Exam 3 Flashcards
Components of the Sec complex
SecYEG = channel, SecDF and YajC = membrane accessory proteins for the channel
SP1 and SP2 = signal peptidases that cleave signal peptides off preprotein
SecA = motor protein that pushes preprotein through channel
SecB = chaperone protein that brings preprotein from ribosome to SecA
Type I Signal Peptide
Positively charged hydrophilic n terminus (n region), hydrophobic core (h region), neutral hydrophilic c terminus (c region)
AXA motif marks cleavage site
Proline and glycine at regional boundaries to prevent helical structure (better able to be recognized)
Recognized and cleaved by SPase I
What types of proteins have their signal peptides cleaved by SPase II?
Lipoproteins
YidC
Helps to insert membrane-targeted proteins into membrane during secretory pathway, thought to be via a “gate opening” mechanism in SecYEG channel
Essential protein; G+ bacteria have two copies (YidC1 and2)
How to tell from an amino acid chain that a protein is bound for the membrane?
Alternating hydrophilic and hydrophobic sequences
Cotranslational secretion mechanism
Signal recognition particle binds signal peptide, then either binds and activates cytoplasmic FtsY receptor or travels to membrane to connect with self-activated membrane FtsY, carrying protein and ribosome (ribosome nascent chain complex) over to SecYEG for protein translocation
Protein expression on G+ cell wall
Sortase enzyme cleaves preprotein at LPXTG motif (between T and G) and covalently bonds to Threonine
Lipid II uses pentaglycine crossbridge to nucleophilically attack the sortase-protein C-T bond and protein becomes attached to lipid II
Lipid II gets converted to PG during cell wall synthesis mechanism
Examples of proteins expressed on G+ cell wall
Protein A - binds antibodies to prevent optimization and limit phagocytosis (eg in staph aureus)
Clumping factor A - binds host fibrinogen to promote bacterial adhesion
Various pili proteins like SpaA pilus
Spore forming proteins (e.g. in bacillus anthracis)
Why might you artificially add or remove a signal peptide?
Target protein to a less complex compartment for easier purification so you can obtain a sample of purified protein for studying
Type I secretion system
Inner membrane bound ABC transporter and Outer membrane bound OMP channel connected by membrane fusion protein (MFP) that spans the periplasm
Substrates are large and often associated with virulence (eg HlyA hemolysin), and contain a Cterminal secretion signal and GGXGXDXUX sequence (where U is a large hydrophobic amino acid)
Secretion is one step from cytoplasm to extracellular, and sec independent
Type II secretion system
Protein platform lodged in inner membrane, with attached pseudopilus that acts as a piston to push substrates thorough a channel in the outer membrane (made of oligomeric secretin and a tiny Pilotin)
Unfolded Substrate enters periplasm via Sec complex, is folded there, then enters type II system and is pushed though outer membrane channel by the pseudopilus
Type III secretion system
Spans inner, outer membrane and plasma membrane of host cell
Chaperones target effector proteins through a sorting platform (cytoplasmic), basal body (periplasmic), and secretory needle apparatus(through outer membrane and extra cellular) to translocon which forms a a channel for entry into host cell
Proteins have signal sequences and are secreted unfolded
Type IV Secretion System
Sec dependent OR independent, can be used to excrete proteins or DNA (conjugation)
Substrate either enters periplasm via Sec and then is extruded through OM channel and pilus, or is taken all the way through entire channel through IM, OM, and then through pilus to plasma membrane
Type V Secretion System
Auto transportation by a special protein with an amino terminal domain that recognizes Sec complex, an internal alpha or “passenger” domain, and a carboxyl terminal beta or “helper” domain
Sec transport of protein into periplasm, then protein itself forms channel and pushes itself through
Helper domain inserts into outer membrane and forms beta barrel channel, then passenger domain passes through
Passenger is either cleaved from helper domain by extra cellular proteases or remains membrane bound by the helper domain
Type VI secretory system
Needle sheath with piston that ejects effector proteins into other bacterial cells (competition)
Type VII secretory system
Used for transport across cell wall
Multimolecular base complex in the inner membrane allows proteins to traverse into periplasm, and from there they travel via an undetermined mechanism across the peptidoglycan, arabinogalactan, and mycolic-acid-containing outer membrane
Two components of the two component sensory system
- Sensor/receptor (eg histidine kinase, contains conserved histidine residue that becomes autophosphorylated upon stimulus binding)
- Effector/response regulator (cytoplasmic substrate for kinase, receiver domain contains an aspartate residue that is phosphorylated by histidine kinase, allowing operator domain to exert downstream effects)
Phosphorylation of the receiver domain in the response regulator leads to a conformational change in what domain?
