Exam (after Midterm 1 Content) Flashcards
What was the purpose of the Griffith Experiment and how was it performed
Evidence that DNA may be the hereditary material
When killed smooth and live rough streptococcus pneumoniae were separately added to separate mice, they both lived
Live smooth killed the mouse
When killed smooth and live rough we’re added together, the mouse died
Rough cells converted to smooth
Avery, MacLeod, and McCarty experiment: steps and purpose
Purpose: is DNA, RNA, or protein the causative agent of transformation
Heat killed smooth strain was treated with enzymes that killed proteins, DNA or RNA, and then were incubated with living rough cells
Rough cells do not grow on agar
All plates showed growth except one treated with DNase
DNA is the transforming principle
Packaging of DNA in the 3 domains
Bacteria: singular circular chromosome, supercooled
Archaea: singular circular chromosome, histones
Eukarya: multiple linear chromosomes, histones
Replication initiation in E. coli
DnaA molecules interact with the oriC bp repeats forming a complex, DNA is unwound
DnaC delivers the DnaB helicase to the replication complex
Single stranded binding proteins present
Initiation of DNA replication in eukaryotes
Multiple origins of replication on each chromosome
Origin replication complex (orc) binds Automatically replicating sequence (ARS)
Proteins such as Cdt1, Cdc6 and MCM are recruited
DNA is unwound and SSB proteins keep strands apart
Replication elongation in bacteria
DNA polymerase III adds nucleotides to RNA primers that are synthesized by RNA primase
Continuous and discontinuous strands (leading and lagging)
Clamp keeps DNA polymerase III bound to DNA
RNA primers removed and replaced with DNA by DNA polymerase I which has 5’ to 3’ exonuclease activity
Sealed by DNA ligase
Clamp processivity factor
Found in all 3 domains
Beta clamp in E. coli
Circular Replication termination
Tus proteins interact with DnaB to open DNA then they bind ter sites stopping elongation
Topoisomerase is recruiter
Chromosomes disentangled
Linear replication termination
RNA primer removed, produces 5’ end that cannot be extended by DNAP
telomerase binds and extends the 3’ end using an RNA template
Meselson and Stahl
Add N15 labelled cells to N14 medium and grow for 4 gen
Results consistent with semi conservative replication
Generation 2 had one N15 and one N14 strand
Approximate length of mRNA
500-10,000 nt
Typical gene size of bacteria
1 kb
mRNA can be _______________, meaning it encodes several proteins
Polycistronic
Do bacterial genes contain introns
No
Length of tRNA
75-100 nt
tRNA orientation
3’ to 5’
rRNA size
1500 to 1900 nt for small subunit
2900 to 4700 nt for large subunit
Eukaryotic ribosome subunit size
Small 40S
Large 60S
Total 80S
Bacteria ribosome subunit size
Small 30S
Large 50S
Total 70S
Small subunit rRNA in eukaryotes
18S
Small subunit rRNA in prokaryotes
16S
Which has more rRNA? Pro or eukaryotes
Eukaryotes
More proteins or rRNA in the ribosome?
