TEST 2 Flashcards
decontamination
treatment of object to make safe to handle
disinfection
targets death of pathogens not all microbes or endospores
sterilization
kills all microbes and viruses
heat sterilization
wet heat better than dry heat
pastuerization
-uses heat to reduce microbial load but not all microbes
-kills pathogens
UV sterilization
used to sterilize food surgical tools lab equipment
D10 value
ionizing radiation needed to reduce bacteria 10 fold
filter sterilization
-used on heat sensitve liquids and gases
-pores are to small for microbes to go through
membrane filters
common for liquids
nucleopore filters
thin irradiated film used to visualize microbes for electron microscopy
-cidal
kills microbes
-static
stops growth of microbes
sanitizers
reduce microbial number but doesnt sterilize
antiseptic
kills/inhibits growth of microbes nontoxic to living tissue
purines
G A 2 ringed
pyrimidines
T C 1 ring
GC bond
3 h bonds
TA bond
2 h bonds
-lytic
lyses all cells destroying even dead cells
negative supercoiling
coils increase becoming tightly wound
positive supercoiling
coils decrease becoming loosely wound
topoisomerase
inserts and removes supercoiles
DNA gyrase
introduces supercoils via double strand breaks
3 rnas and functions
mRNA- carries info of DNA gene to ribosome
tRNA-brings amino acid to mRNA converting it into amino acid sequence
rRNA-ribosomal rna, ribosome component
3 stages of biologic information flow
replication
transcription
translation
eukaryotic transcription
1 gene to 1 mRNA
mRNA exported to cytosol
prokaryotic transcription
several genes to 1 mRNA
transcription and translation in cytosol
plasmids
circular and double stranded
transposable elements
-segments of DNA inserted into other DNA molecules
-moves site to site
R plasmids
grants antibiotic resistance to bacteria
bacteriocins
proteins killing closely related species of same species
can be encoded by plasmids
rhizobia
plasmid encoded function is to fix nitrogen
semiconservative dna replication
2 of the 4 DNA strands in the new cells are the DNA strands from the original cell
DNA pol Family A
-DNA repair and Okazaki fragment maturation
-exonuclease activity
DNA pol Family B
-Main polymerase in eukaryotes
-exonuclease activity
DNA pol Family C
-Main polymerase in bacteria
-exonuclease activity
DNA pol Family X
-monomeric
-fills gaps for DNA repair
DNA pol Family Y
-low fidelity, translesion synthesis
-no exonuclease activity
DNA pol Family RT
-reverse transcriptase
-uses RNA to make DNA to produce more RNAs
Archaeal DNA polymerases
Family B&D
PolB3 present in all archaea
PolB1/2 in some archaea
virus DNA polmerases
-DNA viruses utilize host DNA polymerase for proliferation
DNA helicase
unwinds DNA for replication
Prokaryote vs Eukaryote origin of replication
-prokaryotes-have one origin of replication on circular chromosome
-eukaryotes- have multiple origins of replications on 1 linear chromosome out of all of the linear chromosomes
DNA ligase
seals the nicks in the DNA backbone after primers have been removed and filled with DNA
leading strand
continuous
5>3 strand synthesis
1 primer
lagging strand
discontinuous
5>3 strand synthesis
multiple primers
replisome
large replication complex of proteins
DNA vs RNA
-DNA-
deoxyribose-2’ H bond
thymine
-RNA-
ribose-2’ OH bond
uracil
primosome
helicase and primase subcomplex within the replisome
holoenzyme
RNA polymerase complex of 5 proteins
sigma factor
recognizes promoter sequences on DNA
pribnow box
TATA
promoter sequence
number of Eukaryotic RNA polymerases
3
number of archaeal RNA polymerases
1
B recognition element
-BRE for short
-upstream of TATA box
-the binding of the transcription factor to the BRE allows for initation of RNA transcription
termination method
inverted repeats of TA, creates loops of RNA that fall off of DNA strand
exons
gene coding element of RNA
introns
nongene coding element of RNA
splicing
removal of introns
in eukaryotes this happens in nucleus
capping
-addition of methylated guanosine to end of mRNA, other end will start translation