alpha4-beta5-alpha5 domain (within the effector portion of the molecule)
Signal transduction for bacterial motility behaviour
Positive stimulus leads to activation of Kinase receptor and phosphorylation chain leading to counterclockwise rotation of flagella and a “run” behaviour
Negative stimulus leads to phosphorylation chain leading to a clockwise rotation of flagella and a “tumble” behaviour
Pho P/Q regulon
Two component sensory system in salmonella
Upregulated by low Magnesium concentration (stimulus), also stimuli specific to macrophage inner environment, and anti microbial peptides
Supports intracellular survival in macrophages, resistance to antimicrobials, and Mg uptake
Also upregulates salmonella pathogenicity islands that code for type 3 secretion systems that promote intramacrophage survival and invasion of epithelial cells
Discovered by inserting GFP in salmonella cells, allowing them to enter macrophages, and assessing which bacteria cells survived in the macrophages by counting green glowing macrophages with flow cytometry
VicR/K system
Two component regulatory system of Streptococcus mutans
Oxidative stress leads to a chain reaction including the production of sugars that then trigger biofilm formation
Reason why sugar causes cavities is that the sugar triggers biofilm formation by S mutans in the mouth which leads to dental plaque and eventually tooth decay
Discovery method: GFP reporter strain placed after VicRK promoter lead to increased fluorescence intensity under oxidative stress (indicating gene upregulated under oxidative stress), then knocked gene out and found that mutant was more sensitive to oxidative stress than wild type (indicating gene up regulation helps with protection against ox stress)
Cpx A/R system
Two component system of actinobacillus pleuropneumoniae
Detects envelope/membrane/periplasm stress (eg misfolded proteins, surface adhesion), and responds by activating genes for proteases and enzymes (breakdown misfolded proteins, build peptidoglycan to repair cell wall) and repressing genes for pili and flagella (expensive, need to conserve energy and resources)
CovR/S System
Two component system of group A streptococci, an invasive pathogen
Activated genetic responses to adapt to host condition and promote colonization (capsule biogenesis, surface proteins like adhesions, secreted proteins like cytolysins and anti phagocytic factors, etc)
Discovery: microarray study of gene expression in wild type vs mutant - full genome on microchip, probe with RNA from WT and mutant and look for binding differences
AlgZ/R system
Two component system of pseudomonas aeruginosa
Coordinates expression of type IV pili and alginate
ChIP analysis to determine that it also upregulates mucR expression, increasing c-di-GMP synthesis, supporting biofilm formation
ChIP analysis
Chromatin immunoprecipitation
Investigation of transcriptional regulators
Express protein of interest, allow it to bind DNA, add chemical to stabilize bond between protein and DNA, add antibody that recognizes protein, fragment DNA and remove DNA-protein complexes, add proteases to degrade proteins and isolate DNA, sequence DNA and identify genes regulated by the protein
Microarray study
Put genome onto microchip, probe with RNA from both a wild type and a mutant organism, look for binding differences to determine differences in gene expression between the two organisms
GFP reporter study
Add gene for green fluorescent protein between the promoter and gene sequence for gene of interest
Cells that fluoresce green are expressing that gene
Could use facs to sort them based on gene expression
Knockout study
Knock out a gene, observe effect on phenotype
Extracellular biofilm matrix composition
Proteins, polysaccharides, lipids, nuclei acids
Matrix specific proteins (eg RbmA) have fibronectin domains that mediate interactions with surface receptors
Robust, protease resistant amyloid-like fibres provide matrix scaffold (assembly is energy independent!)