Proteins
————- factors bind bacterial promoters to allow RNAP to recognize them
Sigma
Positions of consensus regions in E. coli
-10 and -35
What is the main sigma factor in E. coli
RpoD
Transcription initiation elements in eukaryotes bound by transcription factors
Octamer box
CAAT box
GC box
TATA box (-20)
Different sigma factors in bacteria can_______________________
Direct RNAP to different sets of genes that are co-regulated
How many RNA polymerase in bacteria
1
How many RNA polymerase in eukaryotes
3
This protein interacts with the TATA box
TATA Binding protein (TBP)
What is TFIIB
A transcription factor in eukaryotes which binds the promoter
Transcription initiation in archaea is similar to _______ in regard to transcription factors and similar to ______ in regard to RNAP
Eukaryotes , bacteria
Transcription termination in bacteria can be done by the _____ protein or through ________
Rho
Rho-independent termination
Rho independent termination involves a _______ rich in _________ followed by a ____ rich sequence
A hairpin structure
GC
Uracil
What is the function of the uracil rich sequence in Rho independent termination
Has less H bonds so is easier to separate
3 pots transcriptional modifications in eukaryotes
5’ 7-methyl guanosine cap
3’ polyA tail
Intron splicing
Which enzyme adds amino acids to tRNA
aminoacyl tRNA synthetase
Which nucleotide is responsible for the wobble effect
3rd in codon
1st in anticodon
Which amino acid charges tRNA that initiates translation of AUG in bacteria
N-formyl methionine
Amino acids are added to the __ end of the tRNA
3’
The shine delgarno sequence is
A sequence complementary to the 16S sequence in bacteria
Interacts with 16S in ribosome to initiate translation
In eukaryotes mRNA is bound by ___________ and one binds the ________
Several polypeptides
5’ cap
Eukaryal mRNA is _______
Monocistronic
Charged tRNAs enter the _____ site
A
Termination of translation
Release factors when stop codon is reached
_______ help proteins fold
Chaperones
Protein modifications in eukaryotes include
Phosphorylation
Acetylation
Methylation
Glycosylation
______________ direct proteins to appropriate locations
Signal peptides
Short AA sequences at N terminus
How many genes in human cells that encode proteins
Approx 22,000
When does regulation of gene expression occur
During transcription
Translation
Post translation
Composition of lactose
Galactose and glucose
Linked by beta 1,4
Function of beta galactosidase
Cleaves beta 1,4 linkage in lactose
Substrate for the detection of beta galactosidase
ONPG
Lactose permease function
Transports lactose into cell across cell membrane
When e.coli are grown with glucose and lactose, which is consumed first
Glucose
Lac Operon not expressed until
All glucose is used up
LacI function
Codes for lacI repressor which binds the operator inhibiting transcription
CAP site function
Activator binding site
DNA site bound by activator
Promoter function in lac operon
Bound by RNAP to direct initiation of transcription
LacO function
Operator
Bound by repressor
Effector function lac Operon
Binds activator or repressor proteins to modify gene activity
Inducer function
Increases transcription levels by enabling activator or disabling repressor
Corepressor
Decreases transcription by binding repressor
LacZ codes for
Beta galactosidase
LacY codes for
Lactose permease
When is lacI transcribed
Always
At a low basal level
What recognizes the allosteric site on the laci repressor
Allolactose
The trp operon encodes for
Proteins in the tryptophan synthesis pathway
When trp is not present
It cannot bind the trp repressor, so it cannot bind the operator
Transcription continues
When trp is present
It binds the repressor so it can bind the operon
No transcription
When glucose levels are high and cAMP levels are low
cAMP cannot bind the CRP activator
RNAP has a low affinity for the promoter and will not bind
When glucose levels are low and cAMP levels are high
cAMP bind CRP
The CRP-cAMP complex binds the activator binding site increasing RNAP affinity for the promoter
Sigma 70 (RpoD) function
Housekeeping
Sigma 32 (RpoH) function
Heat shock
Sigma 54 (RpoN) function
Nitrogen starvation
Sigma-38 (RpoS) function
Stress
Purpose of SOS response in bacteria
Allows cells to recognize and respond to serious DNA damage
SOS response experiment with E. coli
Bacteria exposed to UV light and infected with damaged phages repaired these phage but they had high rates of mutation (more phage, more mutations)
Bacteria that were not exposed to UV and that were infected with damaged phages could not repair them as efficiently and lower mutation rates (few phage, few mutations)