-needed for translation
3 protein functions
-catalysts-enzymes
-structure-integral membrane proteins
-regulatory-DNA binding
peptide bond
carboxylic acid bonded to nitrogen
degenerate code
64 codons to 20 amino acids
shine-delgaro sequence
ensures proper reading frame in bacteria and archaea
open reading frame
start codon- AUG
followed by a number of codons then the stop codon
Subunits of ribosomes in prokaryotes
-has 30s and 50s subunits to from 70s subunits
-needs GTP
chaperones
catalyze molecular folding of proteins
found in all domains
heat shock proteins
attempts to refold partially denatured proteins for reuse before proteases destroy them
cold shock proteins
prevent secondary structure formation in RNA or refold cold-sensitive proteins
Translocases
transport proteins into or through bacterial & archaeal membranes
translocation systems
sec and tat system
sec system
unfolded proteins to be exported are recognized by
-SecA (periplasmic)
-signal recognition particle (SRP;
for membrane-inserted proteins
tat system
TatBC recognizes signal sequence, carries folded protein to TatA
membrane transporter
common DNA binding protein characteristic
binds to major groove
homodimeric
structure of DNA binding protein
helix turn helix
zinc finger
DNA-protein binding technique utilizing a zinc ion
leucine zipper
DNA-protein binding technique
-Contains regularly spaced leucine residues
-Hold two recognition helices in the correct orientation to bind DNA
transcription factor
Proteins that control the rate of transcription by binding to specific DNA
activator protein
turns on transcription
repressor protein
turns off transcription
Allosteric proteins
Conformation altered when effector
molecule binds
Effectors
Small molecules that control binding of activators and repressors
Inducers
turn on transcription
corepressors
turns of transcription
Enzyme repression
-preventing the synthesis of an
enzyme unless the product is absent from culture medium
-excess of product decreases enzyme
synthesis
Dual functionality
used as positive and negative control
two component regulatory system components
Sensor kinase
Response regulator
how does the 2 component regulatory system terminate the responce?
A phosphatase removes phosphate from the response regulator
sensor kinase
-detects environmental signals and autophosphorylates at
specific histidine residue
-integral to cell membrane
response regulator
-DNA-binding protein that regulates transcription
-receives phosphate from sensor kinase
-in cytoplasm
what is this picture depicting?
-This picture shows the 2 component regulatory system
-the sensor kinase gives its phosphate to the response regulator which then blocks transcription of certain genes
Methyl-accepting chemotaxis proteins (MCPs)
proteins that sense attractants and repellents and interact with cytoplasmic sensor kinases
chemoreceptors
clusters of thousands of MCPs
2 component regulatory system and flagella
counterclockwise-run
clockwise-tumble
-When MCPs bind repellent or release
attractant, a kinase is phosphorylated
interacts with flagellar motor to induce clockwise rotation and tumbling
-When MCPs bind attractant or release repellent, a kinase is unphosphorylated and not bound to the flagellar motor, resulting in
counterclockwise rotation and running
Quorum sensing
regulatory mechanism by which
Bacteria and Archaea
assess their population density near themselves
autoinducer
-molecule that indicates to a cell that other cells are nearby
-moves freely about membranes of cells
-reaches high concentrations in a cell when around other cells
-binds to sensor kinases which activates specific genes
Acyl homoserine lactone (AHL)
autoinducer
gram negative only
archaea uses what as an autoinducer
short peptides
Global control systems
regulates transcription of many
different genes in more than one regulon
lac operon
when in absense of glucose, this operon activates its genes to be able to use lactose as main energy source
diauxic growth
