Midterm 2 Flashcards
how has the study of bacterial genetics evolved over time?
it went from just looking at pathogens that make us sick (microbes of practical importance) to trying to understand the genetic potential of all microbes
bacterial growth is _______
increase in number of cells NOT size
why are bacteria ideal candidates for genetic research?
only have one chromosome so easy to detect mutations
if you only have one copy of a gene then the effect of a mutation cannot be muted
how do we know bacteria swap their genes?
experiment with prototrophs and auxotrophs (lederberg’s experiment) where they breed and you see the phenotype in the child
auxotroph
mutant strain that has nutitrional needs additional to those of the normal organism
prototroph
can grow on a normal medium
lederberg expt
- met
- pro
- his
plates that dont have those things do the bacteria still grow?
when there is just a single mutation for one of these mutants it is possible that the gene reverts
however some strains that lacked all three nutrients still grew how? it meant that they were exchanging genetic material with other microorganisms in the environment
organization of bacterial genomes
single chromosome and plasmid (if any)
majority is transcribed unlike eukaryotic dna
plasmid copy number is closely regulated in the cell
replicon
bacterial plasmid or chromosome can originate from a single origin of replication
t or f: plasmid copy number in a cell is closely regulated
t
pSym plasmid
nitrogen-fixing nodule formation on legume plant roots
found in rhizobium
pTi plasmid
tumor formation on plants
found in agrobacterium
pTol plasmid
toluene degradation found in
found in pseudomonas putida
pR773 plasmid
arsenic resistance
found in e coli
pWR100 plasmid
entry into host cells
found in shigella flexneri
plasmid incompatibility
plasmids are considered incompatable if they cannot exist stably in a population of bacterial cells
when two plasmids use similar origins of replication, replication control will treat two plasmids as a single plasmid. in doing so, one plasmid loses out and is not replicated
essentially one daughter cell would not get a copy of the second plasmid
mutant
cell or strain possesses a mutation or change to dna seq in comparison to wild type strain
bacteria have _____ of a gene
ONE COPY
how to write name of gene versus protein
gene is italic followed by letter
protein is capitalized first letter and no italics
how can you tell there are changes in genes in bacteria usually?
changes in genes are often visible by changes in phenotype or growth pattern
CFU stands for
colony forming units
how do we detect mutants? Phenotypic selection:
use of a growth medium that will inhibit microbes lacking the desired genes
ex. antibiotic selection
basically use a culture sensitive to antibiotic, grow it in a medium containing antibiotic, and then only mutants would grow on that medium and you can harvest it
which is the most common method of phenotypic selection on mutants?
antibiotic selection
duplicate plates in phenotypic screening
the first plate has full nutritional support
the second plate lacks a particular nutrient
where a colony grows on a fully supported plate but doesnt grow on a partial support plate a mutation has occurred or vice versa depending on if youre looking for auxotrophy or antibiotic resistance
replica plating in phenotypic screening
imprint colonies from master plate onto velvet, then transfer colonies onto replica plates from the velvet. then incubate and grow the replica plates
this is so you can study a high number of colonies
lenski showed ________
mutations can be advantageous to the point that the mutant cells can outgrow the wild type cells
lenski experiment
used e coli and showed their evolution.
culture were allowed to grow and given fresh nutrients and space. cultures after this extended generational time were compared to original cultures that didn’t have these conditions. the ability to grow in culture was enhanced over time
every 75th day they took samples by mixing the ancestral culture and the evolved culture (the one that was left to sit for 75 days). then we plate the sample and see how many grow. compare to the initial ratio of growth
red is evolved white is old. if mutation is better than red would go faster than white
can mutations occure spontaneously or do they have to occur due to selective pressure?
SPONTANEOUS
esther lederberg
used replica plating to show spontaneous mutation without selective pressure
restriction enzymes
recognize specific dna sequence and cut at restriction sites.
molecular scissors
often the cuts are asymmetrical
similar ends of cut dna can be paired together. the paired ends can be tied or ligated by dna ligase
EcoRI restriction site
GAATTC
CTTAAG
cut at first on top and last on bottom
BamHI restriction site
GGATCC
CCTAGG
cut at first on top and last on bottom
HindIII restriction site
AAGCTT
TTCGAA
cut at first on top and last on bottom
SmaI
CCCGGG
GGGCCC
cut down middle
DNA ligase ________ sticky ends
reconnects
restriction enzymes can be used to make _________
recombinant dna molecules
cloning vectors
used to insert recombinant dna molecule into recipient host bacterial cell.