lexA function
DNA binding transcriptional repressor of the SOS genes
Binds the operator and shuts of transcription
recA function
Recombination and regulation of the SOS response, binds ssDNA
Becomes a protease which cleaves lexA so the genes can be expressed
SOS regulon no DNA damage
LexA keeps SOS regulon genes repressed
RecA is inactive
SOS regulon DNA damage
RecA cleaves the LexA repressor
SOS genes expressed
Quorum sensing definition
Chemical signalling system that controls gene expression
What is the function of autoinducers
Cells release them into the environment and as the population density increases in an area the concentration of autoinducer does too
Detecting changes in autoinducer levels causes regulation of gene expression
The lux quorum sensing system is found in this bacteria
Vibrio fischeri
Where do Vibrio fischeri live in
Hawaiian bobtail squid
When do Vibrio fischeri emit light and what enzyme do they use
When in the light organ of the squid
Luciferase
What is the autoinducer made by Vibrio fischeri
AHL
This activator interacts with AHL when it reaches a high concentration
LuxR
The AHL LuxR complex binds the _________ for activation
Lux box
At high AHL concentration
AHL and LuxR interact and transcription is activated transcribing luxA and luxB which are needed for luciferase production
At low AHL levels
AHL and LuxR do not interact
Lux box is not bound for activation of transcription
Light and luciferase is not produced
Mechanisms controlled by quorum sensing
Motility
Conjugation
Biofilm formation
Pathogenesis
Components of the two component regulatory system
A sensor kinase and a response regulator
Sensor kinase function
Often a histidine protein kinase (HPK)
Located in plasma membrane
Detect environmental stimulus
Response regulator function
Inside the cytoplasm
Regulate transcription
Two component regulatory system in Agrobacterium tumefaciens
Genes expressed under similar conditions to plant wound
virA is an HPK protein which interacts with sugars and phenolic compounds at low pH
virG is an activator RR protein for other vir genes
Regulatory RNAs
Small non coding RNA that control gene expression at transcriptional or translational levels
General attenuation mechanism
If the ribosome quickly follows RNAP, a terminator hairpin will form in the leader sequence and RNAP detaches
Stalling out of the ribosome on the leader sequence allows transcription to continue
Can attenuation occur in eukaryotes? Why or why not?
No, because transcription and translation occur separately
High levels of tryptophan: attenuation
Terminator loop forms (regions 3 and 4) and stops transcription, tryptophan not made
Ribosome stops at stop codon
Low levels of tryptophan: attenuation
Terminator loops does not form and transcription continues
Tryptophan made
Ribosome stops at trp codons
Region 2 anneals to 3
Where in bacterial mRNA are riboswitches found
The 5’ UTR
Riboswitch function
Acts as a binding site for ligands which influence downstream structure regulating transcription and or translation
Transcriptional: terminator loop
Translational: ribosome binding site
Intrinsic factors that effect food spoilage
Water activity Osmolarity Nutrient content pH Antimicrobial constituents Biological structures
Extrinsic factors that effect food spoilage
Temperature
Humidity
Gases
Adding solutes to water ———- it’s water activity
Decreases
Bacterial pathogens cannot grow at a water activity _______ and yeasts and moulds ________
<0.86
<0.65
Reducing water activity by:
Drying Adding solutes (sugar or salt)
Most microorganisms require a water activity greater than
0.99
Canning process
Heating food to 100 degrees for extended period under pressure
Pasteurization process
Heating liquids to 63 degrees Celsius for minimum 30 min
Kills 99% of organisms
Pickling process
Using acid (vinegar)
Modified atmosphere packaging
Vacuum seal
Change atmosphere to inhibit microbial respiration (oxygen depletion)
Chemical preservatives
Lower pH
Interfere with cellular respiration
Irradiation for food preservation
Ionizing radiation causes oxidative damage and toxic free radical production
UV radiation causes thymine dimers
Hurdle technology
Apply multiple constraints to more effectively preserve food
Ex temp, ph, aw, etc.
Clostridium botulinum
Gram positive
Obligate anaerobe
Produces botulism neurotoxin (most potent neurotoxin known)
Associated with improperly canned food
How to control botulism
Pickling, pH less than 4.6
Salt curing, aw less than 0.96
High oxygen
Temp less than 4.4 deg
What was the bacteria responsible for the Romaine lettuce infection outbreak?