Phages in biofilm
Prevent new bacteria from joining/invading
Ability to kill bacteria depends on how long bacteria has been with biofilm (with time will get enveloped and receptors will no longer be exposed to phage)
Strategies to limit biofilms
Matrix degrading enzymes
Immunotherapeutics (antibodies bind and sequester matrix stabilizing proteins, bind adhesins to block attachment)
Develop inhibitor molecules small enough to infiltrate the biofilm
Signalling pathway disrupters to prevent bacteria from communicating with each other
Quorum sensing
Bacteria produce auto-inducers
Critical concentration of autoinducers indicates large enough population of bacteria is present - phosphorelay signal cascade is initiated that affects gene expression, often followed by an additional TCS system with histidine kinase and RR components
Example V cholerae phosphorelay system for quorum sensing
Low conc autoinducer leads to production of sRNAs that block the action of HapR
HapR itself blocks regulators vps and aphA that regulate polysaccharide production, therefore polysaccharide production is allowed when autoinducer is low conc (fewer bacteria = need more polysaccharide to build matrix and form biofilm)
High conc autoinducer results in no sRNA production, allowing HapR to function and inhibit polysaccharide production (lots of bacteria = biofilm already formed, don’t need more polysaccharide material to be added)
Bacterial genome shape
Circular
Experiment to determine replication pattern of DNA
Meselson and Stahl
Grew E. coli in media with 15N isotope rather than the more abundant 14N
Transferred E. coli with only 15N DNA moved to media with 14N
First replication: produced DNA sample with 100% intermediate weight (between 15N and 14N) - excluded conservative theory
Second replication: produced DNA sample with 50% intermediate weight and 50% 14N weight - excluded dispersive theory
Conclusion: semi conservative replication
Mechanisms contributing to low error rate in DNA replication
Discriminatory base selection by the polymerase that adds bases (10^-5 errors per base per round of replication)
Editing of misinserted bases by the 3’->5’ exonuclease associated with the polymerase (10^-2)
Removal of remaining mismatches by postreplicative DNA (10^-3)
Total error rate = 10^-10 errors per base per round of replication
DNA Pol adds nucleotides to which end of DNA strand?
5’
Direction of DNA synthesis
Begins at origin, proceeds bidirectionally, 3’ to 5’ prime meaning one direction will be continuous (leading strand) and the other direction will be in fragments (lagging strand)
Role of primase (DnaG) in replication
An RNA polymerase; makes the RNA primer
Role of polymerase III in DNA replication
“Workhouse” adds nucleotides (5’ to 3’) and proofreads (3’ to 5’)
Role of DnaA in replication
Initiator; Binds origin (at A-T rich sequence) and begins the opening of the replication bubble and recruitment of other replication proteins
Role of SSBs in DNA replication
Single stranded binding proteins, keep open complex open (strands separate)
Role of polymerase I in DNA replication
Replaces RNA primer ribonucleotides with deoxyribonucleotides
Role of DNA ligase in DNA replication
Links Okazaki fragments together on lagging strand
Role of Helicase (DnaB) in DNA replication
Unwinds DNA ahead of the replication fork
Role of Tus in DNA replication
Binds termination sequence, blocks replisome until other one catches up, counters helicase action to help end DNA replication
Replisome
Complex of proteins that make up the replication fork in DNA replication
Role of HU in DNA replication
Histone-like protein that helps DnaA bind the origin
General steps in DNA replication
Formation of open complex
Prepriming complex
Priming
Replication
Termination
DNA replication in depth steps: formation of open complex and prepriming complex
DnaA binds specific sequences in oriC, with help from HU
DNA unwinds (ATP dependent)
SSBs bind to keep complex open
DnaB (helicase) binds to both ends of open complex
DNA replication in depth steps: priming and synthesis
Primase binds and makes 12nt piece of RNA, allowing for addition of DNA nts
DNA Pol III adds DNA nts one at a time to 5’ end of chain (continuous extension on leading strand, in Okazaki fragments on lagging)
RNA primers are removed and replaced with DNA by DNA Pol I
Ligase joins Okazaki fragments together
Who discovered Okazaki fragments?
Tsuneko and Reigi Okazaki (husband and wife)
Who discovered the DNA polymerases?
