Lectures 34-43 Flashcards
How much % of the world’s DNA belongs to bacteria?
30%
What makes up a significant part of the body?
Bacteria
Why is bacteria a good model organism?
Haploid (1 copy of each gene) - easy to study
Asexual reproduction - easier to understand
Short generation times - grow quickly
Grow on plates with defined media
Easy to store
Easy to genetically manipulate
Bacterial genome
Single circular double stranded DNA chromosome
Less space between genes (inter-gene space)
Rare introns
Functionally related genes grouped - operon
Plasmids - extracellular chromosomal DNA replicate independently
Binary fission
Bacterial asexual reproduction
Common in prokaryotes
Elongates, contents increased, DNA replicated + segregated —> 2 identical daughter cells
Septum forms in middle, grows from both sides of cell
E.coli does this in 20 min
E.coli
Can grow on simple inorganic nutrients + carbon source
Need glucose, phosphate, pH control, nitrogen, trace metals
Prototroph - doesn’t require nutritional factors (opposite to auxotroph)
Biosynthetic auxotroph
Need additional nutrients, usually AA
Catabolic auxotroph
Lost ability to degrade/catabolise carbon source
Conditional lethal mutants
Genes essential for survival don’t work under certain conditions
But under some conditions can still make functional proteins even if still mutant
Example of conditional lethal mutant
Temperature sensitive mutant - only grow at permissive temp e.g mutant protein folds correctly at lower temp due to lower E in system
Wild type
Normal species
Mutant
Genome carries mutation with respect to wild type
Mutation
Inheritable change in gene sequence of nucleic acid
Allele
Sequence variant of a gene
Mutagenesis
Process by which mutants are produced
Mutagens
Chemical and physical agents which cause mutations
Shared pathways
Some produce metabolites as precursors for more than 1 pathway
Loss of 1 enzyme leads to requirement for more than 1 AA
Purines
pur
gua, ade
Pyrimidines
pyr
thy, cyt, ura
Vitamins
biotin (bio) riboflavin (rib) NAD (nad) thiamine (thi) pyridoxine (pdx) pantothenic acid (pan)
rpoA
Encodes alpha-subunit of RNA pol
polA
encodes DNA pol I
polC
encodes DNA pol III
sugars
arabinose (ara) mannose (man) xylose (xyl) galactose (gal) melibiose (mel) lactose (lac) rhamnose (rha) maltose (mal)
Drugs + bacteriophage resistance
azide (azi)
rifampicin (rif)
streptomycin (strA)
phage T1 (tonA)
Nonsense suppressors
suppressor (sup)
super/sub script
Temp sensitive (ts) Cold-sensitive (cs) amber mutation (am) ochre mutation (oc) amber mutation (um)
stop codons
amber UAG
ochre UAA
opal UGA
leuA-
mutation
Requires leucine
leuA+
Not wild type but not require leucine
Triangle symbol
Deletion
R
Resistant
( )
Lysogenised by bacteriophage
/F’
Carries F’ plasmid
Lamarckian evolution
vital force
Luria-Delbruck experiment (1943)
early belief: add toxic agent to bacterial culture and entire culture becomes resistant so agent makes cells resistant (Lamarckian)
hypothesis: if Darwinian-random mutations prior to selective agent, if Lamarckian-mutants after selective agent
L model prediction: no mutations till after T1, same no. mutations every time
D: random mutations at any generation so diff no. in diff plates
method: E.coli plated with T1 phage, start with Tonˢ (T1 sensitive) then some Tonᴿ grow
results: big variation in no. resistant colonies so Darwinian
conclusion: variations because mutate at diff times in diff generations so had diff length of time to grow
Newcombe experiment
start with Tonˢ on 2 plates
a: spread bacteria around b: leave
spray both with T1
more colonies on plate A because respreading means little pile of resistant bacteria gets spread and each give rise to own resistant colonies
not spreading means pile of resistant gets bit bigger
Lederberg x2
replica plating
pick out phenotypes that can’t easily select for
master plate with E.coli Tonˢ and made lots replica plates
sprayed with phage
position of Tonᴿ colonies same on each plate so phenotype present before env. change of introducing T1
replica plating
plate put onto cloth, imprint of what on plate onto cloth, new plate onto cloth so transferred onto new plate, exact copy
point mutation
change to 1 base pair
substitution, deletion, insertion
indel mutation
insertion and deletion
transition mutation
1 purine to another purine or pyrimidine to another pyrimidine
trasnversions
purine to pyrimidine or other way round
consequences of point mutation
in promoter: can affect transcription
in coding region: silent/missense/nonsense