E.coli O157:H7
What toxin was responsible for the Romain lettuce infection and what is its function
Shiga toxin
Inhibits protein synthesis in target cells
How did E.coli O157:H7 acquire the shiga toxin gene
Horizontal gene transfer from a prophage
Which bacteria was responsible for the listeria outbreak of 2008
Listeria monocytogenes
Gram positive firmicute
Why can listeria monocytogenes evade control in human foodstuffs
Can grow at temperatures as low as 0 deg C
Can move within eukaryotic cells by polymerization of actin
Facultative anaerobe (can grow with or without oxygen)
Which bacteria is one of the most common causes of food poisoning
Camplyobacter jejuni
Gram negative bacteria
Spiral shaped
Which bacteria grows best in aerobic environments but can also grow anaerobically, and causes food poisoning
Staphylococcus aureus
Lactic acid bacteria characteristics
Gram positive
Ferment glucose to lactic acid
Grow anaerobically but can grow with oxygen
Lack catalase (have alternative enzymes)
Lactic acid bacteria must grow in:
Environment where sugars are present
Complex media that fulfills nutrition requirements
Some LAB cause disease, most notably members of the genus ________
Streptococcus
Why are LAB so important for food fermentation
Taste and texture
Inhibit food spoilage by producing lactic acid and growth inhibiting substances
Preparation of yogurt involves:
Pasteurization, adding starter cultures, and fermentation
What is the purpose of starter culture in food
Helps control fermentation
Produces acid lowering pH and preventing spoilage
_________ is used in koji fermentation to produce products like soy sauce
Mold
Koji is the starter culture for various molds
Kombucha starter culture
SCOBY
Starter culture of yeast and bacteria
How to make mash for beer
Barley malt added to warm water, enzymes activated, and starch is broken down by amylase to maltose creating wort
Boiling step of beer making
Boiling kills unwanted microbes
Hops are added to add flavour and act as an antibacterial
Beer fermentation
Wort is cooled and yeast is added
Maltose is converted to ethanol and co2 which carbonates beer
How is vinegar produced
Acetic acid bacteria metabolize ethanol to acetic acid
Ethanol sprayed on wood chips with a biofilm of bacteria
Which bacteria are used to make vinegar
Gluconobacter and acetobacter
Foodborne intoxication
Microbial toxins in food, symptoms appear quickly
Foodborne infection
Contaminating microbes in food, delay in symptoms (after growth)
Goals for treating wastewater sewage
Remove total organic carbon
Remove harmful organisms
Reduce inorganic compounds
Reduce persistent organic pollutants
Wastewater treatment steps
Pre treatment: remove large objects
Primary treatment: remove sediments and grease
Secondary treatment: uses activated sludge (aerobic), microbes to break down organic compounds
Tertiary treatment: filtration of nitrogen and phosphates
Disinfection: chlorination, UV, ozonation
Drinking water purification steps
Screening Flocculation Sedimentation Sludge treatment Filtration Disinfection Storage
The casein milk protein family contains ________ and _________ at pH 4.6
Phosphorous
Precipitates
The serum whey proteins do not contain ________ and _________ at pH 4.6
Phosphorous
Does not Precipitate
Which protein family provide a good source of calcium for milk consumers
Casein
Carbon cycle in plants, algae and photosynthetic bacteria
CO2 to C6H12O6
Carbon cycles in eukaryotes, bacteria and archaea
C6H12O6 to CO2
Decomposition
Carbon cycle in methanogens
C6H12O6 to CH4
Carbon cycle in methanotrophs
CH4 to C6H12O6
The carbon cycle summary
Incorporation of carbon from the atmosphere into biomass and release of carbon into the atmosphere through decomposition of biomass
Photosynthesis and respiration
Methanogenesis
Metabolism of organic matter or CO2 to methane under anaerobic conditions
Methanogens rely on ______ to produce __________ for methanogenesis
Other organisms
Simple organic substrates
Methanogens form __________ with other organisms
Syntrophic relationships
What sources produce methane
Termites Wetlands Landfills Rice cultivation Domestic animals Fossil fuels Oceans
Type I methanotrophs utilize the ____________ pathway
RuMP
Type II methanotrophs utilize the _________ pathway
Serine
Are methanotrophs aerobic or anaerobic
Can be both
