(24) Signaling and Signal Transduction

Lecture 24

prokaryotes regulate cellular metabolism in response to

environmental fluctuations (e.g., temperature, pH, O2 availability, nutrient availability)

some signals are small molecules that enter the cell and

function as effectors (inducers and corepressers)

in other cases, the signal is external and not directly transmitted to the regulatory protein but is detected by a sensor that transmits the signal to the regulatory machinery

this process is called signal transduction

most signal transduction systems are comprised of two parts so are called

two-component regulatory systems

two-component regulatory systems include

• specific sensor kinase proteins (usually located in the cytoplasmic membrane)

• response regulator protein (usually located in the cytoplasm) = DNA-binding protein that positively or negatively regulate transcription

kinase =

enzyme that phosphorylates compounds typically using phosphate from ATP

sensor kinases detect

signals from the environment and phosphorylate themselves (autophosphorylation) at a specific histidine residue (so are called histidine kinases)

sensor kinase then transfer the phosphate to the

response regulator → repressors or activates transcription

two-component regulatory systems have a feedback loop to

terminate the response

• a phosphatase removes the phosphate from the response regulator, resetting the system

two-component regulatory systems are common in

bacteria

• but rare in archaea and bacteria that live as parasites

two-component regulatory systems examples include

systems that detect and regulate expression in response to phosphate limitation, nitrogen limitation, and osmotic pressure

chemotaxis =

movement towards or away from a chemical (attractant or repellent)

chemotaxis - organisms sense the change in

concentration of a chemical over time (temporal) rather than the absolute concentration of the stimulus

chemotaxis - employs a modified two-component regulatory system to regulate

the direction of flagellar rotation

• this means that the activity of preexisting proteins is what is being regulated, not the transcription of genes

regulation of chemotaxis steps

• repose to the signal

• controlling flagellar rotation

• adaptation

regulation of chemotaxis step 1: response to the signal - methyl-accepting chemotaxis proteins (MCPs) bind

chemicals → change conformation → interact with the Chew

regulation of chemotaxis step 1: response to the signal - CheA (sensor kinase) becomes

autophosphorylated with the help of CheW

regulation of chemotaxis step 1: response to the signal - increase in attractant causes a

decrease in the rate of CheA phosphorylation

regulation of chemotaxis step 1: response to the signal - decrease in attractant, causes

an increase in the rate of CheA phosphorylation

regulation of chemotaxis step 1: response to the signal - CheA passes the phosphate to

CheY, which is the response regulator that controls flagellar rotation

regulation of chemotaxis step 2: controlling flagellar rotation - CheY controls the

direction of flagellar rotation

regulation of chemotaxis step 2: controlling flagellar rotation - once CheY is phosphorylated, it interacts with

the flagellar motor to induce clockwise rotation → induces a tumble

regulation of chemotaxis step 2: controlling flagellar rotation - when CheY is unphposrylated, it cannot

bind to the flagellar motor → flagellum rotates counterclockwise → causes the cell to run

regulation of chemotaxis step 2: controlling flagellar rotation - CheZ dephosphrylates

CheY, allowing the cell to run

regulation of chemotaxis step 2: controlling flagellar rotation - decreases in attractants leads to an

increase in the level of phosphorylated CheY → cell tumbles

regulation of chemotaxis step 2: controlling flagellar rotation - in contrast, if the cell is swimming towards attractants, higher attractant levels leads to

lower level of phosphorylated CheY suppresses tumbles and promotes runs

regulation of chemotaxis step 3: adaptation

once an organism has reposed to a stimulus, it must stop responding and reset the system

regulation of chemotaxis step 3: adaptation - employs a feedback loop

• when MCPs are fully methylated, they no longer respond to attractants

• when MCPs are unmethylated, they response strongly to attractants

regulation of chemotaxis step 3: adaptation - MCPs are methylated by

CheR and demethylation by phosphorylated CheB

regulation of chemotaxis step 3: adaptation - if attractant is high, CheA autophosphorylation will be

low → leads to unphosphorylated CheY and CHeB → run

regulation of chemotaxis step 3: adaptation - methylation of MCPs increases because