-two separate growth phases of bacteria
-once better energy source is expended a different energy source is utilized
lac operon regulation sequences
-lactose must be present to stop lac repressor from being present (negative control)
-cAMP must be present to bind to lac operon to activate (positive control)
heat shock responce
global control mechanism to
protect cells from protein denaturation resulting from heat, high solvent levels, osmotic stress, UV light
heat shock proteins
proteins that counteract damage of denatured
proteins and help cell recover from stress
ncRNA
RNA that does not code for a gene
rRNAs, sRNA, tRNA
small RNA
regulates gene expression
mechanisms of sRNA
1) decrease expression of mRNA
2)decrease mRNA degradation
4)increase mRNA stability through not letting it express
4)open up a ribosome binding site
ribozymes
RNA that acts as a catalyst
riboswitches
RNA that regulates gene expression
mechanisms of riboswitches
-binds to RNA
-controls at transcription or translation
-changes secondary structure of RNA which can control if its translated or not
- remove stem-loop that terminates
transcription, increasing mRNA and protein levels
Aptamer region
-riboswitch control region
-recognition domain that
binds small molecules
Feedback Inihibition
mechanism for temporarily turning off the reactions in a biosynthetic pathway
End product of the pathway binds to an early enzyme in the pathway, thus shutting down the pathway because no intermediates are generated
Reversible reaction
once levels of end product are limiting, pathway functions
the feedback inhibition enzyme has two sites, what are they?
active sites, allosteric protein site for feedback inhibition
isoenzymes
different proteins that catalyze
feedback inhibition but are subject to different
regulators
post translational regulation for proteins
phosphoylation, methylation, covalent modification (AMP,ADP)
PII Signal Transduction Proteins
-found in Bacteria & Archaea
-epigenetic attachments affect activity
-regulates nitrogen metabolism TFs, enzymes, and transport proteins
Anti-sigma
factors can inactivate sigma factors
Super-resolution microscopy
-Super-resolution microscopy
-Observes dynamic behaviors in real-time
Fluorescent Tagging
-Reporter genes encode proteins that are easy to detect or
assay and fused to genes of interest
-Green fluorescent protein used
Photoactivated localization microscopy
maps the movement of individual molecules
hemimethylation after DNA replication
-only parental strand of DNA is methylated
-methylation allows for a protein to bind to the origin of replication to block DNA synthesis through binding of DNA initiating proteins
par system
partitions the correct amount of DNA strands into two separate daughter cells
Par S
centromere like sequence near oriC
Par A
ATPase
Par B
binds the Par complex to the DNA
Pop Z
localized to old pole of DNA
anchors the DNA to one spot
Decatenation
separation of
replicated sister chromosomes
peptidoglycan synthesis
begins with preexsisting peptidoglycan
-rod shaped-
synthesis begins at several points along cell wall
-coccus-
cell walls grow in opposite directions outward
from the FtsZ ring
Bactoprenol
has a major role in precursor
insertion of peptidoglycan synthesis
biofilm formation steps
Attachment, colonization, development,
and dispersal
attachment stage of biofilm
-Random collision accounts for the initial attachment
-Facilitated by flagella and pili or by cell surface proteins
-Attachment is a signal for expression of biofilm-specific genes
-Once committed to biofilm formation, the cell loses flagella and becomes nonmotile
how does the metabolism change when switching to a biofilm?
Signals guide bacteria in transitioning from planktonic growth to life in a semisolid matrix
-Switch to biofilm growth triggered by accumulation c-di-GMP
c-di-GMP
-Widely distributed only in Bacteria
-c-di-GMP removes cell surface proteins like flagella and pili
-initiates synthesis of peptidoglycan
what do antibiotics target?