copies of recombinant dna molecules that can be put into a host cell
if we can put a vector inside a bacteria with the recombinant molecule, bacteria multiply and so does the recombinant molecule
ex.
plasmids
phages
cosmids
plasmid
ds dna molecule present in a lot of bacteria
plasmid cloning vectors first used in 1970s by cohen at stanford
how did cohen use plasmids as cloning vectors?
he cut fragments from two plasmids carrying antibiotic resistance genes with the same restriction enzyme followed by ligation with dna ligase
after inserting the recombinant plasmid into bacteria the strain exhibited traits from both plasmids
how do we know that a plasmid vector worked?
we need to choose a gene like antibiotic resistance to a specific one so that you can see that it grows in the presence of the antibiotic
then you know your gene of choice is there
limitations with plasmid vector insertion:
1) the recipient dna molecule can religate to itself after being cut instead of recombining with the plasmid insert. can combat this with phosphatase
2) the plasmid insert can insert itself into the DNA in either direction (left to right or right to left) and we cnat control that
properties of a plasmid we need to have
- origin of replication
- selectable marker gene (so that you can tell where your plasmid is)
- multiple cloning site (spots for restriction enzyme to cut)
- small size
-high copy number (you would have multiple copies of your gene of interest and more protein from your gene of interest as a result)
selectable marker gene
a gene that allows us to identify which bacteria have the plasmid
ex. antibiotic resistance
copy number
the number of copies of a particular gene in the genotype of an individual.
blue white selection (x gal system)
you dont need a selectable marker to tell where the plasmid is contained.
we use a special strain of bacteria that make lacZ omega. the plasmid insertion site is in the middle of the lacZ gene.
if plasmid is inserted into the gene then it will be disrupted so no lacZ alpha will be made. when this happens beta galactosidase wont be made. the colonies will appear white instead of blue
phage vectors
mix viral DNA with a vector of interest
in blue white selection, _______ colonies contain your gene of interest
white
what does lacZ gene do?
codes for lacZ alpha which complexes to form beta galactosidase and brings blue color
what is the advantage of using a phage vector rather than a plasmid?
no selection markers required
phages can carry much larger DNA fragments UP TO 20 KB
how many DNA fragments can a plasmid hold?
15 kb
cosmid
mix of a plasmid and a phage
phage genome that omit nearly all of the phage dna leaving room for the fragment
- only the critical phage cos packaging recognition sites are left
- other elements include multiple cloning site and an antibiotic selection marker
can take 35-45 kb fragments
how many fragments can a phage hold?
24 kb
how many fragments can a cosmid hold?
45 kb
genome
complete set of organisms dna
genetics
study of individual genes and their functions
genomics
collective properties and quantification of different genes
walter gilbert developed _______
a chemical degradation method for genome sequencing
Sanger developed ________
dideoxy sequencing
Sanger dideoxy sequencing requires 3 steps, what are they?
1) cloning of the gene fragment to be sequenced (use this clone as a template for step 2)
2) DNA synthesis
3) electrophoresis
DNA polymerases require a ________ to continue DNA synthesis. This is the basis for _________
free 3’ hydroxyl group
sanger sequencing
T or F: RNA is less stable than DNA
true
phosphodiester linkage between two ribonucleotides can be broken by ________. whereas the linkage between two deoxyribonucleotides are more stable
alkaline hydrolysis
how do we end the sanger seq method?
place dideoxynucleotides (lack the free 3’ OH group) into the DNA synthesis mixture. when it gets up to this poin the process is terminated with a distinct labeled endpoint nucleotide
Sanger method
1) prepare four DNA polymerization rxn mixtures containing the template, complementary oligonucleotides primer, DNA polymerase, and dGTP, dATP, dTTP, and P-dCTP
2) Add a different ddNTP to each of hte four reaction mixtures to terminate the elongation reaction
3) Lead the reaction products on the polyacrylamide gel, then visualize the bands by exposure to x-ray film
where does the primer bind in the Sanger method?
