1
Q
Define: Oligotrophic Environment
A
Oligotrophic Environment: Low in nutrients
2
Q
What are 2 consequences/causes of an oligotrophic environement?
A
- Nutrients are quickly depleted
- Lots of competition - Nutrients may be in forms that resist breakdown
- Could cause long generation time
3
Q
What activates a cells stringent response?
A
Nutrient limitation activates a cells stringent response
4
Q
What are 4 stringent responses?
A
- Overall cell metabolism decreases
- Genes for growth are down regulated
- Stress response genes are unregulated
- Make proteins that protect DNA, cell wall ect
5
Q
What promotes the formation of persister cells?
A
- starvation
- stress (if under normal conditions)
6
Q
Properties of persister cells
A
- growth arrested bacteria
- genetically identical to “normal” cells
- small subset of the population
- less susceptible to antibiotics
7
Q
Why are persister cells less susceptible to antibiotics?
A
Antibiotics work best against actively growing cells. For example, an antibiotic might target the ribosome, but if the cell isn’t using the ribosome then can’t kill.
8
Q
How do persister cells lead to recurrent infections?
A
Persister cells are in a dormant state, therefore, after the antibiotic is removed these cells can reestablish the population
9
Q
Persister cells vs Endospores
A
- Persister cells are growth arrested cells and they make up a small portion of the population. They are genetically identical to “normal cells”. Promoted by starvation.
- Endospores can also be formed due to starvation. They have a different composition than “normal cells” but the same DNA. Also metabolically inactive. Form inside vegetative mother cells
10
Q
Define: Endospore
A
Endospore: spores that are formed within vegetative mother cells to ensure the cells survival in adverse environmental conditions.
11
Q
Endospore - General Overview
A
Endospore - General Overview
Caused by environmental stress like starvation. Mostly gram-positive bacteria which have a thicker peptidoglycan cell wall. They have a different structure compared to “normal” cells, but the same DNA. They are metabolically inactive like persisters.
12
Q
What are endospores resistant to? How to kill?
A
Resistant to:
- heat
- UV light
- Desiccation
- chemicals
- antibiotics
- phage
To kill:
- autoclaving
12
Q
How are endospores released from the inside of vegetative mother cells?
A
Endospores are released via lysis
12
Q
What is the endospore life cycle?
A
- Invagination of the membrane
- Septum formation and forespore development
- Engulfment of forespore
- Cortex formation
- Coat synthesis
- Lysis of sporangium, endospore liberation
12
Q
Describe the structure of an endospore:
A
Endospore Structure:
- Core: contains nucleoid, ribosomes ect. Contains the proteins that protect the DNA from heat, UV…
- Cortex: surrounds the core (peptidoglycan)
- Coat: protein layers
- Exosporium
outside in = outer coat, inner coat, cortex, core, nucleoid
- note: core wall and coat are impermeable
13
Q
What are 2 heath implications caused by endospores
A
Botulism
- found in home-canned foods
- spores germinate to produce botulinum toxin
Anthrax
- caused by bacillus anthracis spores
- cutaneous anthrax = skin infection
- pulmonary anthrax = spores inhaled (untreated - deadly)
14
Q
Define: Biofilm
A
Biofilm: communities of cells embedded in slimy matrix. Have EPS = extracellular polymeric substances found in the matrix and provides compositional support and protection
14
Q
Define: planktonic
A
Planktonic: free floating bacteria, don’t live in community
15
Q
Define: Quorum sensing
A
Quorum sensing: is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density
16
Q
What molecule do cells in a biofilm secrete, and what does it do?
A
Cells in a biofilm secrete autoinducer (AI) molecules, these signaling molecules are produced in response to cell population. A low concentration of AI signals the cell to work alone. High concentrations signals that the cell is not alone, can work together. AI concentration controls gene expression. At high AI bacteria might become more adhesive (ex. make pili), produce EPS.