-Inhibition of protein synthesis
-Many antibiotics target DNA replication, RNA synthesis, and translation
-peptidoglycan synthesis
(targets DNA gyrase and topoisomerase to prevent DNA unwinding)
(targets RNA synthesis by blocking RNA polymerase active site or RNA elongation)
(can bind to ribosomal binding site to prevent translation)
(can bind to ribosomal subunit leading to error filled proteins)
Endospore formation
-triggered by damaging external events
-monitors external environment through 5 sensory kinases (resembles 2 component regulatory system)
-initates when Spo0A gets phosphorylated a lot
proteins emmited during endospore formation
-toxic protein that lyses other cells for nutrients
-protein that delays sporulation in other cells
-antitoxin protein to protect themselves from other sporulating cells
3 stages of endospores
activation, germination,
outgrowth
Germination Receptors
-within the inner membrane surrounding the endospore core exsists these receptors
-senses and binds nutrients
Activation of endospore steps
-release of DPA
-rehydration of core
-transcription and translation increase
Germination of endospore steps
-removal of the cortex is a
major event
-full rehydration of core
-metabolically active
Outgrowth of endospore steps
-elongates allowing escape of spore from mother cell
-fully metabolically active
heterocyst
-dedicated cells to nitrogen fixation
-anoxic
heterocyst formation
-inactivation of photosystem
-grows thickened cell wall to prevent 02 diffusion
-expresses nitrogenase
-triggered by limited amount of fixed nitrogen
Pseudomonas aeruginosa biofilm technique
-quorum sensing through acyl homoserine lactones in large amounts needed to begin colony growth
Vibrio cholerae
-quorum sensing opposite of pseudomonas aeruginosa
-must be in low population density area to establish colony
aquiring antibiotic resistance
-horizontal gene transfer through plasmids
-chromosomal mutations
efflux pump
-transport molecules, including antibiotics, out of the cell
-lowers intracellular concentration allowing cell to survive at higher external cellular concentrations
Selectable mutations
gives advantage
(ex antibiotic resistance)
requires screening to find the genes
Auxotroph
has an additional nutritional
requirement for growth compared to prototroph
prototroph
wild type bacteria, no additional requirement for life
Complementation
isolation of several strains
followed by comparative genetic analyses
Replica plating
-screens for nutritionally defective mutants
-transfers colonies from main plate
-if colony is unable to grow on medium
lacking a nutrient indicates mutation
Spontaneous mutations
-Occur without external intervention
-most result from occasional errors by DNA
polymerase during replication
Induced mutations
-Caused environmentally or deliberately
-Can result from exposure to natural radiation or chemicals that chemically modify DNA
point mutations
-Change only one base pair
-Occurs via single base-pair substitution
-Phenotypic change depends on the exact location of the mutation
Silent mutations
do not affect the sequence of encoded polypeptide or phenotype
Missense mutation
changes the sequence of amino acids in
polypeptide
Nonsense mutation
mutates a stop codon into the amino acid chain
Transitions
-base pair substitution switches it with another base pair of its own kind
-purine to purine
-pyrimidine to pyrimidine
Transversions
-base pair substiution switches it with a base pair of a different kind
-purine to pyrimidine & vice versa
Frameshift mutations definition
single base pair deletions
or insertions that result in a shift in the reading
frame
frameshift mutation effects
-changes entire polypeptide sequence downstream
-can gain/lose thousands of codons
-can be lethal
Mutagens
chemical, physical, or biological agents that
increase mutation rates, induce mutations
Nucleotide base analogs
-resemble nucleotide bases
but have faulty base pairing
chemical mutagens
intercacalating agents
alkylating agents
Nonionizing radiation
-base pairs absorb UV
-forms pyrimidine dimers
-UV kills cells due to its effects on DNA
Ionizing radiation
-more powerful than nonionizing (UV)
-creates free radicals which are damaging
-creates double/single stranded breaks in DNA backbone
SOS repair system
-Initiates many DNA repair processes
-Also allows DNA repair to occur without a template (translesion synthesis; high error rate)