3’ hydroxyl group
we use ______ labels in sanger seq
fluorescent
much safer cheaper and easier than radioactive
names for sanger seq
chain termination
dideoxy method
primer walking
way to obtain longer sequences using repeated rounds of sequencing with primers comlpetementary to the end of the last seq of dna
whole genome shotgun sequencing
physically shear the dna and sequenced. computer aligns them and gives you whole sequence.
may need 10x total genome length to do this successfully
get genome in one shot
pyrosequencing
- detects addition of a nucleotide to the end of a syntehsized strand of DNA by production of light
- faster and cheaper than Sanger method
one dNTP is added complementary to the template strand. if it is the polymerase will bring it over. if it is complementary the pyrophosphate will be released and will react with APS already in the mixture. this produces light and we will know which nucleotide was complementary.
next generation sequencing (high throughput methods)
can produce small fragments (25-500bp). requires a lot of computing is the downside
bioinformatics
analysis of large data sets of sequencing data
predict where a protein will start or end by identifying open reading frames (orfs)
open reading frames
ORFs allow us to better determine the start and stop points for a given gene
functional genomics
find the role of unknown genes that are newly identified through bioinformatics
genomic library
collection of cloned dna fragments that represent entire genome of organism
the number of cloned fragments needed to encompass an entire genome can be determined by the formula
N = ln (1-P) / ln (1-f)
the number of cloned fragments needed to encompass the entire genome of an organism
N = number of clones required P = the desired probability of generating a complete library F = the average size of each clone divided by the total size of the genome
pathogenesis
processes used by pathogens to produce disease
key aspects of bacterial pathogenesis:
attach to the host tissue and gain access
damage tissues to obtain nutrients and replicate
avoid host defense
_________ is a principal feature in pathogen evolution
genetic mobility
virulence factors
pathogen products that enhance their ability to cause disease
gonorrhea virulence factors
fimbriae –> attachment and evasion
IgA protease –> destruction of IgA antibody
LOS (endotoxin) –> evokes inflammatory damage
steptococcus pyrogenes virulence factors
capsule –> anti phagocytic
M protein –> prevents binding by antibody
hyaluronidase –> degrades connective tissue
steptokinase –> degrades fibrin clots
escherichia coli virulence factors
intimin –> attachment
Tir –> receptor for attachment
Type III secretion system –> injects tir for attachment
Shiga toxins –> stops translation in host cells
heliobacter pylori virulence factors
urease –> neutralizes gastric acids
vacuolating cytotoxin A (VacA) –> host cell death, inflammation
flagella –> transport through mucus
cytotoxin-associated antigen (CagA) –> disrupts host cell cytoskeleton
CagA type IV secretion system –> injects CagA
bordetella pertussis virulence factors
fimbriae–> attachment
pertussis toxin –> disrupts cell ion balance
invasive adenylate cyclase –> disrupts cell ion balance
fimbriae
specialized pili with adhesive tip
both host cells and bacterial cells have negative charge on their surfaces, so they repel each other. we believe the fimbriae spread the distance of repulsion
attachment factors
factors pathogen uses to attach to host
bacterial toxins
endotoxins and exotoxins
endotoxins
endotoxins are a part of the cell wall structure and induce inflammatory responses
LPS on gram negative cells
LTA on gram positive cells
exotoxins
released outside of the producing cell
a-b toxins
cytotoxins
superantigens
a-b toxins
type of exotoxin, b subunit binds to host celll receptor. a subunit has a negative action inside the cell
a subunit has the toxic enzymatic activity
b subunit binds cell receptor
cytotoxins
type of exotoxin, toxins that directly act on host cells
cytolysins specifically act on plasma membrane
superantigens
nonspecifically stimulate t cells to secrete large amounts of cytokines
LPS endotoxin
most common endotoxin
has 3 parts:
1) o antigen (often strain specific can be used for serotyping)
2) core polysaccharide
3) lipid A (inflammatory inducing portion)
o antigen in LPS
repeating units of polysaccharide that are strain specific. target of immune response. used for serotyping
used to evade the immune system
core polysaccharide in LPS
various sugars with side chains
genus or species-specific
lipid A in LPS
innermost component of lps
hydrophobic nature allows it to anchor the LPS to the outer membrane
lipid component of an endotoxin responsible for the toxicity of gram negative bacteria
unusual fatty acids
inflammatory component
WOULDN’T BE TOXIC WITHOUT LIPID A
some lps don’t have ______
o-antigen
helps them evade immune system
LPA endotoxin
similar to lps but on gram positive
capable of inducing inflammation
corynebacterium diptheria a-b toxin
a subunit inactivates EF2, abolishes translation so proteins are not synthesized
1) b binds receptor,
2) ab complex enters cell through endocytosis, 3)acidification occurs dissociating b subint from receptor and channel formation
4) a subunit enters cytoplasm through channel
5) a subunit inactivates translation factor EF2 abolishing translation
shiga a-b toxin in ecoli
animals can carry these strains of bacteria without becoming ill
diptheria toxin causes ______
shut down of protein synthesis
pertussis toxin and cholera toxin cause ______
h2O and ion imbalance
SNARE proteins
used to release neurotransmitters in host cells.