17
Q
Describe the formation of biofilm
A
- Planktonic phase: planktonic bacteria adhere to a surface
- Irreversible attachment: sessile state
- Growth and microcolony formation
- biofilm grows
- cells stick to each other
- cells stick to EPS
- other bacteria can join - Maturation
- Dispersal
18
Q
Describe factors of bacterial attachment in regards to biofilms.
A
- Bacteria can attach to host cells
- Use adhesions (eg. pili)
- Specific - Can attack to abiotic surfaces
- non specific
- uses bacterial components
(ex. lipoplysaccharide)
- Surfaces can be conditioned (there might be things there to help the bacteria grow - can attach to early colonizers, EPS
19
Q
Describe: Extracellular Polymeric Substances (EPS)
A
- Slime-like matrix
- glycoproteins, polysaccharides, DNA…
- some are secreted others are released by cell death
- EPS helps bacteria stick to biofil
- EPS trap nutrients
- channels distribute nutrients
- retains secreted digestive enzymes near cells
- highly hydrated (holds onto water)
20
Q
Describe the environment biofilms create for bacterial growth (ie inside vs near surface)
A
- The structure creates a nutrient oxygen gradient
- Cells near the surface have access to more nutrients
= more metabolically active
= similar to planktonic cells
= waste more easily disposed - Cells near middle have limited nutrients/O2
= dormant (persisters)
= favors anaerobic
= waste accumulates
21
Name 3 benefits of biofilm
1. Nutrition:
- can grow in nutrient poor environment bc it traps nutrients and other digestive enzymes, waste, lysed cells
2. Genetic Diversity
- can acquire new DNA
3. Protection
- EPS protects from predators, UV, desiccation ...
22
What % of bacterial infections are caused by biofilms, name 4.
> 65%
- endocarditis
- UTI
- Corneal infection
- Dental plaque
23
What are 2 ways in which a biofilm protects itself against infection
1. Protect from antimicrobials
- get stuck in EPS
- persister cells (middle) are less susceptible
2. Protect from immune system
24
Innate vs Adaptive Immune Response
Innate
- nonspecific
- first line of defense
Adaptive
- specific
- slower to response to unfamiliar pathogens
25
Name 3 components of innate immunity
1. Physical barriers = skin, mucous membrane
2. Cells, tissues = phagocytes engulf and destroy bacterial cells, recognize common features
3. Chemical Mediators = antimicrobial peprides,
- complement (promotes inflammation, forms holes in cell walls, helps phagocytes recognize bacteria (opsonization))
26
What is opsonization?
Opsonization: an immune process which uses opsonins to tag foreign pathogens for elimination by phagocytes
27
Describe the process of opsonization and phagocytosis
- complement protein C3b binds to surface of bacterium
- phagocytes have complement receptors
- leads to phagocytosis
- taken into phagosome
- fuses with lysosome
- bacterium is degraded
28
Adaptive immunity - describe
- Activated by innate response,
- Dendritic cells (DC) a type of phagocyte degrade bacterial proteins (antigens) and display them on their suface
- DCs activate T cells and B cells (B cells make antibodies that bind antigen)
- Antibodies bind to bacteria, are recognized by phagocytes
29
Commensals vs Pathogens
Pathogens: bacteria that causes infection/disease
Commensals: usually beneficial, can compete for nutrients, space, produce inhibitory substances, alter conditions, making it harder for pathogens to grow
- commensals can cause disease, opportunistic pathogens,
Ex. E.coli is good in gut, if migrates to urinary tract causes UTI
30
Define: Virulence
Virulence: severity of disae
30
Describe virulence factors
They enhance the ability of pathogens to cause infection
- ex. cell structures, toxins ect
30
Infection Component - Adhesion and Colonization
- pathogens enter via skin, inhalation, ingestion...
- body can clear bacteria = swallowing, mucociliary escalator, urination
- pathogens must adhere, colonize to infect (use adhesions like pili or capsules, biofilms)
30
Infection have 4 major components:
- Adhesion
- Proliferation
- Invasion
- Tissue damage
30
Infection Component - Toxins
Endotoxin vs Exotoxin
Endotoxin
- lipopolysaccharide (LPS)
- released if cell is lysed
- systemic effect (fever, shock)
- activates severe response, may lead to septic shock
Exotoxin
- secreted
- target host cell function, metabolism
- may be tissue specific
30
Infection Component - Invasion
Why?