-E Coli’s SOS system controls ~40 genes
3 mechanisms of bacterial genetic exchange
Transformation, transduction,
conjugation
Homologous recombination
-A process that results in genetic exchange between homologous DNA from two different sources
3 fates of bacterial genetic exchange
-uptake of the genetic info as separate from host genome
-degredation of genetic info
-integrated into host genome
Endonuclease
-nicks one strand of the donor molecule for crossing over of genetic info
Strand invasion
-single-stranded DNA molecule pairs with and displaces a similar or complementary strand in a double-stranded DNA molecule
-occurs in genetic recombination
heteroduplex
-double-stranded DNA molecule formed by the base pairing of two complementary single-stranded DNAs from different chromosomal sources
-can differ slightly in sequence, leading to mismatches within the heteroduplex
-observed during genetic recombination
Merodiploid
-strain carries two copies of a chromosomal segment
-(usually one copy on the chromosome, other on plasmid or phage)
Complementation
-occurs if a functional wild-type copy is
supplied on a plasmid or bacteriophage, restoring wild-type phenotype
Transformation
-Genetic transfer process where
DNA is incorporated into a recipient cell and brings about genetic change
Competent
-a cell that can take up DNA and be
transformed; genetically determined
-this is regulated
Gram negative sex pilus
-proteins within pilus recognize
and bind extracellular DNA, pilus retraction pulls DNA in
Gram positive sex pilus
-pili or secretion system binds DNA and bring it in
virus
genetic element that can multiply
only in a living (host) cell
Obligate intracellular parasite
Needs host cell for energy, metabolic
intermediates, protein synthesis
virion
singluar virus outside of a cell
virus structure
head & tail pilus
attachment of virus
-requires complementary receptor
(proteins, carbs, other cell structures)
Virulence
-relative ability of a pathogen to cause
disease
virulence factor
-toxic/destructive substances produced by the pathogen
-enhances invasiveness
Transduction
transfer of DNA from one cell to another by a
bacteriophage
Generalized transduction
-DNA from any portion of the
host genome is packaged inside the virion
-Donor genes cannot replicate independently
-Will be lost without recombination
Specialized transduction
-DNA from host genome is integrated directly into the virus genome
-typically replacing some viral genes
what genes can be tranduced
all of them
transducing particle
-host DNA packaged into a phage produces this
-makes the DNA defective, wont be able to lead to viral lytic infection
-can be transduced into host cells genoome
Gene Transfer Agents (GTAs)
-Defective bacteriophages that transfer DNA between prokaryotic cells
Conjugation
-Horizontal gene transfer that
requires cell-to-cell contact
Donor cell
contains conjugative plasmid
Recipient cell
does not contain plasmid
F plasmid
-Contains genes that regulate DNA replication
-contains transposable elements that allows plasmid to integrate into the host chromosome
-contains tra gene that encode transfering functions
mechanism of conjugation
-starts w/cell to cell contact
-tra gene nicks plasmid
-plasmid is transferred and replicated in both cells
-takes place in favorable conditions
Mobile DNA
-segments of DNA that move from one location to another in other DNA molecules
(TRANSPOSABLE ELEMENTS)
-utilizes inverse repeats for easy movement
transposase
enzyme required for transposition
insertion sequences
-simplest transposable element
-1000bp
-10-50 inverted repeats
-only gene codes for transposase
transposons
-genes vary widely on transposons
-transposase recognizes inverted repeats and moves the transposon
-can create mutant
transposase functions
-recognizes cuts and ligates DNA
Conservative transposition
-Transposon is excised from one
location and reinserted at a second location
-no duplicates of transposon made
Replicative transposition
-A new copy of the transposon is
produced and inserted at a second location
-2 transposons are present, one at old and new spot
Sequencing
determining the precise order of
nucleotides in a DNA / RNA molecule
Genome annotation
converts sequencing data into a list of genes & functional sequences present in the genome
Bioinformatics
storing and analyzing sequences
and structures of nucleic acids and proteins
first generation of DNA sequencing
sanger sequencing