Arthur Kronberg discovered Pol I
His son Thomas discovered II and III
(His other son Roger studied RNA polymerases)
Paula De Lucia isolated the gene for Pol I, named Pol A after her
DNA replication in depth steps: termination
Tus binds termination sequence (Ter)
Ter allows one way replication
Ter stops replisome, believed to counter helicase action to stop its movement
Other replisome catches up and dislodges Tus - replication complete
Daughter chromosomes end up “tangled” - resolved through decatenation by Topoisomerase IV and XerC/D
Resolution of chromosome dimers after replication
During chromosome replication, homologous recombination between sister chromosomes can result in a chromosome dimer
FtsK lines up dif sites at the septum where XerC/D (a recombinase) and topoisomerase IV can resolve the chromosomes into two separate daughters
DNA helix size and shape
10.5 bases per turn, right handed helix
DNA supercoiling
Supercoiling = storing energy in DNA as tension (twists)
Superhelical tension can be used to aid processes (packaging, open complex formation, etc)
Positive supercoiling = overwinding
Negative supercoiling = underwinding
DNA gyrase introduces negative supercoils
Topoisomerase removes supercoils
Topoisomerase action on DNA
Topo I removes supercoils by breaking and rejoining DNA backbone
Topo IV removes catenations to help resolve dimers of replicated chromosomes
Gene architecture
Coding strand (resembles the mRNA strand except with T instead of U) Template strand (strand that is read during transcription to form the mRNA)
RNA Polymerase subunits
Holoenzyme: 2 alpha subunits, 1 beta, one beta’, one omega and one sigma subunit
Core enzyme: holoenzyme without the sigma subunit (sigma helps to guide enzyme to initiation sequence, then leaves)
Housekeeping sigma factor
Sigma 70 - binds most initiation sequences
Alternate sigma factors have a specific sequence or small subset of sequences they recognize (more specialized)
Sigma 54
Holoenzyme with sigma 54 is Not independently competent for transcription - requires activator sequence NtrC at far upstream enhancer sequence which is activated via a TCS and loops over to the enzyme to activate it
Transcription promoter sequence
Two sequences at -10 and -35 (with space between)
Stronger the closer it is to consensus sequence (TTGACA-17-TATAAT)
More nt differences = weaker promoter
Transcription initiation steps
RNA Polymerase Holoenzyme binds promoter (closed complex)
DNA unwinds to form open complex
Ribonucleotides begin being incorporated
Transcription elongation steps
Sigma subunit dissociates
RNA nucleotides added by core enzyme
Transcription termination steps
Factor independent:
- secondary structures in newly formed mRNA cause polymerase dissociation
Factor dependent:
- Rho proteins bind newly made mRNA and move up chain to Pol-DNA complex
- Pol reaches Rho dependent termination sequence and stalls
- Rho catches up and dissociates the polymerase using ATPase driven energy
Operon
Cluster of genes encoding for proteins with related functions
One promoter used for transcription of all genes in the cluster into one long mRNA strand that will code for separate proteins
“Polycistronic mRNA”
Ribosome
Made of RNA and proteins
Small subunit binds mRNA, large subunit provides enzymes needed to form peptide bonds
2 sites: A (aminoacyl) site = where new charges tRNA binds; P (peptidyl) site = where amino acid is joined to peptide chain via peptide bond and where empty tRNA leaves from after losing its mRNA
Translation steps: initiation
Ribosome binds mRNA at Shine Delgarno sequence, recognized by 16sRNA in the small subunit (30s) of the ribosome
Ribosome scans until it finds start codon (AUG)
fMet tRNA binds start codon
AARS
Aminoacyl-tRNA synthetase
Adds correct amino acid to tRNA
Translation steps: translocation
Peptide bond is formed between tRNA in A site and peptidyl-tRNA in P site
Uncharged RNA leaves the P site, ribosome moves so next codon is in A site
Translation steps: termination
Release factor binds stop codon and dislodges growing polypeptide chain
RF1 recognizes UAG and UAA
RF2 recognizes UGA and UAA
Attenuation
Coupling transcription to nutrient supply (don’t make molecules that are already abundant, vice versa)
Common mode of regulation for amino acid synthesis
Attenuation at the trp operon
High trp levels = ribosome moves quickly (not waiting for trp to be available), favours formation of RNA 3-4 terminator structure
Low trp levels = ribosome moves slowly (waiting for trp), favours formation of RNA 2-3 anti terminator structure
Also: repressor molecule can bind operator, but only when bound to trp. therefore high levels of trp lead to repression of the trp operon
Jacques Manod
One of the founders of molecular biology
Figured out gene regulation of the lac operon and trp operon
Nobel laureate
Anti-nazi-occupation activist
lac operon
Operon encoding enzymes that help with uptake and breakdown of environmental lactose
No lactose in environ: lacI repressor binds operator, preventing RNA Pol from binding promoter and initiating transcription of the enzymes
Lactose in environ: lacI repressor binds lactose, can’t bind operator, RNA Pol is able to bind promoter and initiate transcription
IPTG = artificial inducer (mimics effect of lactose but isn’t broken down by enzymes - continual activation of operon)
Lac Z
B-galactosidase; cuts disaccharide lactose into glucose and galactose
Lac Y
Permease; lets lactose into cell
LacA
B-galactosidase transacetylase
Control of flagella expression
flhC and flhD = early genes
Bind to promoter to turn on middle genes
Middle genes code basal body and hook, during construction flgM is bound to sigma28. When basal body and hook is complete, flgM leaves sigma factor and is the first molecule to be excreted through the structure.