Which pathway do aerobic methanotrophs use
Either the RuMP or serine
Which enzyme do aerobic methanotrophs use to oxidize methane
Methane monooxygenase MMO
What process converts biologically inaccessible N2 to accessible NH3
Nitrogen fixation
Nitrogen fixation is generally under ____ conditions but can be under _____
Aerobic
Anaerobic
Ammonia is used to:
Make amino acids or by lithotrophs as energy
Denitrification
Removes nitrate and converts it to nitrogen gas
As nitrogen is converted from nitrate to ammonia, it’s oxidation state:
Decreases
Which enzyme reduces N2 to ammonia
Nitrogenase
Very sensitive to oxygen
Nitrification
Oxidation of NH3 to nitrite to nitrate
Nitrifying bacteria are:
Chemolithoautotrophs
Ammonia oxidizers harvest energy from oxidation of ammonia using
AMO and hydroxylamine oxidoreductase
Nitrifiers _______ from the oxidation of nitrite to nitrate using ______
Harvest electrons Nitrite reductase (NOR)
Which process accounts for 50% of loss of nitrogen from marine environments
Denitrification
Bacteria and archaea use ___ and ____ as terminal electron acceptors in anoxygenic environments
Nitrogen and sulfur compounds
Chemical fertilization through the _____________ has nearly doubled flux of reactive nitrogen into the environment
Haber-Bosch
Ecosystem
Interactions and exchange of materials between organisms and their environment
Success of microbes in a niche depends on
Their ability to obtain nutrients and produce biomass
Formation of a biofilm
Adhesion by a primary colonizer
Cells divide
Microcolony produces EPS
Introduction of secondary colonizers
EPS/slime function in a biofilm
Protects biofilm
Forms water filled channels for transport of nutrients and wastes
What percentage of microbes can be cultured in a laboratory
0.1%
Enrichment cultures
Promote growth if desired microbes over undesired cells
Winogradsky column
lake sediment in a cylinder
Carbon and sulfur added
Placed in sunlight
Creates layers of different microorganisms
Direct sequencing
Extract DNA from environmental sample
PCR using universal primers for rRNA and sequencing
Compare to known databases
Fluorescent in situ hybridization
Labeled oligonucleotide probes bind specific RNA or DNA in a mixed population of cells
Visualized under microscope
Metagenome library
DNA from an environmental sample used to construct a genome library
Primary producers
Capture light through photosynthesis
Inorganic carbon to organic carbon
Consumers
Heterotrophs that ingest organic carbon molecules
How much of the marine biomass is made up of microbes
Over 98%
These microbes are often present in marine environments
Oligotrophs
Low levels of nutrients including N,P, Fe
Nutrient influx effects
Farmland runoff adds N and P to the water
Phytoplankton proliferate
Heterotrophic microbes feed on phytoplankton and consume too much oxygen
Heterotrophs die due to low oxygen levels
Coastal dead zones
Hypoxic
N and P stimulate growth of algae and Cyanobacteria and upon their death heterotrophic organisms use all of the oxygen
How to enrich for nitrogen fixing bacteria
Medium without nitrogen containing organic material
Can use nitrogen gas from air to convert to usable molecules
Photosynthetic microbes in the ocean produce how much of the atmospheres oxygen
1/3
Surface photic zone
Penetrated by light
Populated by photosynthetic phytoplankton And Cyanobacteria
Non photosynthetic zooplankton
Pelagibacter (SAR11) present in very high quantities
Pelagibacter
Small genome, energetically efficient
Highly specified for the environment, little junk DNA
Dark mid water zone
Fed by organic matter produced in upper surface water
Deep sea zone
Barophiles and pliezophiles which withstand high pressure
Dilution to extinction method
Inoculate very dilute sea water into autoclaved sea water followed by incubation
25-30% of cells were SAR11 proteobacteria
Proteorhodopsin
Light driven proton pump
Genes that encode for it are present in SAR11
Many sequences found in aquatic sample similar to proteorhodopsin
What nutrients are often limiting in the ocean
Fe and P
How do soils form
Microbial decomposition of plant and animal matter combines with abiotic minerals and nutrients excreted from plant roots
Rhizosphere
Area around the plant root
Supports growth of microbes
Can life exist in the absence of photosynthesis driven primary production?