able to sequence ~800 bp
second generation of DNA sequencing
pyrosequencing- ~700 bp
Ilumina method- ~100bp
SOLiD method- ~100bp
Ion torrent- 300bp
third generation of DNA sequencing
Pacific Biosciences SMRT- 2500-3000bp
Oxford nanopore- ~9000bp
Functional Open reading Frames
-encodes a protein, can be
identified by computer
Codon Bias
Some codons are used more than others
Hypothetical proteins
uncharacterized open reading frames
-protein likely exsists, function unknown
minimum number of genes for a viable cell
250-300
Archaeal Genome composition
-higher # of genes devoted to energy and coenzyme production compare to bacteria
-fewer genes for carbohydrate metabolism and membrane functions
Metagenomics
-analyzes DNA or RNA
from environmental sample containing organisms which have not been isolated/identified
Metagenome
total gene content of microbial
community
Heterologous expression
Expressing a gene in
a different host
Genetic engineering
-using in vitro techniques to alter genes in the laboratory
Thermocycler
automated PCR machine
Quantitative PCR (qPCR)
-variation of pcr technique that quantifies initial amount of DNA
Process of PCR
1) Denature
2)DNA polymerase extends primers using the original DNA template
3)Heat again with the target in twice the original amount, cool, and repeat 20–40 times, yielding a 106-108- fold increase
gel electrophoresis
-uses agarose gel to separate nucleic acids by size and charge
-smaller nucleic acid chains move further up the gel
Molecular cloning
-Movement of a gene from the original source to a small and manipulable genetic element
palindrome
inverted repeats
cloning vector
-Plasmids designed specifically for cloning DNA
products made by Taq in PCR
YACs
yeast artificial chromosome
cloning vector
for cloning into yeast
T7 expression vector
-cloned genes are place under this promoter to be able to control transcription/translation highly
-T7 under same controls as lac operon
cloning genes via mRNA
-modify the gene expressed in mRNA
(easy to find due to polyA tail)
-use RT-PCR to create desired gene
Fusion proteins
-joining target and carrier proteins can
simplify purification
Reporter gene fusion
-Coding sequence from reporter is fused with regulatory region from another source to form hybrid gene
Reporter gene
-Encodes protein easy to detect and
assay
Gene Fusion
- DNA segments from 2 different genes are fused
-promoter can be changed
Northern Blot Test
-Detects RNA in a sample
Southern Blot Test
-Detects DNA in a sample
vaccine creation
-virulence factor removed
-retains immune responce
Polyvalent vaccine
-single vaccine that immunizes
against two different diseases
Subunit vaccines
-contain only a specific protein from a pathogenic organism
vaccinia virus
-used to prepare vaccines
-takes genes as a vector to insert DNA from pathogen that its immunizing against
-tdk (thymidine kinase) gene is used
Commensal Bacteria
bacteria innate to the host organism
non pathogenic
antibody as anticancer therapy
-antibody complex binds to receptor
and is taken up by cancer cell which triggers immune responce to destroy cell
-uses antibodies to target cancer cells to trigger apoptosis
-antigen has been engineered to carry anticancer antibody
Gene mining
-process of identifying and isolating
potentially useful genes from the environment without culturing the organisms that contain them
BACs
-bacterial artificial chromosomes
-used for large DNA inserts
nonspecific immunity
innate immunity to pathogen
specific immunity
adaptive immunity
Phage exclusion
-variant of restriction enzyme systems
that recognize and modify incoming foreign DNA, preventing replication
Abortive infection
-triggers host suicide, leads to
programmed cell death by toxin–antitoxin systems
(prevents viruses from infecting wider community)
CRISPR domain
-domain of DNA where foreign infectious DNA is kept with spacers inbetween each infectious DNA portion
CRISPR domain is used for waht
memory bank of DNA to prevent re-infection
adaptive immunity
Cas proteins
-endonuclease activity
-Mediate defense and incorporate new spacers into CRISPR region
PAM
-protospacers adjacent motif
-the DNA inbetween the spacers in the CRISPR domain but on the invading pathogen
cas function
-cleaves the PAMs on the invading pathogen breaking up its DNA
Pre-CRISPR RNA
-binds to cas protein
-acts as a complementary DNA strand to the pathogenic DNA, when it binds the protein destroys the DNA
