Stationary phase - stress response L8 Flashcards
how do bacteria effect protein production
Bacteria activate and induce systems to make new proteins to carry out new functions switch off systems don’t need to adapt to environment
how are bacteria grown in a batch culture
Cells are inoculated into a sterile broth, incubated under permissive growth conditions
when does cell mass increase in batch culture growth
Cell mass increases rapidly as cells enter exponential phase of growth, then reach stationary phase
when does cell mass stop increasing
until conditions inside flask limited
why is it important for single cells to monitor nutrients
needs to be able to monitor level of nutrients and respond that will run out - no point continually running processes if these processes can’t run to completion, need to start to ‘turn things down’
when do toxic products start to accumulate in batch growth, effect
Will get a change in environment, pH tends to drop, oxygen lowers, toxic by-products will start to accumulate– protect against harsh conditions
Cell growth stops, and cells go into a dormant state
what is the stringent response
Fast cell growth requires high levels of protein synthesis, so cell needs lots of ribosomes
Under starvation conditions, when amino acids levels are limited, cell reduces number of ribosomes - would use a lot of energy to make all proteins that wouldn’t be used
how is regulation achieved
Regulation achieved by stringent factor acts with GTP in synthesis of ppGpp
stringent factor (RelA protein)
how can ppGpp be synthesised
Carbon limitation can also cause synthesis of (p)ppGpp
what does ppGpp do
down regulates the rRNA synthesis of genes = fewer ribosomes being assembled
- ppGpp down regulated tRNA synthesis
Everything in the cell is being down regulated
ppGpp and protein production
signal to RNA polymerase to stop making proteins, needs to switch off transcription – need to turn things down till have nutrients available again
what is ppGpp
an alarmone
how is ppGpp an alarmone
involved in the stringent response in bacteria, causing the inhibition of RNA synthesis when there is a shortage of amino acids present
what causes increased ppGpp
nutritional stress
how does transcription reprogramming occur
ppGpp will bind to the RNAP sites, causes “reprogramming” of transcription
what transcription changes occur during reprogramming in E.coli
results in changes in ~700 genes now changed in their gene pattern of regulation
- Most of them are down regulated – rRNA (to make ribosomes, fatty acids for membrane synthesis), some specific stress genes are up-regulated
how do bacterial cells in stationary phase differ to those in growing cells
- shorter and rounder (if rod shaped) (spheres would be smaller)
- altered metabolism (recycle molecules from other things)
- change in membrane and cell wall structure as defines shape
- increase resistance to number of different stresses e.g. starvation, osmotic shock – cell prepared self for ‘survival mode’
what are sigma factors like
interchangeable, control which sets of genes are expressed (differential expression)
what is core polymerase
enzyme that makes RNA in cell carries out transcription, when it binds to a promoter – core polymerase is directed where to start transcription by the sigma factor
where will the sigma factor bind to
-10 -35 motif
what are the motifs in bacteria
motifs differ in different sigma factors
what must a bacteria have to be expressed all the time
sigma 70 motif, can switch on others when they’re needed
how may names does each sigma factor have
two as discovered in two ways
what is the primary site of control of prokaryotes
transcription
how are prokaryotic genes arranged
operons
what is monocistronic
1 protein
what us polycistronic
more than one protein
what drives the expression of genes
promoters
are bacteria monocistronic or polycistronic
Bacteria have option to go from monocistronic to polycistronic and translated into separate proteins
how can genes and operons that are spatially separate on chromosome be controlled
by same regulatory mechanism (regulon)
e.g. stress response controlled by RpoS
what does RpoS control
novel gene expression at onset of stationary phase
how many genes are identified that can change pattern of expression
> 100 genes identified that change their pattern of expression – form a ‘regulon’ of genes
what are many genes needed for
stress survival
in regulon are genes down or up regualted
Some down regulated and others are up regulated – don’t have to go the same way in regulon
what molecules are needed for RpoS control of cell shape
bolA
dacC
ftsQAZ
what does bolA do
- morphogene controlling pattern of peptidoglycan synthesis and determines cell shape
- over expression causes rounder cells
what does dacC do
- transpeptidase in peptidoglycan synthesis
- crosslinking in peptidoglycan thickens cell wall - stronger
- over expression causes increased cell wall strength
what is dacC
penicillin-binding protein 6
what does ftsQAZ do
- cell division genes up-regulated
- increased cell division
- over expression causes cells to be rounder and shorter as not grown much before divided
what molecules does RpoS need for metabolism control
otsAB arcAB glg genes pex appA
what does otsAB do
- synthesis of trehalose (compatible solute) in cytoplasm
- protects cell against thermal and osmotic stress (dehydration) hold proteins in complex, more stabilised
what does arcAB do
- adaptation to anaerobiosis
what does glg genes do
- synthesis of glycogen (storage granules)
what does pex do
- carbon starvation proteins (utilisation of alternative C sources)
what does appA do
- uptake of organophosphates (phosphate starvation)
what molecules are needed in RpoS resistance control
katG
katE
dps
what is katG and katE
catalase genes
what are katG and katE like
resistant to oxidising agents
what is dps
(upregulated) structures the nucleoid
> small DNA binding histone-like protein
what does dps do
compacts DNA in nucleoid (if not growing, dividing can be compact)
protects DNA from damage
when is RpoS induced
ANY condition that interrupts cell growth will induce RpoS expression (often at the end of stationary phase)
when is RpoS switched off
Responding to environment they switch RpoS off again
how is it tested if RpoS is needed
Transient thing that the cell tests out if RpoS needed - very responsive to environment
e. g. nutrient or physical changes during exponential growth result can also in induction of RpoS regulation
- starvation
- acid shock
- oxidative stress
how much RpoS is present when in exponential growth phase
RpoS protein barely detectable in exponential phase growth when cells are growing fast and nutrients aren’t limited
what are the first two genes in the 2 gene operon
first - nlpD (lipoprotein of unknown function)
second - RpoS
how many promoters does RpoS have, name
Pr1
Pr2
what occurs in RpoS operon
Always make basal levels of gene expression occur from Pr2
> sigma 70 - dependent expression and NOT growth-phase regulated
Pr1 expression growth-phase induced
> sigma 70 - dependent expression
When things are bad, second promoter- Pr1 (in response to stress)
if nplD not present what happens
cannot get RpoS
how is RpoS gene regulation balanced
Lots of different cellular signals act as positive and negative signals to create balanced gene control
- cells don’t want to induce stress response unless condition are becoming extreme
what is up regulated in RpoS
ppGpp(p), pheromones
what is down regulated in RpoS
H-NS, cAMP, UDP-glucose
what is the effect of RpoS induction
regulon induction by any one stress will lead to cross-protection against other stresses
- i.e. stationary phase cells are more resistant to challenge
what is an examples of cells inducing genes for survival aid
Biofilm formation regulated by rpoS
effect of pathogenic bacteria on rpos induction
Pathogenic bacteria become primed for infection of hosts
- induction of many virulence genes
what are the features of stressed cells that are like spores
- condensed DNA
- small rounded cell shape
- synthesis of protective molecules
- gene expression controlled by alternative sigma factors
what is the stress response like in other bacteria - eubacteria
RpoS equivalent found in all eubacteria
- Mechanism of control is NOT always the same
what is the stress response in gram-positive
Equivalent system identified in Gram-positive bacteria = SigB
- Mechanism of control is very different, involving multiple anti-sigma factors
what is ppGpp
guanosine 5’-diphosphate, 3’-diphosphate