Sigma 28 now free to activate late genes, which code for the filament
Cis sRNA
Regulatory small RNA molecule adjacent to the gene they control (often in plasmids), they have extensive homology with gene target and are very specific
trans sRNA
Regulatory small RNA molecule separate from the gene they control, they have limited homology with gene target and thus require the aid of Hfq protein to stabilize the RNA-RNA interaction
Riboswitches
Secondary Structure of gene sequence itself regulates its transcription
Eg at certain temperature, strand changes structure to one that is more/less permissible to the action of RNA Pol
Mechanisms for bacteria to destroy/block incoming foreign DNA
- Restriction modification systems (eg methylate host DNA to mark it as “self”, incoming DNA will be unmethylated and marked for destruction
- block phage attachment (eg through receptor mutation)
- block phage infection (less well known mechanism(s))
- CRISPR (insert copy of foreign DNA into own genome, recognize it in future and destroy fast)
CRISPR locus
Interspaced repeats with DNA homologous to that of phages and plasmids between them
A few adjacent genes associated: CAS genes (code for enzymes that process the locus)
CRISPR stages
Adaptation/immunization: new foreign sequence is acquired and put into the CRISPR locus
crBiogenesis: locus is transcribed and the Cas proteins process the crRNAs
Interference: crCRNAs guide an endonuclease to a target DNA that is then cleaved
Importance of PAMs in CRISPR
Sequence next to target sequence that is NOT taken up into the CRISPR locus
Only when the crRNA-endonuclease complex sees the target sequence WITH the PAM sequence will they attack it, they won’t attack if I’m they see the sequence surrounded by the CRISPR repeats - protects the self genome that has had the foreign DNA added to it
Types of CRISPR
Three types (I, II, V) Classified based on the way the crRNAs are processed Most commonly discussed is type II, which uses the cas9 endonuclease
Lytic cycle of phage
Injection of phage DNA into cell
Replication of DNA within cell, synthesis of new phage particles
Packaging of DNA into phage heads
Lysis of cell, phages find more cells to infect and repeat cycle
Hershey chase phage experiment
Looking to see whether DNA or Protein was the genetic material being injected by phages into bacteria
Labeled phage protein capsule with sulfur 35, labeled phage DNA with phosphorus 32. Let phage infect bacteria, then separated phages from bacteria through centrifugation
Sulfur was detected in the phage sample (protein was not deposited) and phosphorus was detected in the bacteria sample (DNA was deposited)
Proved DNA was the genetic material
Lysogenic cycle of phage
Phage infects cell
Phage DNA becomes incorporated into host genome
Cell divides, prophage DNA is passed on to daughter cells
Under stressful conditions, phage DNA is excises from the bacterial chromosome and enters the lytic cycle (phage DNA replicated and phage proteins are made, phages are assembled and then released upon cell lysis)
Phage plaques
Zone of lysed bacteria in culture is a plaque
Lysogenic phage cycle will result in turbid plaques (bullseye shape due to Lysogenically infected bacteria still alive in the middle, surrounded by lysed bacteria around it)
Prophage
Dormant phage within a host bacterium
No genes expressed except one: cl, which codes for a repressor
This repressor also prevents other phages from infecting the same bacterium, giving the bacterium the appearance of immunity (but really it is already infected)
Lysogen
Host bacterium containing a dormant phage
“Immune” to further phage infection (superinfection) due to already being infected and repressor gene from phage dna being expressed
Cl and Cro
Genes controlling phage life cycle
Cl predominates: repressor levels high, cro gene inactive, levels of cro protein low, lysogenic
Cro predominates: levels of cro protein high, cl gene inactive, levels of repressor low, lytic. Cro then triggers production of other phage genes needed to continue lytic growth
Phage-infected bacteria response to DNA damage
RecA protease activated, which then cleaves the phage Repressor protein
Inhibition of cro and stimulation of repressor expression are interrupted
Increased level of cro leads to lysis
Phage gene expression timing
Very early: only N (regulator of early genes) and cro are on
Early: N, cll (regulator of cl repressor and integrate synthesis), cro, replication and recombination genes are on
Late:
- lytic: head, tail, and lysis genes on. Early genes off
- lysogenic: cl (repressor) and int (integrase which allows chromosome to enter E. coli genome) are on, then once chromosome is integrated only cl is on
Phage genome integration into bacterial chromosome
Phage DNA is circularized
AttP (phage site) and attB (bacteria site) recombine to form attL and attR half-sites at either end of prophage
Reaction catalyses by integrase, a phage encoded factor
Phage genome excision from bacterial genome
