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BIO 315 > Midterm 2 > Flashcards

Midterm 2 Flashcards

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1
Q

how has the study of bacterial genetics evolved over time?

A

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

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2
Q

bacterial growth is _______

A

increase in number of cells NOT size

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3
Q

why are bacteria ideal candidates for genetic research?

A

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

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4
Q

how do we know bacteria swap their genes?

A

experiment with prototrophs and auxotrophs (lederberg’s experiment) where they breed and you see the phenotype in the child

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5
Q

auxotroph

A

mutant strain that has nutitrional needs additional to those of the normal organism

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6
Q

prototroph

A

can grow on a normal medium

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7
Q

lederberg expt

A
  • 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

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8
Q

organization of bacterial genomes

A

single chromosome and plasmid (if any)

majority is transcribed unlike eukaryotic dna

plasmid copy number is closely regulated in the cell

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9
Q

replicon

A

bacterial plasmid or chromosome can originate from a single origin of replication

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10
Q

t or f: plasmid copy number in a cell is closely regulated

A

t

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11
Q

pSym plasmid

A

nitrogen-fixing nodule formation on legume plant roots

found in rhizobium

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12
Q

pTi plasmid

A

tumor formation on plants

found in agrobacterium

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13
Q

pTol plasmid

A

toluene degradation found in

found in pseudomonas putida

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14
Q

pR773 plasmid

A

arsenic resistance

found in e coli

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15
Q

pWR100 plasmid

A

entry into host cells

found in shigella flexneri

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16
Q

plasmid incompatibility

A

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

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17
Q

mutant

A

cell or strain possesses a mutation or change to dna seq in comparison to wild type strain

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18
Q

bacteria have _____ of a gene

A

ONE COPY

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19
Q

how to write name of gene versus protein

A

gene is italic followed by letter

protein is capitalized first letter and no italics

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20
Q

how can you tell there are changes in genes in bacteria usually?

A

changes in genes are often visible by changes in phenotype or growth pattern

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21
Q

CFU stands for

A

colony forming units

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22
Q

how do we detect mutants? Phenotypic selection:

A

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

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23
Q

which is the most common method of phenotypic selection on mutants?

A

antibiotic selection

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24
Q

duplicate plates in phenotypic screening

A

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

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25
Q

replica plating in phenotypic screening

A

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

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26
Q

lenski showed ________

A

mutations can be advantageous to the point that the mutant cells can outgrow the wild type cells

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27
Q

lenski experiment

A

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

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28
Q

can mutations occure spontaneously or do they have to occur due to selective pressure?

A

SPONTANEOUS

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29
Q

esther lederberg

A

used replica plating to show spontaneous mutation without selective pressure

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30
Q

restriction enzymes

A

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

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31
Q

EcoRI restriction site

A

GAATTC
CTTAAG

cut at first on top and last on bottom

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32
Q

BamHI restriction site

A

GGATCC
CCTAGG

cut at first on top and last on bottom

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33
Q

HindIII restriction site

A

AAGCTT
TTCGAA

cut at first on top and last on bottom

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34
Q

SmaI

A

CCCGGG
GGGCCC

cut down middle

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35
Q

DNA ligase ________ sticky ends

A

reconnects

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36
Q

restriction enzymes can be used to make _________

A

recombinant dna molecules

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37
Q

cloning vectors

A

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

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38
Q

plasmid

A

ds dna molecule present in a lot of bacteria

plasmid cloning vectors first used in 1970s by cohen at stanford

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39
Q

how did cohen use plasmids as cloning vectors?

A

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

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40
Q

how do we know that a plasmid vector worked?

A

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

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41
Q

limitations with plasmid vector insertion:

A

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

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42
Q

properties of a plasmid we need to have

A
  • 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)

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43
Q

selectable marker gene

A

a gene that allows us to identify which bacteria have the plasmid

ex. antibiotic resistance

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44
Q

copy number

A

the number of copies of a particular gene in the genotype of an individual.

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45
Q

blue white selection (x gal system)

A

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

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46
Q

phage vectors

A

mix viral DNA with a vector of interest

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47
Q

in blue white selection, _______ colonies contain your gene of interest

A

white

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48
Q

what does lacZ gene do?

A

codes for lacZ alpha which complexes to form beta galactosidase and brings blue color

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49
Q

what is the advantage of using a phage vector rather than a plasmid?

A

no selection markers required

phages can carry much larger DNA fragments UP TO 20 KB

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50
Q

how many DNA fragments can a plasmid hold?

A

15 kb

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51
Q

cosmid

A

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

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52
Q

how many fragments can a phage hold?

A

24 kb

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53
Q

how many fragments can a cosmid hold?

A

45 kb

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54
Q

genome

A

complete set of organisms dna

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55
Q

genetics

A

study of individual genes and their functions

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56
Q

genomics

A

collective properties and quantification of different genes

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57
Q

walter gilbert developed _______

A

a chemical degradation method for genome sequencing

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58
Q

Sanger developed ________

A

dideoxy sequencing

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59
Q

Sanger dideoxy sequencing requires 3 steps, what are they?