Yes
Nutrients can be carried from primary producers to areas where sunlight cannot reach
Microbes in harsh, deep surface environments depend on ___________ for production of organic compounds
Chemolithoautotrophy
454 sequencing
DNA or RNA sheared into small fragments
Adapter sequences ligated to the fragments which each anneal to one bead
Fragments amplified by PCR and each bead is placed in the well of a slide containing DNA sequencing reagents
Each of the 4 dNTPs is added sequentially to the wells sequencing the fragments through fluorescence
Based on pyrosequencing
Illumine sequencing
4 Fluorescently labelled nucleotides and terminator added one cycle at a time to a slide
Terminator and previous base removed after each cycle
Nanopore sequencing
Guides ssDNA through protein pore that reads bases as they exit
Reads longer sequences than illumina
PacBio sequencing
High accuracy and longer sequences
Can explore epigenetics
Horizontal gene transfer
Movement of DNA between organisms rather than from parent to offspring
Can be found by comparing GC content of organisms
Proteomics
Separate proteins on a gel
Split proteins into smaller peptides and determine molecular weight using mass spec
BLAST to sequence
Symbiont definition
An organism that has entered a long-standing and intimate relationship with an organism of another species
Syntrophy
Provision of nutrients from a microbes that the organism is unable to synthesis themselves
In syntrophic relationships, both partners depend on each other for:
Energetic reasons
Perform a fermentation process neither could run on their own
Ectosymbionts
Live on the surface of their host
Endosymbionts
Live within the body or cells of a host
_______ and __________ live on the human skin and play a role in immunity
Firmicutes and actinobacteria
What is the most enriched part of the human body with anaerobic bacteria
The intestinal tract
What is the order of microbes in the small intestine (10^?)
10^10
How do herbivorous vertebrates break down cellulose from food
Depend on their symbiotic microbes for most of their caloric intake
Don’t posses enzymes to break down cellulose
Humans are __________ fermenters, where the primary site of fermentation is in the _______
Colonic
large intestine
Cecal fermenters: definition and examples
Enlarged cecum that acts as primary fermentation site for breakdown of plant tissue
Rabbits, mice, herbivorous birds
Rumen fermenters: definition and examples
Evolved a specialized organ (the rumen) as the primary site of microbial fermentation
Deer, cattle, sheep
Primary endosymbionts of insects show evidence of:
Co-speciation
Primary endosymbionts of invertebrates are found exclusively in:
Specialized host cells called bacteriocytes which are transmitted from mother to offspring
Can be required for survival or fertility
Secondary endosymbionts live:
Can live outside of the host and aren’t restricted to specialized cells
Not always present or maternally transmitted
Example of primary and secondary symbionts of invertebrates
Primary: Buchneria with sap-feeding aphids
Secondary: Wolbachia can colonize many insect species, can reduce dengue virus spread in mosquitos
What is lichen composed of and what is the role of each component
Fungi (mycobiont) and and a photosynthetic microbe (photobiont, which can be Cyanobacteria or algae) in a symbiotic relationship
Photobiont performs photosynthesis and supplies C and N
The mycobiont provides inorganic nutrients and a protected environment
~85% of plant families participate in symbiosis with:
Mycorrhizal fungi which colonize roots
Roots provide fixed carbon, and fungus aids in nutrient acquisition from soil by penetrating the plant cell wall with its hyphae
What is the only way nitrogen can enter the biosphere
Nitrogen fixation by bacteria or archaea
What is the nitrogen fixing bacteria that forms a symbiotic relationship with legumes
Rhizobium
Legume forms a root nodule for the bacteria and they fix and provide nitrogen to the plant
Legume provides fixed carbon
What protects the oxygen sensitive nitrogenase in rhizobium
The root nodule and leghemoglobin of the legume plant which tightly binds oxygen
How do rhizobia infect the legume
Attach to the root hair, formation of a Shepards crook and an infection thread
Infectious disease definition
Disease caused by a microbe that can be transmitted from host to host
Zoonotic disease definition
Infectious disease in animals that can cause diseases when transmitted to humans
Ex. Rabies
Pathogen definition
Microbes that can cause disease
Primary pathogen
Produce disease readily in healthy hosts
Opportunistic pathogens
Only cause disease when displaced to an unusual site or when host has weakened immune system
Exotoxin
Secreted proteins that negatively affect the host cells
Endotoxins
Part of the microbial structure itself
Do viruses produce toxins?