some ab neurotoxins cleave the snare proteins preventing neurotransmitter release
botulism toxin prevents _______
muscle contraction. prevents acetylcholine release from snare protein
flaccid paralyiss
tetanus toxin produces __________
continuous muscle contraction. acetylcholine secreted.
spastic paralysis
how does botulism toxin work?
the a subunit attacks snap25 breaking the connection between synaptobrevin and syntaxin which renders the vesicle with ac inside unable to reach the plasma membrane and release the ac to the muscle cell receptors outside of the cell
how does acetylcholine get released in a normal motor neuron?
ac enters motor neuron. synaptobrevin interacts with snap-25 interacts with syntaxin which leads the vesicle to its correct location to release ac outside of the cell to the muscle cell receptors.
how does tetanus toxin work?
tetanus toxin enters the motor neuron in a similar way to glycine and gaba. it then uses the motor neuron to gain entry into CNS in order to go to the inhibitory neuron. inside, the a and b subunit separate and the a subunit breaks the synaptobravin, breaking the chain and glycine and gaba can no longer be released.
cytolysins
work on plasma membrane of cells
membrane damaging toxins
more than 1/3 of all bacterial protein toxins
botulism toxin facts
300,000 times more toxic than snake venom. protease like enzyme that can break proteins
tetanus toxin facts
second most toxic
Ld50 = amount that is required to kill 50% = 3-5 ng/kg body weight
hemolysins
example of cytolysins that break red blood cells
alpha, beta, and gamma hemolysis
alpha hemolysis produces a zone with a greenish tinge because itsin the middle
beta hemolyiss produces a clear zone because they dont break
gamma hemolysis produces a red zone because everything lyses
alpha, beta, and gamma hemolysis
alpha hemolysis produces a zone with a greenish tinge because itsin the middle partial hemolysis occurs
beta hemolyiss produces a clear zone because everything lyses
gamma hemolysis produces a red zone because nothing lyses
cytolysins that attack eukaryotic cells bilayer membranes by dissolving their phospholipids
c perfringens alpha toxin, s aureus beta toxin
cytolysins that attack the hydrophobic regions of membranes and act like detergents
g toxins from straphylococcus aureus, bacillus subtilis toxin and the cytolysin from pesudomonas aeruginosa
cytolisins that are pore forming toxins
perfringiolysin o from clostridium perfringensbacteria, hemolysin from ecoli
3 types of cytolysin
1) attack bilayer by dissolving phospholipids
2) attack hydrophobic regions of membranes and act like detergents
3) pore forming
PFCs
pore forming cytolysins. make up largest portion of cytolysins
produced by bacteria fungi and plants
form pores at the target cell membrane
lethal effects performed by causing influx and outflux disorder
what is the lethal effect of pfc due to
influx and outflux disorder
cholesterol dependent cytolysin (CDC)
exist in many gram positive bacteria. require the presence of cholesterols on target cell membranes. large poresize (25-30 nm) due to oligomeric process
superantigens
exotoxins that act on helper t cells (CD4)
cause massive release of nonspecific cytokines and induce a system inflammatory response (normally its fewer t cells produced that are specific)
cytokines are known as ___________
immunomodulators
toxic shock syndrome and food poisoning (enterotoxins) are examples of ________
super antigens
enterotoxigenic
means microorganisms that produce enterotoxins, a protein toxin that is produced by strains of s aureus
can superantigens and endotoxins act together?
yes! syngeristically and induce shock and death,
each is capable of inducing inflammation individually
strep pyogenes facts
primary residence is the pharynx
opportunistic pathogen that produces a variety of diseases
it possess numerous virulence factors
-removing one factor doesn’t prevent pathogenicity
how does step pyogenes access host tissues
surface components attach to host fibronectin and collagen
how does strep pyogenes overcome host defenses
capsules prevent phagocytosis, capsules inhibit antibody/complement binding
several methods for preventing complement activation are present
some strains of strep pyogenes can induce ___________
post infection siculi
glomerulonephritis, scarlet fever, rheumatic heart disease, cellulitis, necrotizing fascitis
M protein in step pyogenes
a virulence factor produced by cert species of streptococcus
anti phagocytic and is major virulence factor
150 types
antibodies against one does not protect against another M protein, therefore reinfection can occur
matches with heart muscle myosin protein so if we dont treat within 7 days (usually we do) then antibodies would bind to our heart muscle
horizontal gene transfer
evolution by quantum leaps
antibacterial resistance- highly unlikely to occur from random mutations over time
rapid swapping of genetic information including antibiotic resistence genes
examples of horizontal gene transfer
- conjugation
- transposable elements
- plasmids
- temperate (lysogenic) phages
Microbes in _______ are foundation of microbio
pure culture
BUT in real life microbes dont exist in pur culture.
