PRP-OMP
One of the three conjugated Hib vaccines introduced in 1992. It uses a meningococcal outer membrane protein (OMP) carrier.