silent mutations
code for same AA
usually 3rd pair substitution
missense mutations
amino acid substitution
usually 1st or 2nd pair changes
nonsense mutations
leads to stop codon
inverions
change orientation, flips around
tandem repeats
genome duplicated and inserted
can lead to overproduction of proteins
transposons
nucleotide sequences that can move themselves around
have encoded mechanisms that allow to cut out and insert elsewhere
reversion
usually point mutation
results in restoration of original sequence
tautomer
isomers that exist together in equilibrium
base can switch to tautomer so pair with different base (enol-rare to keto-normal) - isomerisation switch
suppressor mutation
2 mutations but restores phenotype
intragenic: 2nd suppresses 1st
frameshift suppression: most sequence okay
intergenic: 2nd mutation in diff gene e.g. nonsense
nonsense suppression: mutation to tRNA, inserts AA instead of stop codon so can get back to original sequence except for 1 AA
supF
suppresses amber mutations
insert glycine at stop codon site
example of mutagens
nitrous acid reactive oxygen species alkylating agents intercalating agents UV light
mutation rates
frequency per generation
can’t record silent mutations
deamination of bases
removal of amine group (NH2 replaced by =O)
caused by nitrous acid
cytosine converts to uracil, guanine to xanthine (not problem), adenine to hypoxanthine (pairs with C so problem)
reactive oxygen species
natural side product of aerobic R
from chemical reactions caused by UV light/ionising radiation
cause changes to DNA (oxidation and addition to double bonds)
so can change base pairings
alkylating agents
chemicals that react with DNA adding alkyl groups (CH3CH2-)
e.g. EMS often used in chemotherapy - changes base pairings
affect coiling because extra bulky group
affect how proteins bind to DNA
intercalating agents
flat multiple ring structures so squeeze into DNA
binds between base pairs so leads to frameshift
stretch and distort helix
UV light causes 2 pyrimidines to form dimers
kink in DNA so point mutation or polymerase falls off
segregation of mismatched base pairs
deamination of 1 strand so 1 daughter cell wild type and 1 mutatn so culture is a mixture of diff genotypes
phenotype lag
phenotype not seen for several generations e.g. resistance to T1 because protein that phage binds to decreases over generations till none so then resistant
select mutants
easy for drug/phage resistance on plates
can’t see replication errors because all dead so need conditional lethal mutants
cross feeding
blocked metabolic pathways, provide each other with metabolites so depend on each other to grow
look like prototrophs but aren’t
ames test
identify mutagenic chemicals
plate w/ or w/o chemical
difference in no. bacteria if mutagenic
BUT metabolite may be mutagenic instead of chemical itself
operon
in prokaryotes
group of genes under control of same promoter
regulated together
different places where translation can start so more than 1 protein
housekeeping genes
required to be active all the time (constitutively expressed)
not all genes are like this because switch off when not needed to save energy
Lac operon
not constitutively transcribed
breaks down lactose
diauxic growth
2 growth phases
1st: glucose used up
lag phase: E.coli can’t grown so turn lac genes on
2nd: lactose used
LacY
LacZ
LacA
β-galactosidase permease - lets lactose enter cell
β-galactosidase - cleaves glycosidic bond, to glucose and galactose
galactoside acetyl-transferase - transfer acetyl group to galactosides and glucosides
default when glucose used
when use lactose
LacI protein binds to operator so blocks promoter so RNA pol. can’t bind to operon off
allolactose (comes from LacZ- small amount always present) is inducer, disables repressor protein LacI by binding to it so RNa Pol. binds promoter and makes mRNA of lac genes
other processes involved in glucose and the lac operon
glucose inhibits adenylatee cyclase enzyme which makes cAMP, so CAP in certain conformation
no glucose means CAP changes conformation so binds to promoter and helpds RNA Pol. bind so transcription is enhanced
competence
ability of bacerial cell to take up extracellular DNA from env.
artificial transformation
electroporation: DNA into bacterial cell with electric pulse by creating pores in membrane
natural transformation
1 cell releases, other cell takes up
horizontal gene transfer
transformation
transduction
conjugation
transposition
when are cells naturally competent?