AttL and attR sites reform attP and attB sites of the phage and bacterial sequences
Catalyzed by integrase and Xis, two phage-encoded factors
Plasmid
Extrachromosomal DNA
Usually circular, but can be linear
Can encode factors that allow growth in new environments (eg antibiotic resistance and virulence)
Can be shared horizontally (within same generation)
Colicins
Toxins encoded on plasmids used by bacteria to kill other bacteria that don’t have the same plasmid as them (plasmid that encodes the toxin also encodes a toxin neutralizer)
Usually kill by forming pores or entering bacteria and acting as nucleases
High copy vs low copy plasmids
High copy: “relaxed” (made in abundance), small size, replication unlinked to cell division, random partitioning. Eg ColE1 plasmid (makes colicins that kill species related to E. coli)
Low copy: “stringent” (make few copies), large, replication linked to cell division, directed partitioning, often conjugative. Eg R100 plasmid
Regulation of plasmid copy number in E. coli
RNA I = antisense RNA that binds RNA II, the promoter for plasmid replication
Rop = protein that helps with pairing of RNA I to RNA II
When plasmid number increases, so does level of Rop and RNA I —> they then act to stop plasmid production
Regulation of R100 plasmid copy number by antisense RNA
RepA = required for initiation of replication at oriV
CopB = repressor of repA, encoded by copB gene
CopA = Gene that encodes RNA which is antisense to the RepA sequence
When plasmid enters cell there is no copB and little copA so RepA is expressed until plasmid copy number reached
When copy number reached, CopB and CopA repress expression of RepA and following plasmid gene
Plasmid incompatibility of R100 plasmid
R100 can’t coexist with related plasmids because the copA regulatory genes are similar. Therefore they will repress the replication of the related plasmid
Regulation of R100 plasmid replication by iterons
Iterons = repeated sequences of DNA
RepA binds iterons and “handcuffs” two plasmids together, preventing further replication
Can also handcuff related plasmids (more plasmid incompatibility)
Plasmid incompatibility
Plasmids can regulate their own regulation, as well as that of related plasmids in the same cell
In these cases, only one of the plasmids will be inherited
These plasmids are in the same “incompatibility group”
Factors causing instability of plasmids: Plasmid integrity
Plasmids often have insertion sequences or other recombination “hotspots” that allow for deletions or inversions
One gene may be intact but others are lost or have a changed orientation
Can cause problems, eg antitoxin genes are usually less stable than toxin genes, so if a plasmids antitoxin gene is lost, the bacterium will die from the toxin
Factors causing plasmid instability: partitioning systems
ccdB-ccdA toxin-antitoxin system encoded on some low copy plasmids
If plasmid partitioning doesn’t go right, cells that don’t inherit the plasmid will die because they don’t have the gene coding for the antitoxin ccdA
Hok-sok toxin-antitoxin system: Hoi encodes toxic protein, sok encodes antisense RNA that suppresses hok translation. if plasmid is lost, Hok is made and cell dies
Factors that cause instability of plasmids
Plasmid integrity
Partitioning of plasmids
Differential growth rates (plasmid encodes genes that are not being used, gets lost to decrease burden)
Transformation
Uptake of “naked” DNA that is free in environment
Competence = ability of organism to take up DNA
DNA enters as a ssDNA molecule
Naturally competent organisms become competent in response to growth phase (late log phase) or diffusible factors from quorum sensing
Competent organisms may have proteins that bind preferred DNA sequences (eg Neisseria and Haemophilus)
Lab methods to encourage transformation (DNA uptake) in less competent organisms
Treat with CaCl - pokes holes in membrane, encouraging DNA entry
If that doesn’t work: Electroporation (shock in DNA using capacitor, more common) Biolistic transformation (shoot DNA in on gold particles, less common)
Self-transmissible plasmids vs mobilizable plasmids
Self transmissible plasmids encode the machinery necessary to initiate conjugation of themselves
Mobilizable: can be conjugated but require Tra functions supplied in trans
Transconjugant
Plasmid recipient following successful conjugation
Plasmid conjugation mechanism
Single strand break in donor plasmid at oriT via endonuclease action of Dtr “relaxase”, rolling circle replication, plasmid that is transferred is a copy of original
Donor plasmid contains transfer (tra) genes with Dtr and Mpf component. Donor forms pilus via expression of Mbf genes and brings cells in close contact, pilus forms bridge to transfer DNA
Non conjugating plasmids can also be transferred if it contains mob genes and non site (oriT), by utilizing the pilus formed by the other plasmid
Argobacterium
Plant pathogen
Uses type IV secretion to transfer DNA from bacteria to plant host
Causes tree tumours!