Typically no, replication typically induces cell death via immune responses to viral spread
What is the purpose of mucus in the inner surfaces of the body
Sticky layer that traps microbes, which is then swept out by cilia
Innate immune response
All multicellular eukaryotes
Immediate and nonspecific immune response
Inflammatory response recognizes PAMPs which are detected by TLRs
TLR 1 and 2
Recognize lipoteichoic acids
TLR 2 and 6
Recognize lipopeptides
TLR 4
Recognize LPS
TLR 5
Recognize Flagellin
TLR 10
Ligand unknown
Adaptive immune response
In vertebrates
A specific immune response
B cells and T cells coordinate to recognize and remember specific antigens
Three steps for a microbe to cause disease
Gain access to host tissues
Overcome host defences
Obtain nutrients from host
Virulence factors purpose
Help the pathogen gain access to the host tissues, evade host defences, and access limiting nutrients
What is fibronectin
A large glycoprotein found in plasma and in extracellular matrix
Prime target for pathogen binding
How does gonorrhoea attach and invade host cells
Uses fimbriae to attach
Endocytosis and exocytosis
Replication and spread of infection
Fimbriae
Specialized pili used for attachment
Binds to receptor on host cell
Can be altered to evade immunity
Special adherence proteins
E.coli can engineer attachment with virulence factors Tir and Intimin protein
Tir is made by bacteria and excreted through the type III secretion system
Intimin produced by bacteria and interacts with Tir to act as a landing pad for bacterial cell
What microbe causes gastric ulcers
Helicobacter pylori
Effect of LPS on mammals
An endotoxin
Toxic to mammals, released upon bacterial death triggering inflammation
Can lead to septic shock or death
What can act as an endotoxin in gram positive bacteria
Lipoteichoic acids
Components of LPS
Lipid A: toxic activity
Core polysaccharide
O-antigen
AB toxin structure
A subunit has toxic enzymatic activity
B subunit binds the cell receptor
Diphtheria, pertussis, and cholera toxins work by ______
ADP ribosylation of target molecules (EF2 and Cyclase)
Function of shiga toxins
Cleaves rRNA preventing ribosome synthesis
Function of botulism and tetanus toxins
Inactivate motor neuron proteins involved in activation of halting of muscle contraction
Results in paralysis
Cytolysins
Toxins that act on the plasma membrane to encourage cell lysis to provide nutrients for pathogens
Hemolysins
Cytolysins that target and lyse red blood cells to get iron from host
alpha toxin and listerolysin are:
Pore forming cytolysins
Cause apoptosis and membrane disruption
Lecithinases are:
Membrane degrading cytolysins
How does an egg yolk plate work
The egg yolk contains lipids that when degraded produce a precipitate
Superantigen function
Act on helper T cells, over activating them and causing a systemic inflammatory response which does not efficiently target the pathogen
The type III secretion system is similar to _______
Flagella
Type IV secretion system is similar to ________________
Bacterial conjugation machinery
Spans inner and outer membrane and injects effectors into host
_______ systems are associated with pathogenesis
Type III and IV secretion
Capsules as virulence factors
Extracellular polysaccharide matrix used to evade the host immune response and hide from receptors on immune cells
Can also play a role in attachment
Siderophores
Iron binding molecules that compete with host iron binding proteins
Mechanisms to obtain iron from host cells for pathogens
Siderophores
Bacterial transport proteins
Lower pH
Hemolysin
How do bacteria become pathogens
Horizontal gene transfer
Uptake of DNA from the environment, conjugation, transformation
Pathogenicity islands
Can encode various virulence factors
Usually have low GC content
Flanked by repeat units and enzymes for movement
Often adjacent to tRNA genes
Lysogenic phages can _____________ by transduction and incorporation into genome
Transfer pathogenicity genes to bacteria
What is the natural role of restriction enzymes in bacteria
To protect against viruses
Will cleave unmethylated phage DNA while methylated bacterial DNA is protected
CRISPR cas function
