Vernon's lectures Flashcards
why do bacteria have transport systems and how does this show in their genome?
cell survival requires the generation and maintenance of electrical and chemical concentration gradients across the generally impermeable cell membrane
5-10% of genome dedicated to transport i.e. huge amount of energy goes into controlling it
what are the three main classes of bacterial transport system?
ATP-binding cassette (ABC) superfamily
major facilitator superfamily (MFS)
group translocation e.g. phosphotransferase system (PTS)
what is the ATP-binding cassette (ABC) superfamily?
a transport system superfamily which performs primary active transport i.e. transports molecules or ions from low–>high conc. against gradient by coupling ATP to movement of these molecules/ions across cell membrane
ABC transporters have two forms allowing them to be closed at all times - has binding proteins that bind molecule, these then bind ABC transporter triggering break of high energy ATP phosphate bond which forces structural change allowing it to pass through transporter
e.g. maltose
what is the major facilitator super family (MFS)?
class of bacterial transport system that facilitates diffusion of ions/solutes across cell membrane
can be passive (high–>low) or secondary active (co-transport using electrochemical gradient)
for secondary example is lacY requiring H+ ion to transport lactose against conc. gradient
outline the group translocation e.g. phosphotransferase system (PTS)?
like what it does and what it allows
performs active, energy dependent transport - couples transport with chemical modification of substrate
e.g. as consequence of enzyme system bringing glucose into cell it get phosphorylated to glucose 6-P
group translocation allows nutrient uptake under low cell energy conditions allowing for re-initiation of growth and survival in hostile environment
outline the PTS system?
aka phosphoenylpyruvate (PEP): sugar phosphotransfer system
catalyses the transport and phosphorylation of sugars across cell membrane in preparation for catabolism and energy generation
what are the three main components of the PTS system?
EI and Hpr - the two conserved non-specific components utilised for uptake of all PTS sugars
EII - the specific component of which there is a diff EII transporter for diff PTS sugars (e. coli has 21), is a complex of sugar-specific proteins A, B and C; C embedded in membrane and A and B in cytoplasm
PEP located near EI
how does group translocation occur in the PTS system?
phosphate group (from PEP–>pyruvate) gets picked up by EI
once EI phosphorylated it phosphorylates HPr then to EIIA then to EIIB and as glucose moves through EIIC it gets the phosphate group from EIIB in the process
so direction of phosphotransfer opposite to direction of glucose transport
why is the PTS system an important regulator of cell function?
PTS sugars control catabolite repression, gene transcription regulation, virulence, inducer exclusion and many other important things
outline how the PTS system is an important regulator of carbon metabolism and what this allows you to ultimately regulate?
not efficient for cell to make all the proteins required for all sugars all the time
carbon flow important for making amino acids needed for cell growth - diff PTS sugars go through diff pathways to feed into basic carbon flow (glycolysis, TCA cycle)
by regulating uptake of sugars you can regulate this carbon flow and ultimately cell growth
why and how is the PTS system a regulator of carbon metabolism through carbon catabolite repression (CCR)?
why: allows bacteria to rapidly select a preferred carbon source i.e. adapt quickly to changing environments
how: CCR inhibits synthesis of enzymes involved in catabolism of secondary carbon sources through:
- altering activities of specific regulators
- activation of global control proteins
CCR leads to selective utilisation of carbon sources; mode of CCR action varies between gram neg and gram pos
what is diauxic growth in e. coli?
a classic example of carbon catabolite repression (CCR)
in growth medium with glucose and lactose available; glucose used initially (exponential growth phase) until it runs out and e coli enters short lag phase then starts growing again but using lactose
shows that something is regulating this switch
why is glucose the most preferred carbon source?
high availability
low energy expenditure
high growth
what are the three main systems/mechanisms by which PTS-mediated carbon catabolite repression (CCR) occurs in e. coli?
transcriptional regulation
inducer exclusion
transcription factors
what are the main players for PTS-mediated CCR occur in e coli through transcriptional regulation?
EIIA - a component of the PTS
adenylate cyclase - catalyses conversion of ATP –> cAMP
cyclic AMP (cAMP) - secondary metabolite that is an indicator of cellular carbon and energy levels
crp/CAP - dimeric transcriptional activator
discuss the phosphorylation state of EIIA during high and low/no glucose?
during high glucose phosphorylation of EIIA is transient i.e. essentially de-phosphorylated
during low/no glucose phosphotransfer cascade becomes saturated = EIIA phosphorylated
phosphorylation of EIIA leads to activation of expression for genes responsible for uptake/break-down of alternative carbon sources
how does phosphorylation of EIIA result in uptake/break-down of alternative carbon sources?
when phosphorylated EIIA binds C-terminal of adenylate cyclase activating it so it converts ATP to cyclic AMP resulting in increase in cellular cAMP levels
cAMP binds CRP and together they act as the global transcriptional activator of genes involved in catabolism of alternative carbon sources (so they pre much turn on expression of those genes)
what is the structural mechanisms behind PTS-mediated CCR in e coli by transcriptional regulation?
cAMP-CRP bind specific DNA sequences in the promoter regions of genes
they bend DNA to facilitate recruitment of RNAP (by making promoter accessible for interaction with RNAP)
this can occur by two diff mechanisms (class I and class II) depending on where it binds in promoter region i.e. where it binds determines the effect it has on diff genes
discuss PTS-mediated carbon catabolite repression in e coli via inducer exclusion?
inducer exclusion is a form of repression where an inducer is excluded from the cell to prevent it from functioning
EIIA is an allosteric regulator of proteins involved in the utilisation of alternative carbon sources
outline PTS-mediated CCR in e coli in the lac operon?
lactose required for activation of lac operon
lactose permease (LacY) responsible for uptake of lactose in e coli
in the presence of glucose lactose uptake and catabolism inhibited by inducer exclusion
non-phosphorylated EIIA binds lacY locking it in inactive conformation so that it blocks lactose entry to cell and thus lac operon expression cant be induced
outline gram-positive bacteria?
lack an outer membrane, thick peptidoglycan layer of the cell wall (biosynthesis of this a major AM target)
includes clinically relevant genera e.g. enterococcus, staph, listeria, strep
outline the PTS in gram-pos bacteria (B. subtilis)?
very similar to in e. coli, genes for secondary C sources not expressed when preferred C sources available
but there are key regulatory differences in carbon catabolite repression (CCR)
E. coli CCR - prevents activation of catabolic genes in presence of glucose
B. subtilis CCR - negative regulation via repressor protein in presence of glucose (when glucose present something actively blocks prod of genes rather than turning off/on)
what are the major players in PTS-mediated CCR in gram-pos bacteria via transcriptional regulation?
Hpr - component of PTS
HPrK - a bifunctional kinase/phosphatase
Fructose1,6-biphosphate (FBP) - glycolytic metabolite: indicates high glycolytic activity (glucose present)
Pi - inorganic phosphate: indicates nutrient limitation
catabolite control protein A (CcpA) - dimeric transcriptional repressor
the P state of _____ is central to CCR transcriptional regulation in gram pos bacteria?
HPr