Exploited mechanism to transfer DNA into fungi, malaria, etc
Recombination frequency
Probability that recombination will occur at a DNA site
Fairly constant except for some hotspots
Increased homology between sequences increases chance of recombination
Greater distance between the two DNA sequences increases chance of recombination
RecA
Mediates all bacterial homologous recombination events by pairing homologous DNA segments together so they can exchange strands
RecA monomer binds a single strand of DNA (activated by presence of ssDNA eg during replication - particularly stalled replication), more RecA are recruited to make a nucleo-protein filament, this filament then goes looking for homology to pair with and recombine
RecA mutant will not be able to perform homologous recombination
F plasmid
Conjugative plasmid (contains oriT and tra genes) Can integrate into chromosome (contains IS 2 and two copies of IS 3)
Cells described as F+ or F- depending on if they have f plasmid or not
Chance for homologous recombination between plasmid and chromosome DNA
Hfr strains
Bacteria with an F plasmid inserted in their chromosome that results in lots of homologous recombinations
Hfr = high frequency of recombination
F’ plasmid
Imprecise excision of F plasmid from chromosome leads to inclusion of small piece of chromosome in the plasmid - now called an F’ plasmid
These plasmids are useful for constructing partial diploids (merodiploids)
Hfr mapping
Mate Hfr strain with various markers, stop chromosome transfer at different times by shearing cells apart
Order and timing of gene transfer directly reflects the order of genes on the chromosome
Insertion sequence
Sequence that codes transposase flanked by inverted repeats
Duplicate random target sequence and insert themselves into sequence between the two duplicates
Transposons
Like insertion sequences but have selectable marker
Noncomposite transposon: transposon gene flanked by inverted repeats (eg tn3)
Composite transposon: transposon gene flanked by insertion sequences (eg tetracycline resistance gene)
Regulation of Transposition
Unregulated Transposition would be lethal
Transposase binds poorly to methylated DNA - takes advantage of hemimethylated DNA during replication to perform Transposition then
Preferentially acts in cis (unstable and doesn’t accumulate to high levels, binds DNA as soon as it is made therefore more likely t bind it’s own site)
Lydia and Delbruck Fluctuation Test
Determining answer to the question does environmental pressure cause mutations (Wallace’s theory) or does it simply select for existing mutations (Darwin’s theory)?
Exposed multiple cultures of bacteria to toxic phage and measured proportion of resistant mutants obtained each time
If proportion of mutants was the same every time, would support directed mutation theory; if number was variable, would support spontaneous mutation theory
Results supported spontaneous mutation theory
Point mutation
Base substitution (replace one base with another)
Silent mutation
Codon change doesn’t result in amino acid change Eg UUA (leu) to UUG (leu)
Missense Mutation
Codon change results in one amino acid changing to another
Conserved change: amino acids are similar enough that protein function is unlikely to change (eg hydrophobic amino acid to another hydrophobic amino acid)
Nonsense mutation
Codon changes from amino acid to stop codon, resulting in truncated protein
Frameshift mutation
Addition or deletion of base(s) (not in multiples of 3) that changes reading frame (all following codons affected
Usually hit a stop codon soon after
Null mutation
Full deletion of a gene
Mutation reversion
Second mutation that reverses the first
Insertion mutations can be reverted by precise excision of inserted DNA, deletion mutations can’t be restored by “true reversion”
Mutation notation
Gene is italicizes, protein first and last letter is capitalized
Insertions lacZ::Tn10
Null mutations: delta lacZ
(Pretend lacZ is italicizes in both of the above)
Essential gene
Gene where null mutation (full deletion) is lethal to organism
Can be studied by isolating mutations that are dead under one condition and alive under another (eg temperature sensitive mutants will grow at permissive temperature and die at restrictive temperature)
Polarity of mutations
Nonsense and insertion mutations are often polar (impact downstream genes as well)
Potential Reasons:
Insertional mutation inserts a transcriptional terminator
Mutation may disrupt translational coupling
Pseudoreversion
Intragenic suppression (mutation is suppressed) Second mutation acquired that masks the effect of the first Eg amino acid codon changed to stop codon then back to an amino acid codon but a different amino acid than original - it masked the effect of the stop codon without reverting back to original amino acid
Suppression of nonsense mutation by tRNA mutation
Pseudoreversion
Amino acid changed to stop codon (mutation) but tRNA develops mutation that allows it to recognize the stop codon as an amino acid codon (suppression of mutation effect)