CheB-P is not present to demethylate them

regulation of chemotaxis step 3: adaptation - since MCPs no longer respond to the attractant when they are fully methylated, if

the attract remains high but constant, the cell will begin to tumble in order to stay in area of high concentration

Che proteins also play a role in

regulating other forms of taxis

phototaxis =

movement toward light

Che proteins and phototaxis

light sensor replaces MCPs and interacts with cytoplasmic Cheproteins to direct runs or tumbles

aerotaxis =

movement toward oxygen

Che proteins and aerotaxis

redox protein monitors O2 levels and interacts with cytoplasmic Che proteins to direct runs or tumbles

quorum sensing:

mechanism by which Bacteria and some archaea assess their population density

quorum sensing ensures that

a sufficient number of cells are present before initiating a response that, to be effective, requires a certain cell density (e.g., toxin production)

quorum sensing: widespread in

gram-positive and gram-negative bacteria and in some archaea

quorum sensing utilizes an auto inducer for regulation =

specific signaling molecule produced by the participating organism

auto inducer for regulation

• freely diffuses across the cell envelope in both directions

• only reaches a high concentration in the cell when the cell density is high

autoinducer binds to a

specific activator protein or sensor kinase, triggering transcription of specific genes

several classes of autoinducers exist, including:

• acyl homoserine lactones (AHLs) - gram-negative bacteria

• autoinducer 2 - gram-negative bacteria

• short peptides - gram-positive bacteria

quorum sensing in used for:

• regulating light production in bioluminescent bacteria

• regulating cell differentiation in yeast (single cells vs filaments)

• virulence factor production by pathogenic bacteria (toxins, extracellular peptides that damage host cells)

virulence factor regulation in Shiga toxin-producing E.coli - as the bacterial population increases, AI-3 produced by E. coli plus

epinephrine and norepinephrine produced by intestinal cells acculumulate and bind to sensor kinases in the E. coli cytoplasmic membrane

• leads to phosphorylation and activation of two transcriptional activator proteins

virulence factor regulation in Shiga toxin-producing E.coli - activators activate the transcription of genes encoding

motility functions, toxin secretion functions, and proteins that form lesions in the host intestinal mucosa

quorum-sensing is also used by cells to:

• determine if their population density is sufficient to initiates biofilm formation

• regulate the production of secondary metbolites

• coordinate swarming motility

• detect the presence of other bacterial species

quorum sensing disruptors are potential drugs for

dispersing biofilms and preventing virulence gene expression

organism often need to regulate

unrelated genes simultaneously

global control systems =

regulatory mechanisms that response to environmental signals and regulate the expression of many different genes simultaneously (e.g., lactose operon and maltose regulon)

catabolite repression (“glucose effect”) is an example of global control

• controls which carbon source is used if more then one is present ensures the “best” course is used first

• synthesis of enzymes used in the catabolism of dozens of other sugars (lactose, maltose, arabinose) is repressed if glucose in present in the growth medium/environemtn

catabolite repression can result in diauxic growth =

two exponential growth phases separated by a lag phase

catabolite repression - cells grow first on the

preferred carbon source; genes for catabolizing the second carbon source are not expressed

catabolite repression - after the preferred carbon source is depleted, there is

a lag in growth while the cells start expressing the enzymes needed to catabolize the second carbon source

catabolite repression - exponential growth resumes on the second carbon source after

the cells have synthesized adequate amounts of catabolic enzymes

catabolite repression relies on an activator protein =

cyclic AMP receptor protein (CRP)

• form of positive control

genes that encode a catabolite-repressible enzyme are only expressed if

CRP binds to DNA in the promoter region; CRP can only binds to DNA when it is bound to cyclic adenosine monophosphate (cAMP)

cAMP is a key molecule in

metabolite control and is considered a regulatory nucleotide (and is an inducer type of allosteric effector)

cAMP is synthesized from

ATP by the enzyme adenylate cyclase

glucose inhibited the synthesis of

cAMP and stimulates its transport out of the cell

when glucose is present, the intracellular concentration of cAMP is low →

cAMP not available to bind to CRP → CRP cannot bind to DNA → cannot help RNAP bind the promotor → no transcription of the catabolite repressed genes

catabolite repression is the indirect result of

the presence of a better energy source (glucose) which reduces the amount of cAMP in the cell → precludes CRP-cAMP activation

the lac operon is controlled by

catabolite repression

for lac genes to be transcribed, two things are necessary:

• cAMP must be available so the CRP-cAMP complex cn bind to the promoter of the lac operon, helping RNAP bind to initiate transcription

• lactose or another molecule that can act as an inducer must be present to prevent the lactose repressor from blocking transcription by binding to the operator

lac operon repression - Lacl (repressor) gene is controlled by

a different promoter than the lac operon genes

lac operon repression - Lacl binds the operator, inhibiting RNAP binding the promoter, unless

allolactose (inducer) is present

lac operon repression - RNAP needs

CRP-cAMP to bind the promoter

lac operon repression - RNAP will bind the promoter and initiate transcription if

glucose is absent (CRP-cAMP is present) and lactose is present (Lacl inactivated)

stringent response =

a widely distributed regulatory mechanism used by bacteria to survive nutrient deprivation, environmental stresses, and antibiotic exposure

triggering the stringent response ultimately leads to a

shutdown of macromolecule synthesis and the activation of stress survival pathways to improve the cell’s ability to compete in nature

strident response - following shift to a nutrient-poor medium , synthesis

of most macromolecules are down-regulated

strident response - synthesis of proteins required to makes

amino acids not present in the poor medium must be induced

strident response - response is triggered by regulatory nucleotides:

guanosine tetra phosphate (ppGpp) and guanosine pentaphosphate (pppGpp) = alarm ones

strident response - alarmones are made by the

RelA protein when uncharged tRNAs (due to low amino acids) enter the ribosome

alarmones inhibts DNA replication and

transcription of most genes, but activate the stress response pathways

stringent response is a global mechanism that balances the

metabolic state of the cell, so the environment of the bacterium ultimately determines the response casade

E. coli voided in feces goes from a nutrient-rich intestinal environment to a nutrient-poor enviornment → initiates

ppGpp synthesis, stringent response occurs

strident response and caulobacter

normally inhabits nutrient-poor freshwater environment; stringent response triggered by carbon/ammonia starvation (instead of amino acid limitation) → guanosine tetra peptide (ppGpp) increases swarmer (motile) cell formation so the cell can hopefully reach a niche with more nutrients

mycobacterium tuberculosis and stringent response

dormant granulomas of the lungs are hypoxic and phosphate-limited → triggers strigent response → converts a subpopulation to dormant persister cells that are resistant to antibiotics and can revert back to infective cells

other well-studied global networks include

the phosphate (pho) region, heat shock response, and RpoS regulon

the phosphate (pho) regulon:

inorganic phosphate is often limiting in many environments; pho regulon evolved in Bacteria to deal with inorganic phosphate limitation

the phosphate (Pho) regulon consists of

a two-component regulatory system that activates genes encoding extracellular enzymes for obtaining Pi from organic phosphates, Pi transporters, and Pi storage enzymes and represses genes involved in antibiotic production and nitrogen metabolism

heat shock proteins:

counteract heat damage (denatured proteins) and help cell recover from termpature stress

three major classes of heat shock proteins:

Hsp70 (DnaK in E. coli), Hsp60 (GroEL in E. coli), and Hsp10 (GroES in E. coli)

Hsp70 (DnaK in E. coli) prevent

aggregation of newly synthesized proteins and stabilizes unfolded proteins

Hsp60 (GroEL and GroES in E. coli)

molecular chaperones, catalyze the correct refolding of unfolded proteins

in many bacteria, the heat shock response is controlled by the

alternative sigma factor RpoH (s32) which regulates the expression of heat shock proteins

RpoH is normally degraded within a

minute or two of its synthesis

however, when cells suffer a heat shock, RhoH degradation is

inhibited → RpoH levels increase → increased expression of heat shock proteins

the rate of RhoH degradation depends on the

level of free DnaK, which inactivate RpoH

in unstressed cells, the level of free DnaK is

high and the level of intact RpoH is corresponding low

however, DnaK binds preferentially to

unfolded proteins so when heat begins to unfold protiens, DnaK is no longer available to degrade RhoH → RhoH can activate shock gene expression

when the stress has passed, DnaK binds

RhoH again → synthesis of heat shock proteins is reduced