A

1) cloning of the gene fragment to be sequenced (use this clone as a template for step 2)
2) DNA synthesis
3) electrophoresis

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60
Q

DNA polymerases require a ________ to continue DNA synthesis. This is the basis for _________

A

free 3’ hydroxyl group

sanger sequencing

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61
Q

T or F: RNA is less stable than DNA

A

true

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62
Q

phosphodiester linkage between two ribonucleotides can be broken by ________. whereas the linkage between two deoxyribonucleotides are more stable

A

alkaline hydrolysis

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63
Q

how do we end the sanger seq method?

A

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

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64
Q

Sanger method

A

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

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65
Q

where does the primer bind in the Sanger method?

A

3’ hydroxyl group

66
Q

we use ______ labels in sanger seq

A

fluorescent

much safer cheaper and easier than radioactive

67
Q

names for sanger seq

A

chain termination

dideoxy method

68
Q

primer walking

A

way to obtain longer sequences using repeated rounds of sequencing with primers comlpetementary to the end of the last seq of dna

69
Q

whole genome shotgun sequencing

A

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

70
Q

pyrosequencing

A
  • 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.

71
Q

next generation sequencing (high throughput methods)

A

can produce small fragments (25-500bp). requires a lot of computing is the downside

72
Q

bioinformatics

A

analysis of large data sets of sequencing data

predict where a protein will start or end by identifying open reading frames (orfs)

73
Q

open reading frames

A

ORFs allow us to better determine the start and stop points for a given gene

74
Q

functional genomics

A

find the role of unknown genes that are newly identified through bioinformatics

75
Q

genomic library

A

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

76
Q

N = ln (1-P) / ln (1-f)

A

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
77
Q

pathogenesis

A

processes used by pathogens to produce disease

78
Q

key aspects of bacterial pathogenesis:

A

attach to the host tissue and gain access

damage tissues to obtain nutrients and replicate

avoid host defense

79
Q

_________ is a principal feature in pathogen evolution

A

genetic mobility

80
Q

virulence factors

A

pathogen products that enhance their ability to cause disease

81
Q

gonorrhea virulence factors

A

fimbriae –> attachment and evasion

IgA protease –> destruction of IgA antibody

LOS (endotoxin) –> evokes inflammatory damage

82
Q

steptococcus pyrogenes virulence factors

A

capsule –> anti phagocytic

M protein –> prevents binding by antibody

hyaluronidase –> degrades connective tissue

steptokinase –> degrades fibrin clots

83
Q

escherichia coli virulence factors

A

intimin –> attachment

Tir –> receptor for attachment

Type III secretion system –> injects tir for attachment

Shiga toxins –> stops translation in host cells

84
Q

heliobacter pylori virulence factors

A

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

85
Q

bordetella pertussis virulence factors

A

fimbriae–> attachment

pertussis toxin –> disrupts cell ion balance

invasive adenylate cyclase –> disrupts cell ion balance

86
Q

fimbriae

A

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

87
Q

attachment factors

A

factors pathogen uses to attach to host

88
Q

bacterial toxins

A

endotoxins and exotoxins

89
Q

endotoxins

A

endotoxins are a part of the cell wall structure and induce inflammatory responses

LPS on gram negative cells

LTA on gram positive cells

90
Q

exotoxins

A

released outside of the producing cell

a-b toxins
cytotoxins
superantigens

91
Q

a-b toxins

A

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

92
Q

cytotoxins

A

type of exotoxin, toxins that directly act on host cells

cytolysins specifically act on plasma membrane

93
Q

superantigens

A

nonspecifically stimulate t cells to secrete large amounts of cytokines

94
Q

LPS endotoxin

A

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)

95
Q

o antigen in LPS

A

repeating units of polysaccharide that are strain specific. target of immune response. used for serotyping

used to evade the immune system

96
Q

core polysaccharide in LPS

A

various sugars with side chains

genus or species-specific

97
Q

lipid A in LPS

A

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

98
Q

some lps don’t have ______

A

o-antigen

helps them evade immune system

99
Q

LPA endotoxin

A

similar to lps but on gram positive

capable of inducing inflammation

100
Q

corynebacterium diptheria a-b toxin

A

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

101
Q

shiga a-b toxin in ecoli

A

animals can carry these strains of bacteria without becoming ill

102
Q

diptheria toxin causes ______

A

shut down of protein synthesis

103
Q

pertussis toxin and cholera toxin cause ______

A

h2O and ion imbalance

104
Q

SNARE proteins

A

used to release neurotransmitters in host cells.

some ab neurotoxins cleave the snare proteins preventing neurotransmitter release

105
Q

botulism toxin prevents _______

A

muscle contraction. prevents acetylcholine release from snare protein

flaccid paralyiss

106
Q

tetanus toxin produces __________

A

continuous muscle contraction. acetylcholine secreted.

spastic paralysis

107
Q

how does botulism toxin work?

A

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

108
Q

how does acetylcholine get released in a normal motor neuron?

A

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.