- Access more nutrients
- Less competition
30
Infection Component - Immune invasion
- pathogen must overcome immune system to invade
- avoid detection
- avoid recognition (decrease amount of antigen, modify antigen structure, hide antigens)
30
Infection Component - Immune Evasion
How could a pathogen interfere with immune response
- degrade antibodies, complement proteins
- hide bound antibodies from phagocytes (capsules, biofilms)
- bind to antibodies (ex. S.aureus protein A binds to Fc region of antibody, no opsonization)
31
Infection Component - Invasion
Active penetration vs Passive penetration
Active penetration = involves virulence factors
- ex. enzymes that degrade extracellular matrix, break cell-cell connections
Passive penetration = follows an unrelated event
- ex. injury causing damage to skin
32
MRSA
- MRSA = methicillin-resistant staphylococcus aureus
- acquired mecA gene which is involved in the cell wall, makes it less penetrable to antibiotics (B-lactams)
- major cause of nosocomial infections
33
Changing DNA - Effects (2)
Changing DNA
- genetic variation , can adapt to selective pressures
- evolution (note: binary fission doesn't generate diversity)
34
How does bacterial change?
- mutations
- acquiring new dna
- recombination
35
Mutations - Spontaneous vs Induced + Impacts
Spontaneous mutations:
- Errors in DNA replication
- Mobile genetic elements (Ex. transposons)
Induced mutations:
- caused by mutagens ( chemicals, UV, stress)
Both can impact:
- gene sequences
- gene expression levels
36
Vertical vs Horizontal gene transfer:
Vertical: get DNA from mother
Horizontal: acquire DNA from other sources
37
Describe the 2 main types of recombination:
Homologous recombination: DNA with similar sequences
Site-specific recombination: DNA without extensive homology
38
Describe the bacterial chromosome
- is usually a single circular DNA molecule
- packaged with proteins in nucleoid
- highly compact
39
What are NAPs
NAPs = nucleoid associated proteins
- cause DNA to bend and fold so it can fit in cell
40
How does DNA fit in the cell??
- NAPs (nucleoid-associated proteins) cause DNA to bend and fold
- Supercoiling which is catalyzed by topoisomerase which breaks and re-ligates DNA
41
Chromosomal Replication - Name the key players
Replisome: the proteins and enzymes used for chromosome replication
Helicase: unwinds double-stranded DNA, forms supercoils ahead of replication fork
Topoisomerase: relieves supercoiling, needed for helicase to keep working
DNA Pol: synthesizes new DNA with help from primase
42
Describe the steps of chromosomal replication:
1. Replisome starts at OriC, an AT rich region which contains some genes
2. Chromosome is replicated bidirectionally via its 2 replication forks
3. Catenated (linked) chromosomes are produced
4. Topoisomerase forms a double strand break to separate the chromosomes, then it repairs the break
43
What is the main action of Quinolones/Fluroquinolones?
Quinolone Mechanism?
- Antibiotics that target topoisomerase
1. topoisomerase binds to DNA and forms double stranded break
2. Quinolones bind to topoisomerase: DNA complex
3. Blocks DNA replication, transcription
- High doses can even lead to chromosome fragmentation, formation of reactive O2 species
44
MGE - " "
Define:
Classified based on:
Types include:
MGE - " Mobile Genetic Elements"
Define: Genetic material that can be transferred between cells (or can move within a cells), contributes to genetic variability
Classified based on:
- how they replicate
- how they move btwn/within cells
Types include:
- plasmids
- transposable elements
- genomic islands
45
Plasmids
Shape:
Size:
Replication:
Copies:
Plasmids
Shape: usually circular
Size: 3 - 100 kb
chromosome: 2-6 Mb
Replication: independently from chromosome (mechs vary ex. rolling-circle replication)
Copies: based on copy numebr
high = 30 - >100 / cell
low = 1 -2
46
What are some essential components of a plasmid?