number of culturable microbes is very low. so we need ways to study microbes that cant be grown within the context of their natural habitats
ecosystem
community of living organisms in conjunction with the nonliving components of their environment interacting as a system
includes biotic and abiotic factors
nonliving can be water, air, soil
guilds
members of communities that can be put into functional groups very similar to each other
niche
specific functional role of an organism within an ecosystem
biofilms
groups of layer of microbes ona surgave that interacft with and support each other
can be found in nature often but can be of practical importance to humans who want to rid a surface of microbes
how are biofilms formed?
begins with appendaged bacteria forming the primary layer of the surface.
secondary colonizers then join the biofilm
microbes secrete exopolysaccharide (EPS) which protects the biofilm but also water-filled channels for transport of nutrients and waste
EPS colanic acid
important to biofilm formation in e coli k12 – it cant without it
enrichment cultures
when microbes can be grown in lab setting, they may grow slowly or may be rare in mixed population
enrichment methods promote growth of desired microbes over undesired cells by adding nitrogen, oxygen air etc. add soil sample and incubate
can test quality of soil.
cultivation independent technique
direct sequencing without growth or cloning of the bacteria
extract dna from environmental sample. followed by pcr and sequencing. sequencing compared to databases of known sequences for identification
cultivation independent tecniques: metagenomics
also known as community genomics
dna from environmental sample is used to construct a genomic library
genes from this community library can be identified screened and compared to other areas
we can either directly sequence or look at functional analysis
marine ecosystems
98% of oceans biomass is microbial
most of these microbes exist in oligotrophy or using nutrients at very low concentrations
overfeeding marine microbes can lead quickly to anoxic water states (dead zone formation)
oligotroph
chracterized by slow growth and low metabolic rate
direct link between _______ and dead zones
agricultural activity
dead zones
areas without enough oxygen to support much eukaryal life
phytoplankton
microscopic marine plants
provide food for sea creatures
account for about half of all photosynthetic activity on earth
their proliferation increases energy and organic carbon in the marine water
N and P from the agricultural run off increase the phytoplankton
how many cells in how much water
10^8 cells in 1 mL water
surface zone
populated by phytoplankton (photosynthetic microbes) contribute to the oxygenation of water
primary production is done via the phytoplankton
carbon and energy are distributed through other levels by zooplanlton feeding on phytoplankton and through viral mediated lysis
dark mid water zone
phytoplankton production drifts downwards to feed zooplankton in the lower levels
viral lysis of phytoplankton helps to release nturients for heterotrophic microbes
deep sea zone
average depth 3,500 m
deepest spot known 11,000 feet
pressure is 1000 at sea level
piezophiles are microbes that can withstand and sometimes require intense pressure to grow
____ of primary production reaches the seafloor
1%
anoxic cold conditions make it unlikely to biodegrade
it forms rich sediment on the sea floor
however few microbes can survive intense conditions
viruses in oceans
probably 10 times more viruses exist in oceans than microbes (bacteria and archaeons)
little is known about these viruses their reproduction cycles and their ecological impacts
viruses of cyanobacteria dominate surface zones
viruses most likely liberate nutrients from cyanobacteria making them available for heterotrophic microbes in deeper waters
how do we cultivate oligotrophic microbes from seawater?
difficult due to heterogeneity of seawater
what do the mcirobes really need in order to grow outside of ocean.
we use dilution to extinction method
dilution to extinction method
inoculation of very diluted seawater samples in autoclaved seawater followed by incubation
the tubes that show growth get subcultured in a larger volume so that cells can be harvested by centrifugation for analysis
biomes
categories of ecosystems based on vegetation characteristics
- plants are dominant primary producers
- soils and soil quality are critical
rhizosphere
- the area of soil immediately surrounding plant roots possessing large amounts of organic carbon
- because of excess carbon the area often has higher numbers of microbes than bulk soil
bioremediation
a process of cleaning contaminants using microbes
xenobiotics do not occur naturally. microbes have not evolved the ability to degrade them efficiently, natural substances can be added to induce degradation
biodegradation is slow and often limited by lack of oxygen
biostimulation
add missing oxygen and limiting nutrients (N and P) to increase activity of resident microbes
bioaugmentation
addition of bacteria known to degrade the contaminant to a contaminated area