109
Q

how does tetanus toxin work?

A

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.

110
Q

cytolysins

A

work on plasma membrane of cells

membrane damaging toxins

more than 1/3 of all bacterial protein toxins

111
Q

botulism toxin facts

A

300,000 times more toxic than snake venom. protease like enzyme that can break proteins

112
Q

tetanus toxin facts

A

second most toxic

Ld50 = amount that is required to kill 50% = 3-5 ng/kg body weight

113
Q

hemolysins

A

example of cytolysins that break red blood cells

114
Q

alpha, beta, and gamma hemolysis

A

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

115
Q

alpha, beta, and gamma hemolysis

A

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

116
Q

cytolysins that attack eukaryotic cells bilayer membranes by dissolving their phospholipids

A

c perfringens alpha toxin, s aureus beta toxin

117
Q

cytolysins that attack the hydrophobic regions of membranes and act like detergents

A

g toxins from straphylococcus aureus, bacillus subtilis toxin and the cytolysin from pesudomonas aeruginosa

118
Q

cytolisins that are pore forming toxins

A

perfringiolysin o from clostridium perfringensbacteria, hemolysin from ecoli

119
Q

3 types of cytolysin

A

1) attack bilayer by dissolving phospholipids
2) attack hydrophobic regions of membranes and act like detergents
3) pore forming

120
Q

PFCs

A

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

121
Q

what is the lethal effect of pfc due to

A

influx and outflux disorder

122
Q

cholesterol dependent cytolysin (CDC)

A

exist in many gram positive bacteria. require the presence of cholesterols on target cell membranes. large poresize (25-30 nm) due to oligomeric process

123
Q

superantigens

A

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)

124
Q

cytokines are known as ___________

A

immunomodulators

125
Q

toxic shock syndrome and food poisoning (enterotoxins) are examples of ________

A

super antigens

126
Q

enterotoxigenic

A

means microorganisms that produce enterotoxins, a protein toxin that is produced by strains of s aureus

127
Q

can superantigens and endotoxins act together?

A

yes! syngeristically and induce shock and death,

each is capable of inducing inflammation individually

128
Q

strep pyogenes facts

A

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

129
Q

how does step pyogenes access host tissues

A

surface components attach to host fibronectin and collagen

130
Q

how does strep pyogenes overcome host defenses

A

capsules prevent phagocytosis, capsules inhibit antibody/complement binding

several methods for preventing complement activation are present

131
Q

some strains of strep pyogenes can induce ___________

A

post infection siculi

glomerulonephritis, scarlet fever, rheumatic heart disease, cellulitis, necrotizing fascitis

132
Q

M protein in step pyogenes

A

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

133
Q

horizontal gene transfer

A

evolution by quantum leaps

antibacterial resistance- highly unlikely to occur from random mutations over time

rapid swapping of genetic information including antibiotic resistence genes

134
Q

examples of horizontal gene transfer

A
  • conjugation
  • transposable elements
  • plasmids
  • temperate (lysogenic) phages
135
Q

Microbes in _______ are foundation of microbio

A

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

136
Q

ecosystem

A

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

137
Q

guilds

A

members of communities that can be put into functional groups very similar to each other

138
Q

niche

A

specific functional role of an organism within an ecosystem

139
Q

biofilms

A

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

140
Q

how are biofilms formed?

A

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

141
Q

EPS colanic acid

A

important to biofilm formation in e coli k12 – it cant without it

142
Q

enrichment cultures

A

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.

143
Q

cultivation independent technique

A

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

144
Q

cultivation independent tecniques: metagenomics

A

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

145
Q

marine ecosystems

A

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)

146
Q

oligotroph

A

chracterized by slow growth and low metabolic rate

147
Q

direct link between _______ and dead zones

A

agricultural activity

148
Q

dead zones

A

areas without enough oxygen to support much eukaryal life

149
Q

phytoplankton

A

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

150
Q

how many cells in how much water

A

10^8 cells in 1 mL water

151
Q

surface zone

A

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

152
Q

dark mid water zone

A

phytoplankton production drifts downwards to feed zooplankton in the lower levels

viral lysis of phytoplankton helps to release nturients for heterotrophic microbes

153
Q

deep sea zone

A

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

154
Q

____ of primary production reaches the seafloor

A

1%

anoxic cold conditions make it unlikely to biodegrade

it forms rich sediment on the sea floor

however few microbes can survive intense conditions

155
Q

viruses in oceans

A

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

156
Q

how do we cultivate oligotrophic microbes from seawater?

A

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

157
Q

dilution to extinction method

A

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

158
Q

biomes

A

categories of ecosystems based on vegetation characteristics

  • plants are dominant primary producers
  • soils and soil quality are critical
159
Q

rhizosphere

A
  • 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
160
Q

bioremediation

A

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

161
Q

biostimulation

A

add missing oxygen and limiting nutrients (N and P) to increase activity of resident microbes

162
Q

bioaugmentation

A

addition of bacteria known to degrade the contaminant to a contaminated area

BIO 315 (4 decks)

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