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
how does HprK function in high and low glucose levels in gram pos bacteria?
in presence of high glucose acts as kinase; phosphorylates Hpr at ser46
at low glucose it has alternate mode as phosphatase; takes phosphate group back off Hpr ser46
discuss Hpr phosphorylation at low and high glucose?
at low glucose; EI dependent P at his15 by other PTS sugars
at high glucose; HprK dependent P at ser46
can HprK be phosphorylated at his15 and at ser46 at the same time?
no only one at a time
how does FBP and Pi relate to HprK phosphorylating Hpr at ser46?
when glucose present glycolysis occuring so FBP high
if no glucose glycolysis not happening so no PEP or ATP generation and buildup of Pi
Pi targets phosphodiester bond by nucleophilic attack causing dephos of ser46 on Hpr meaning his15 can be phos
but when lots of glucose lots of FBP available and HprK acts as kinase and phos Hpr at ser46 so his15 can’t be phos
what is Pi an indicator of?
nutrient limitation
how does Hpr phosphorylation lead to CCR?
when lots of glucose present infec for gram pos to use alt carbon sources
high glucose = high FBP = HprK-dependent phos of Hpr at ser46
when Hpr phos at ser46 it undergoes small conf change decreasing phos at his15 and allowing Hpr to act as cofactor and bind transcriptional regulator CcpA (transcriptional regulator) which shuts down expression of genes for using alt carbon sources
how does CcpA regulate expression of alternate carbon source genes in gram pos?
CcpA is a dimer that bind two copies of Hpr(ser46) forming a complex of four proteins
this complex can bind conserved cre (catabolite-responsive element) sites which is a sequence in promoter region of bacterial genome
so when glucose present this complex (Hpr(ser46)-CcpR) assembles and can bind cre sites stopping expression of genes responsible for utilisation of alternative carbon sources
how can CcpA act as both an activator and a repressor?
high glucose = high FBP = HprK a kinase - Hpr(ser46)-CcrP dimer binds cre elements
if cre element sitting upstream a lot it acts as activator, if it binds close to ribosome binding site it acts as repressor by physically blocking gene (this is how it causes CCR)
how does Hpr regulate inducer exclusion in gram pos bacteria?
Hpr(ser46) can act as allosteric regulator of proteins involved in utilisation of alt carbon sources
i.e. Hpr(ser46) can bind some of the uptake mechanisms for other sugars (e.g. maltose) blocking them by changing the structure of their transport proteins (e.g. malK)
how does PTS regulate CCR in gram pos bacteria by inducer expulsion?
phosphorylation of incoming sugars prevents them from transport back out of cell
but if glucose reappears you wanna activate CCR
Hpr(ser46) activates part of membrane import system activating phosphotase that strips P off alt sugar forcing it back across membrane/ exporting it (inducer expulsion)
this gets rid of the alt sugar so glucose can get used
outline the expression of the LicT transcription factor in gram pos bacteria?
in B. subtilis beta-glucosides transported by permease BglP; BglP expression induced in presence of beta-glucosides; translation requires TF LicT which is anti-terminator protein i.e. binds specific RNA target in 5’ region of bglp mRNA preventing formation of transcription terminator
LicT has RNA binding domain (CAT) and 2x PTS regulatory domains (PRD1 and 2) with conserved his residues
bglPH operon gets transcribed to mRNA but forms stem loop blocking translation initiation site so not translated - LicT binds upstream regulatory RAT domain of bglPH mRNA inducing conf change resolving stem loop and initiating translation
how is BglP expression regulated by the PTS system?
if EIIBC(bgl) phos it can phos the his residues of PRD1, if HPr phos at his15 (not ser46) is can phos his residues of PRD2
LicT only activated when PRD1 non-phos and PRD2 phos i.e. only when EIIBC(bgl) non-phos and Hpr phos at his15
under what conditions would EIIBC(bgl) be phosphorylated (so PRD1 phos and LicT inactive)
when no beta-glucosides present
under what conditions would HPr be phosphorylated at his15 (and so PRD2 phos and LicT active if PRD1 non-phos)?
when preferred carbon sources have been exhausted
how is PTS important to the virulence of streptococcus?
carbon sources essential for growth during early infection - CCR allows bacterial responses to nutrient availability in diff host tissues
s. pyogenes is strict pathogen of nasopharynx and skin where glucose availability limited but rich in other carbohydrate sources so must be able to use these e.g. with beta-glucoside operon
what is group A streptococcus?
aka GAS - opportunistic pathogen, commensal of mouth, skin, intestine, URT
beta haemolytic i.e. can rupture RBCs; GAS infections cause lots of diff disease; diff nutrient availability so needs good mechanism for controlling metabolism
why have bacterial pathogen adapted to the conditions of the mammalian ECM?
mammalian extracellular matrix rich in beta-linked dissacharide units e.g. beta-glucosides
bacterial pathogens may have have adapted to utilise these during infection
this is the link between sugar uptake (PTS) and virulence in bacteria like streptococcus
outline signal transduction systems in bacteria?
signal transduction systems allow bacteria to sense, respond and adapt to changes in their environment or intracellular state
signal sensing by protein sensors triggers a biochemical cascade known as a signalling pathway
what things can signal transduction systems result in/change?
cellular redox state
quorum signals
nutrients (PTS system)
antibiotics (Stk1, BcrR, CroRS and VanRS)
changes in osmolarity
what are serine, threonine, tyrosine protein kinases?
phosphorylate serine, threonine or tyrosine aa residues on target substrate; hydroxy side chains on these aa are target for phos - enzyme catalysed nucleophilic attack of OH proton on phosphodiester bond
regulate diff functions e.g. antibiotic resistance, virulence, capsule synth, sporulation
some of these kinases target multiple proteins; phos can turn a protein on or off depending on protein
discuss the Stk1 serine/threonine protein kinase in S aureus?