when about to enter stationary phase - when stop growing
quorum sensing
ability to regulate genes based on population density (know how dense culture is)
very dense=take up DNA
B.subtilis mechanism for competence
cells secrete ComX so conc. increases with cell density
bind to ComP which changes gene regulation so becomes competent
RecA
DNA repair protein
involved in homologous recombination to integrate new DNA
how does bacteria distinguish between DNA of diff species?
recognise specific sequences (by sequences in DNA of surface proteins)
transduction
2 types
genetic exchange in bacteria, mediated by bacteriophages
generalised transduction: transfer any DNA, occasional incorrect packaging so package host DNA instead of viral so when infect new cell, insert host DNA
specialised transduction: transfer specific genes (next to phage DNA) by lysogenic phages
phage DNA cuts self out of host by loop, sometimes loop picks up host genes so carry to next host
Lambda phage
double stranded linear DNA
tail important for interacting with E.coli
can be lytic or lysogenic
lytic cycle of λ phage
inserts linear DNA into E.coli and DNA circularises in cell
new virions assembled which lyse the cell and release them
lysogenic cycle of λ phage
inserts DNA which integrates into E.coli genome
transmitted to daughter cells and lives until trigger for lytic cycle
lysogen
strain of beacteria carrying a lysogenic phage
prevents other phages infecting it
prophage
phage in lysogenic state
Lederberg and Tatum
2 cultures opposite in what can grow on, no colonies on minimal media but wild type growth if mixed together
so shows conjugation - mix DNA
Davies
2 strains in glass tube with filter that allows media through but not cells
no growth if on minimal media but growth if remove filter because require cell-to-cell contact to transfer DNA
plasmids
piece of double stranded DNA
most are circular but some linear
incompatible: related plasmids sharing common replication mechanisms can’t coexist
episomes: plasmids that can integrate into host genome
role of plasmids
carry non essential but highly useful genes (controlling replication and copy no.)
some are conjugative - encode tra genes needed for transfer
virulence factors
toxins that increase pathogenicity
bacteriocins
proteins killing/inhibiting closely related species
conjugation
one bacterium transfers genetic material to another through direct contact
process of conjugation
bacteria sends out F pilus (fertility factor) - mating pair connection - unidirectional transfer of DNA
cells pull closer when pilus makes contact, pore made and plasmid transferred
both cells retain plasmid
rolling circle replication in conjugation
DNA nicked at DSO (double stranded origin)
proteins unravel 2 strands so 1 strand goes into new cell and 3’ end recognised by DNA Pol. which synthesises 2nd strand
Hfr strain
high frequency recombination
F plasmid (episome) integrated into genome by recombination
plasmid nicked in chromosome, unravel and transfer single strand, made into double strand
can’t circularise because not all chromosome transferred so most is degraded but sometimes recombination occurs
new strand has only some genes so not whole F plasmid
F plasmid can excise from genome so become plasmid again, incorrectly takes some host DNA = F’ plasmid
recombination
break and rejoin DNA into new combination
2 types: homologous and non-homologous
homologous recombination
switch similar DNA
requires holiday junctions
ALIGNMENT: helices align
BREAKAGE: 1 strand nicked by E.coli enzyme RecBCD at specific sequences
INVASION: free 3’ end pulled off, stabilised by SSB protein and catalysed by RecA, strand invades other double helix because homologous so displacement
CROSS STRAND EXCHANGE: 2nd nick so 2 strands exchange
BRANCH MIGRATION: switch strands, requires RuvAB helicase, cross over=holiday junction - needs to be resolved so rotated to make cross so no crossover
ISOMERISATION: crossing and uncrossing of strands, can result in 2 outcomes, by RuvC nuclease and RuvAB
RecA
essential for DNA repair
bind to ssDNA
stabilise and help displacement
2 binding sites: hold 2 DNA together and catalyses branch migration
RecBCD
nuclease - catalyses single stranded nick
helicase - unwind DNA
non-homologous recombination
insertion sequences (transposons): hop from 1 position in DNA to another (transposition) catalysed by transpotase (encoded by insertion sequence), no other genes in them, have tandem repeats at ends needed for insertion, no new phenotype, can disrupt genes, high degree of reversion
transposons: bigger version of insertion sequences, carries additional genes like resistance, can carry tra genes (make pili) so conjugate
tandem repeats bound by transpotase, cuts out transposon from sequence and repair original sequence, carry transposon to new target sequence
replicative transposition: original copy retained and new copy insets elsewhere
conservative transposition: cuts out inserts elsewhere