- OriC
47
Plasmid Narrow vs Broad Host Range
Narrow Host Range = replicated in closely related species
- very reliant on host proteins
- less encoded in plasmid
Broad Host Range = replicated in many different species
- multiple OriC (diff species use diff ori)
- less reliant on bacterial cells
- encode more proteins for its own replication, transfer
-
48
What are plasmid accessory genes?
Genes that are not essential but often beneficial
Ex. antibiotic genes
49
How are plasmids classified?
Plasmids are classified based on:
- resistance plasmids
- virulent plasmids
50
Describe Virulence Plasmids
Virulent plasmids:
- carry genes for virulent factors
- contribute to ability to cause disease
ETEC "enterotoxigenic E.coli"
- has virulent plasmid
- plasmids encode pili and toxins
- pili to attach to epithelia, toxins cause diarrhea
51
What is the mechanism called by which bacteria transfer plasmids?
Conjugation
52
What 2 components to conjugative plasmids have?
Conjugated plasmids have:
- sex pilus genes
- mobility (MOB) genes
53
What is the purpose of 'sex pilus' in conjugative plasmids?
Sex pilus forms bridges between bacterial cells
54
What is the purpose of 'MOB' in conjugative plasmids?
Proteins encoded for by MOB genes deliver plasmids to pilus, initiate transfer
55
What are the steps of plasmid transfer?
1. Sex pilus attaches to recipient cell
2. Mobility proteins deliver plasmid to base of pilus
3. Plasmid replication begins, and strand of plasmid DNA is transferred thu pilus
56
What are "Molbilizable plasmids", What makes them unique?
Mobilizable plasmids lack sex pilus genes but can still be transferred
- carry MOB genes
- can use sex pilus made by other (conjugative) MGEs
57
What are Transposable Elements (TEs)?
What are the 2 mechanisms?
What are Transposable Elements (TEs)?
Transposable elements are nucleic acid sequences that can move within, between molecules (transposition)
- within chromosome, chromosome to plasmid, plasmid to chromosome...
What are the 2 mechanisms?
Simple - 'cut and paste'
- cut transposable element out and insert into another region
Replicated - 'copy and paste'
- get a new copy of the transposable element and put it elsewhere without moving the original position
58
Type of TE - Insertion sequence
Insertion sequences
- the simplest TE
- only contain elements needed for transposition
- gene encoding transposase
- inverted repeat sequences at both ends as a recognition sites for transposes
- flanked by direct repeats
59
What are the steps of Simple Transposition?
1. Transposable element (TE) produces transposase
2. Transposase cuts DNA at inverted repeats
3. TE excised from DNA
4. "Mobile" complex formed of transposase and TE
5. Target DNA cut, making insertion site
6. Transposase inserts TE DNA into cut target DNA
7. DNA flanking insertion site filled in, repaired
8. Repair generates direct repeats
60
Type of TE - Unit transposons
Unit transposons
- transposase gene
- inverted repeats at both ends
- may have accessory genes btwn IRs (ie. antibiotic resistance)
- may have other genes involved in transposition
61
What is the significance of transposons?
- Te insertion can impact gene function, regulation
( can inactive, change transcription)
- TEs can take flanking DNA, spread to other bacteria
- accessory genes can be beneficial (ie. antibiotic resistance)
EX. Tn1546 transposon:
- protects against vancomycin
- found in VRE
- will remodel the cell wall to protect from antibiotics
62
What are genomic islands?
Genomic islands are region of chromosome that was acquired by HGT
- 10 - > 600 kb
- contribute to genetic diversity
- usually contain genes to improve fitness
- may contribute to antibiotic resistance
63
Conjunctive vs Mobilizable genomic islands
Conjunctive genomic islands: encode excision, conjugation, and integration functions
- makes enzymes that chop islands out
Mobilizable genomic islands:
encode excision, integration, but not conjugation
- conjugative MGE needed for transfer
- can cut self out and integrate in
64
Salmonella Pathogenicity Island
Salmonella Pathogenicity Island
- encodes type III secretion system
- injects proteins into host cells
- helps salmonella invade intestinal cells
- causes inflammation
65
What are the 3 fates for acquired DNA?