Stk1 phos global transcription regulator MgrA at ser100 and ser113; this anatagonises MgrA dimerization preventing DNA-binding so it can’t repress its target genes e.g. efflux pumps (de-repression)
also phos thr18 and thr88 in CcpA helix-turn-helix DNA binding domain disturbing protein-DNA interaction and activating CcpA-repressed promoters resulting in sugar metabolism, biofilm formation
under pressure of antibiotic Stk1 phos mgrA, CcpA resulting in expression of AMR genes
outline one component signal transduction?
one-component systems comprised of one protein with two parts; input domain recognises signal, output domain drives response
dominate signal transduction systems in bacteria and archaea; often cytosolic but can be membrane bound
discuss an example of a one component signal transduction system?
e.g. BcrR - membrane bound one component regulator of high-level bacitracin resistance in e. faecalis
when BcrR recognises bacitracin it forms dimer and then two of those form a dimer of dimers which binds DNA sequences bending the DNA to open up promoter so RNAP can transcribe bacitracin resistance operon
discuss two-component regulatory systems?
transduction of info about status of environment by one protein (sensor kinase) to a second one (response regulator)
found in all three domains of life; the average bacterium has 10-50 tcr systems
two types; we interested in ones w histidine kinase as sensor kinase
discuss the sensor kinase of two-component regulatory systems?
sensing domain binds stimulus
this triggers dimerisation and phosphorylation of his on dimerisation domain by ATP-binding kinase domain (catalytic domain that uses ATP to phos that protein)
this leads to phosphorylation of regulatory domain on response regulator thus triggering response in cell
discuss the structure of sensor kinases?
has multiple conserved domains and is heterogenous in size and aa sequence between bacteria due to detecting a variety of stimuli
N-terminal domain; detection, dimerisation, phosphotransfer
C-terminal domain; catalytic and ATP-binding domain; encompasses a number of conserved sites
how are sensor kinases of two-component regulatory systems recognised by common sequence properties?
they are heterogenous in aa sequence, however have conserved motifs in N, G1, G2 and F boxes which encode key functions - these serve as signatures of these molecules despite them looking like v diff proteins
how well do we understand two-component regulatory systems?
we can easily identify them by sequencing (conserved motifs) and understand their shared properties well
but don’t know what a lot of them do i.e. what stimulus they detect, what the response is etc.
outline how TCS are an important sensor for antibiotic challenge in enterococcus?
gram pos enterococcus has thick outer cell wall which is important for survival and so biosynthesis of this AM target
has a bunch of regulatory systems in cell wall as sensor for antibiotic challenge
outline peptidoglycan biosynthesis as an antimicrobial target?
NAGS and NAMS and peptide chains exported beyond cytoplasmic membrane and transpeptidase (PBP) reaction crosslinks these
vancomycin binds terminal D-ala-D-ala residues in pentapeptide stem of lipid II (essential precursor of peptidoglycan) which stalls biosynthesis inhibiting cell growth
outline vancomycin resistance in enterococci?
is conferred by change in target substrate (D-ala-D-ala) resulting in high-level acquired vanc resistance (D-ala-D-lac) or low-level instrinsic vanc resistance (D-ala-D-ser)
high-level: most common forms in enterococci are vanA-type and vanB-type
low-level: encoded by e. hirae vanC gene
how is high-level vanA-type and van-B type vancomycin resistance regulated in e. faecalis?
by two-component regulatory system VanRS; vanS sensor kinase and vanR response reg
vanS binds vanc resulting in autophos –> phos of vanR resulting in expression of vanc resistance genes vanH, A, X
how do the genes vanH, vanA and vanX actually cause vancomycin resistance?
vanH is a alpha-keto acid reductase (converts pyruvate to d-lactate)
vanA is an ATP-dependent depsipeptide ligase (joins d-ala to d-lac)
vanX is a D-ala-D-ala depsipeptidase (cleaves d-ala-d-ala off pentapeptide)
murF enzyme adds d-ala-d-lac onto pentapeptide (same as it does w d-ala-d-ala but with newly available d-ala-d-lac)
how does the cell wall sense antimicrobial-induced stress in gram pos bacteria?
two-component regulatory systems e.g. vanRS, croRS
outline the croRS TCS in e. faecalis?
is a cell wall stress TCS; CroR is response regulator, CroS is sensor kinase - regulates expression of over 200 genes (downregs or upregs)
enables cell to respond to many environemental stressors e.g. NaCl, glycine, temp
also linked to both antibiotic resistance and antibiotic tolerance
outline how croRS causes antibiotic resistance?
beta-lactams bind transpep blocking it from crosslinking
beta-lactam resistance conferred by expression of alternative PBPs; beta-lactams bind with less affinity
croRS regulates expression of alternative PBP called PBP5
deleltion of croRS genes or pbp5 restores susceptiblity to beta-lactams
how might TCS help in the fight against AMR?
could look at them as an antimicrobial target; which molecule interferes with phosphotransfer