Compensatory mutations
Pseudoreversion of a mutation whereby one mutation prevents/creates one function (eg pathway inhibition) but another mutation affects another function that masks the phenotype (eg activation of different pathway that performs similar function)
Mutator strain
Strain of organism with mutations in error prevention mechanisms - have much higher mutation rates, useful for lab work
Insert plasmid into mutator strain, let it acquire mutations, bring it back out and have a library of gene mutations to choose from
Methods of increasing mutation rate
Treat with ethidium bromide: intercalating agent that inserts between paired bases
Treat with NTG or EMS: alkylating agents, modify guanine to generate O6-methylguanine (polymerase pairs base with thymine instead of cytosine) GC becomes AT
Treat with nitrous acid: deaminates bases
Treat with UV irradiation: causes cross links between adjacent pyrimidines (thymine-thymine dimers) leads to frameshift (polymerase skips over base)
Ames test
Method of determining if substance is a carcinogen (dna mutagen)
Plate His- salmonella typhimurium onto His- media, add suspected carcinogen, count frequency of revert ants (back mutations) that are now His+ (colonies that successfully grow)
Bacterial response to DNA damage
Polymerase stalls, leaving ssDNA exposed
RecA binds ssDNA and is activated
Activated RecA cleaves LexA repressor which results in multi gene activation (excision repair of DNA mutation by Uvr system, activation of alternative Polymerase PolV or PolIV)
PolV and PolIV are error prone so don’t want to use it all the time but it is good because it is not so “perfect” that it will freak out and stall over a mutation like polIII does
Mutations commonly caused by Pol IV and PolV
PolV - base substitution or error free (Ok)
PolIV - frameshift (bad!)
Mut complex DNA repair
During DNA replication, DNA is briefly hemimethylated - MutH binds hemimethylated GATC sequences.
MutS and MutL complex bind mismatched DNA
DNA is threaded through complex in both directions until it reaches MutH protein bound at hemimethylated GATC sequences
MutH cleaves unmethylated DNA
Complex of DNA helicase II and exonucleases degrade unmethylated DNA towards the mismatch
Gap is filled by DNA Pol II and nick is sealed by DNA ligase
Complementation
Restore phenotype by replacing damaged gene with a good copy
Usually done on a plasmid
Used in lab to demonstrate phenotype is due to mutation
Knock out: phenotype shows, knock back in: phenotype goes away
Epistasis
Interaction of genes that are not alleles, in particular the suppression of the effect of one gene by another
Forward genetics
Have a phenotype, need to figure out what gene(s) are involved
Reverse genetics
Have a gene, need to figure out it’s function/effect(s)
16s rRNA gene sequencing (amplicom sequencing)
Technique to identify organisms in a microbiome
Amplify gene for 16s rRNA with PCR, sequence it, compare to known sequences to identify the organism(s)
Meta genomic analysis
Sequence all DNA in a sample
Way more data than 16s rRNA sequencing but tells you about more organisms than just bacteria and presence of enzymes (what organisms are doing)
Complementation group
Different mutations of the same gene that affect same phenotype can’t complement each other (are in the same Complementation group)
However different mutations on different genes with same phenotype affected can complement each other (different complementation groups)
How do you know when you have found all the genes involved in a process?
Classical genetics: isolation of same mutation(s) over and over until you can assume there are no other mutations involved (genetic screen is “saturated”)
Genomics and next gen sequencing platforms allow for much easier saturation
How to introduce a transposon?
Use suicide plasmid - conjugative plasmid with R6K origin of replication (requires Pi protein for replication)
Pi protein is supplied in trans by a temperate phage
Plasmid contains transposon with antibiotic selection
Mate donor (where plasmid replication is permitted) to recipient (plasmid cannot replicate)
Select for antibiotic resistance due to the transposon hopping - these organisms will have added transposon
Signature tagged mutagenesis
Mutants with unique DNA sequence - grown in a pool and DNA isolated from each
Apply selection pressure
Isolate DNA from pool of mutants that made it through, compare to previous DNA sample
Missing mutants: indicate that the gene they were mutated for was essential for survival within the environmental condition
TnSeq
Transposon mutagenesis meets next generation sequencing
Sequence whole genome - know where all genes are
Sequence all mutants, compare to genome to find where the transposon went
Identify all essential genes in one screen
Pool of Tn mutants -> selection pressure -> pool of Th mutants (compare input and output pool to see which genes were required for selection pressure)