1. integrate into genome
2. replicated independently of genome
3. get lost
66
Define conjugation
What is required?
Conjugation: direct transfer of DNA between cells connected by sex pilus
Requires mobile genetic elements (MGEs)
- sex pilus genes, MOB genes ...
67
Describe F Factor Mediated Conjugation
- F+ x F- mating
F+ = donor
F- = recipient
1. Donor sex pilus attaches to recipient
2. Pilus retracts
3. Pilus converts to type IV secretion system (T4SS)
- F Factor contains contains origin of transfer (OriT)
4. Relaxosome cuts at oriT
5. Intact strand undergoes rolling-circle replication
6. Coupling factor recognizes cut DNA bound to relaxase, delivers DNA to T4SS
7. T4SS pumps cut F factor strand, relaxase into recipient
8. Cut strand is replicated forming intact F factor
9. Recipient is now F+
68
What are Hfr cells?
Hfr cells = high frequency of recombination
- When F factor integrates into chromosome get Hfr cell
Note: conjugation can still occur after integration
69
What is F' conjugation
F factor integration is reversible, but errors can occur during excision. If the F factor takes extra DNA makes F' plasmid
F' x F- mating = recipient gets F' plasmid
70
Transformation - experiement
Mouse experiment
- basically the smooth cells were virulent and killed mouse, rough weren't, but rough + dead smooth = dead bc transformation
71
What is transformation?
Why?
- When bacteria acquires DNA from the environment, this process may be selective
Why?
- Genetic diversity
- DNA repair
- Nutrition
72
Define: Competence
Competence: the state of being able to take up DNA
- some cells are constitutively competent, others only competent in certain conditions
73
What triggers competence?
Competence is triggered by:
- Cell-cell signaling
- Nutritional stress
- DNA-damaging agents
74
How is the production of Com proteins regulated in S.pneumoniae?
- transformation requires competence (Com) proteins
- Production of Com proteins is regulated
- Cells secrete Competence-stimulating peptide (CSP)
- if enough CSP is present, receptor is activated
- com genes are transcribed
- Essentially this ensures other cells (and their DNA) are present
75
Describe the 4 significant Com proteins in S.pneuoniae
Transformation pilus
- binds to double-stranded DNA
Membrane receptor
- binds dsDNA at cell surface
Nuclease
- cuts ds DNA
- makes ssDNA
Transport complex
- transports ssDNA
76
Describe the process of transformation in S.pneumoniae
1. dsDNA binds to transformation pilus. Pilus draws dsDNA to surface
2. dsDNA binds to receptor on surface
3. Endonuclease cuts dsDNA into fragments, makes ssDNA
4. Transport complex transports ssDNA into cytoplasm
5. Rec A binds to ssDNA, recombinase catalyzes recombination
6. New DNA is integrated by homologous recombination
77
What are the 2 ways to artificially induce competence?
Chemically competent cells
- usually treated with CaCl, the calcium ion masks DNA's negative charge
- Then heat shock to increase permeability
- DNA can now enter the cell
Electrocompetent cells
- Mix cells with DNA
- Shock, electrical current forms pores
- Cells take up DNA
78
Define: Bacteriophages
Bacteriophages: viruses that infect bacteria, they carry nucleic acids in capsid
79
When phages transfer DNA to bacteria =
When phages transfer DNA to bacteria = transduction
80
Phages that mediate transfer =
Phages that mediate transfer = transducing particles
81
Lytic vs Lysogenic cyles
Lytic cycle: The virus immediately replicates, lyses host cells
Lysogenic cycle:
- viral genome is integrated into chromosome
- bacterium is lysogen; contains prophage (spots with viral dna)
- phage can reemerge undern certain conditions
82
Describe the steps of the lytic cycle
1. Phage absorbs to surface of cell, using specific receptors
2. DNA is injected into cell, but the capsid remains outside
3. Phage DNA directs degradation of host DNA, and replication of phage DNA
4. Phage DNA directs cell to make phage proteins
5. Phage DNA is packaged into capsids
6. New phage particles are assembled
7. Bacterial cell lysed by phage proteins
83
Describe 'Generalized Transduction' in regard to the lytic cycle
- During the lytic cycle bacterial DNA is degraded, but sometimes bacterial DNA can be randomly packaged into the capsid. This forms transducing particles with bacterial DNA.