CRISPR locus is transcribed forming crRNA
Cas genes expressed to form cas proteins
CRISPR cas complex surveys cells for complementary phage DNA targeting the DNA for destruction
What are the 2 main uses of microbes in the pharmaceutical industry
Produce secondary metabolites with therapeutic properties
Hosts for production of human proteins
What was the best selling pharmaceutical in history
Atorvastatin
Antibiotics interfere with
Peptidoglycan synthesis Membrane integrity DNA synthesis Transcription Folic acid synthesis Ribosome function
Basic principle of industrial fermentation
Convert low cost biomass to high value products that are used in medicine and proteins
Primary metabolite
Product of metabolic process associated with growth
Ex. Fermentation byproducts such as alcohol
Secondary metabolites
Non-growth associated metabolic product, often in stationary phase
Ex. Antibiotics
Systems used for industrial fermentation
Fed-batch reactors
Chemostat
How can molecular biology tools be used to improve microbial strains
Random mutagenesis by chemical/radiation exposure followed by screening for mutations
The best antibiotic producers are kept
Drawbacks: possibility of negative mutations, screening can be difficult
Conditions for penicillin production
Aerobic
Various carbon sources
Sterile
Extracted with acetate
Original penicillin strain used __________ yielded 1mg/L, and now ___________ yields 50g/L
Penicillium notatum
Penicillium chrysogenum
How are bacteria used to express desired proteins for pharmaceuticals
An expression plasmid for the production of the a tagged fusion protein is transformed into the bacteria to be expressed, the cells are lysed and the lysate is purified
Arnold’s discovery of directed enzyme evolution
Start with an enzyme with similar properties to the desired ones
Introduce random mutations into the genes and insert them into bacteria to express mutant enzymes
New enzymes are tested and most effective are selected
New mutations introduced and cycle is repeated
Why are microbial enzymes favoured over chemical synthesis
They produce stereospecific reactions
Biotechnology definition
Use of biological processes or organisms for the production of goods or services
Which bacteria allows for the engineering of transgenic plants
Agrobacterium tumefaciens
What process causes crown galls to form
The pTi plasmid contains T-DNA that is transferred into the plant cell through the action of the vir gene products
The DNA is inserted into the plant genome and expression leads to production of phytohormones and opines that result in plant cell proliferation
The bacteria then reside in the tumour and use the opines as nutrients
What triggers the sensor in the bacterial cell membrane to transcribe vir genes for the dna transfer
Phenolic compounds produces from wounded plants
How can plant genomes be modified using the Ti plasmid
Exchange genes in the Ti plasmid with transgene of interest
How does the glyphosate herbicide work
Inhibits EPSP synthase, which is important for amino acid synthesis
How is Bacillus thuringiensis used to engineer traits into plants
The bacteria produce Bt toxins that are harmful to insects from cry genes
Plants engineered to express Bt toxin to be resistant to insects
What is the refuge strategy used for resistance management of Bt crops
Resistant and vulnerable populations of insects will mate and produce offspring that are less resistant than if both parents were resistant
Grown non Bt crops next to Bt crops so there are populations of resistant and non resistant that can mate
Biofortification
Engineering increased nutrient levels in crop plants
Ex. Golden rice
How is the synthetic Mycoplasma mycoides genome synthesized
Small 1kb fragments that overlap are synthesized and then assembled into larger and larger units to produce a synthetic genome
How is the synthetic M. mycoides genome characterized
Rare restriction enzyme cut sites are added to the genome, then electrophoresis is preformed with the synthetic and the original plasmid
Applications of yeast 1.0
Over express single genes
Complex gene modifications
a DNA assembly host
Capture of large DNA fragments