- Transducing particles then inject their DNA into a new cells, but since this is the bacterial DNA not the phage DNA its not lytic and the DNA can integrate into the chromosome
- Note: this DNA was randomly packaged thus its generalized transduction (not specific)
84
Describe the lysogeny
This is when the viral genome is maintained in the bacterial cell
- this can be beneficial if host are in low abundance, the viral DNA can replicate and wait until there is more to infect
- When the phage genome integrates into the chromosome = prophage
- the phage genome is replicated when the bacterium replicates
85
Define: Induction
Describe?
What leads to induction?
Induction: prophage initiates synthesis of phage proteins
- phage genome is excised, replicated
- new phages are assembled
- bacterial cells is lysed, releasing the phage
- Stress to bacterial cells leads to induction
86
What are the steps of the lysogenic cycle?
1. Phage DNA is injected into the bacterium
2. Phage DNA is integrated into the bacterial chromosome
3. The bacterial cells divides, replicating phage DNA
4. A trigger (stress) causes the phage DNA to excise from the bacterial chromosome
5. Lytic cycle initiated
87
Describe "Specialized Transduction"
- Errors can occur when the prophage is excised from bacterial DNA and it can take the DNA next to the integration site
- This mixed genetic material is packaged into transducing particles
- DNA from integration site is transferred to anther cell, this is not random DNA (specialized)
88
Describe Lysogenic Conversion
- A prophage can change lysogen (bacterium) phenotype
- They often modify the cell surface ( They may remove receptors needed for phage infection, thus protecting the prophage DNA)
- Or they may have gene unrelated to replication like toxin production, antibiotic resistance
89
Describe "Extracellular Vesicles"
- Spherical structures surrounded by a lipid bilayer that are released from the surface of bacterium.
- They contain cargo from the cytoplasm or periplasm (proteins, nutrients, DNA...)
- Contribute to HGT
- Vesicles fuse with recipient cell membrane and the DNA cargo is transferred into the cell
- Transferred DNA may have genes for antibiotic resistance, virulence ect
90
During the process of conjugation involving Hfr cells, which of the following is transferred from the donor cell (the Hfr cell) to a recipient cell?
1. Only DNA from the bacterial chromosome
2. DNA from both the conjugative plasmid and from the bacterial chromosome
3. Only DNA from the conjugative plasmid
4. An Fʹ plasmid containing some chromosomal DNA
DNA from both the conjugative plasmid and from the bacterial chromosome
91
What is a HFR cell
A high-frequency recombination cell (Hfr cell) (also called an Hfr strain) is a bacterium with a conjugative plasmid (for example, the F-factor) integrated into its chromosomal DNA. The integration of the plasmid into the cell's chromosome is through homologous recombination
92
Transduction is a mechanism of horizontal gene transfer that involves bacteriophages. Which of the following statements about specialized transduction is correct?
1. After DNA is transferred by a bacteriophage, the DNA must be capable of replicating independently of the chromosome
2. Unlike generalized transduction, specialized transduction involves the transfer of chromosomal DNA from one bacterium to another
3. Bacteriophages which only replicate through lytic cycles cannot contribute to specialized transduction
4. Specialized transduction involves the random packaging of bacterial DNA into a bacteriophage
3. Bacteriophages which only replicate through lytic cycles cannot contribute to specialized transduction
93
PHGY - Midterm
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