Environment Sensing and Response

Question 1

Define diffusion.

Answer 1

Uncharged gases diffuse freely through OM and CM. Driving force is delta C, difference in concentration.

Question 2

Describe porins.

Answer 2

In gram neg bacteria and others with OM, solutes mainly diffuse into periplasm through porins in the OM. A passive diffusion process, dependent on delta C.

Question 3

Describe permeases.

Answer 3

An array of transporters (permeases) moves most solutes across the CM.These transporters are very efficient. A functional consequence of this is that the periplasmic concentrations of transported solutes remain low enough to drive continued, rapid diffusion through porins.

Question 4

Describe facilitated diffusion.

Answer 4

Driven by delta C, glyercol update by E. coli.

Question 5

Describe active transport.

Answer 5

Establishes conc gradients, maintains concentration gradients in which [solute]in is 3 to 6 orders of magnitude greater than [solute]out. Displays saturation kinetics, includes many types of transport, which can be divided into two classes, primary and secondary transport.

Question 6

Describe ABC transporters.

Answer 6

Primary transport. Have ATP-Binding Cassettes, use energy from ATP hydrolysis. Multicomponent complexes, 2 ABCs and 2 transmembrane (permease) domains. In prokaryotes, ABC transporters can behigh-affinity uptake systems. Uptake ABC transporters in gram-negative bacteria are also sometimes called shock-sensitive transport systems, because they have periplasmic substrate-binding proteins (components) which are lost when cells are subject to osmotic shock.

Question 7

What are the steps of ABC transport?

Answer 7

1. solute enters periplasm via OM pore (porin)
2. substrate-binding protein binds solute and undergoes conformational change
3. solute-bound binding protein binds to periplasmic face of permease
4. transporter complex transports solute across cell membrane, with concomitant hydrolysis of ATP
5. transporter returns to unstimulated state

Question 8

Describe group translocators: phosphotransferase system (PTS).

Answer 8

Covalently modify the solute when moving it across the CM, phosphorylates it. Conc gradient not established, but phosphoenolpyruvate (PEP) is used.

Question 9

What are the steps of PTS?

Answer 9

Steps in PTS transport:

1. PEP-dependant phosphorylation of EI
2. EI~P donates phosphoryl group to HPr
3. HPr~P phosphorylates EIIA
4. EIIA~P donates P to EIIB
5. EIIC-EIIB~P complex transports substrate into cell, phosphorylating the substrate

Question 10

Describe the proteins involved in PTS.

Answer 10

There are 16 different EII transporters in E. coli, each specific for a different sugar.
EI and HPr are general PTS proteins, shared by all PTSs and encoded in a single pts operon
EI: Enzyme I (ptsI), soluble protein kinase
HPr: Histidine protein (ptsH), links the EI with the multiple EII components, can phosphorylate any EIIA in the cell
EII components (Enzyme II) are substrate-specific PTS proteins -named according to substrate(e.g., EIIGlc transports glucose), encoded by individual operons, 3 functional domains that may be variously fused as 1 or 2 proteins
EIIC: membrane-bound transporter, binds substrate
EIIB: cytoplasmic, forms a complex with IIC, often fused with IIC as a single protein
EIIA: cytoplasmic, forms a complex with IIB, usually separate protein

Question 11

How much energy does PTS use?

Answer 11

One substrate molecule is translocated per PEP dephosphorylated. This is energetically equivalent to consuming one ATP, since conversion ofPEPto pyruvate in glycolysis (central metabolism) yields one ATP. Note that the glucose-6-P released into the cytoplasm by the PTS is identical to the first intermediate in glycolysis. This obviates the need for the ATP-dependent glucose (hexose) kinase reaction to start glycolysis.

Question 12

Describe rhodopsins.

Answer 12

Light-driven transporters, primary transport. Form a pore in the CM, have a retinal chromophore bound to a lysine residue located in the pore. The retinal is capable of a light-driven trans-cis isomerization that can drive transport. This system transduces energy, converting light energy to a chemiosmotic (electrochemical) gradient.

Question 13

What is the mechanism for bacteriorhodopsin?

Answer 13

a, Light-induced isomerization of the protonated retinal from all-trans to 13-cis triggers the transfer of the proton to aspartate 85, aided by a slight movement of this residue in the L intermediate (b) towards the nitrogen atom. In the M state (c), the deprotonated retinal (yellow) straightens, pushing against helix F and causing it to tilt. This opens a channel on the inner, cytoplasmic side of the membrane through which aspartate 96 is reprotonated (d), having given up its proton to the nitrogen on the retinal. Aspartate 85 transfers its proton through a network of hydrogen bonds and water molecules to the outside medium, past arginine 82, which has moved slightly. Red arrows, proton movements; blue arrows, movements by groups of atoms. Helices D and E are omitted in b–dfor clarity. The ‘paddle’ attached tohelix F represents the bulky side chains, which move to open the cytoplasmic proton channel.

Question 14

What is a halorhodopsin?

Answer 14

Primary transport, light-driven anion pump that can import chloride, other halides and nitrate. The function of this pump may be to maintain the proper osmotic potential in the cytoplasm of halotolerant archaea.

Question 15

Describe ATPases.

Answer 15

Primary transporter, proton or sodium driven. Use gradient to make ATP, use ATP to pump solutes out of cell.

Question 16

What is secondary transport?

Answer 16

Driven by H+ and Na+ gradients, established by primary transport. Symport, antiport, uniport. PMF usually directly or indirectly drives it.

Question 17

Describe the two parts of the PMF.

Answer 17

the membrane electrical potential (delta psi) and the membrane proton gradient (delta pH). For all secondary transport, one or both components may drive transport.If a net translocation of charge occurs, the delta psi is involved; if a net translocation of H+occurs, then the delta pH is involved.

Question 18

What is symport?

Answer 18

Secondary transport. Simultaneous transport of two species in the same direction, one of which flows down a concentration gradient. Neutral solute + H+ symport (e.g., LacY lactose permease) uses both delta psi and delta pH. Neutral molecule + Na+ symport uses only delta psi. Negative molecule + H+ symport uses only delta pH. Negative molecule + Na+ symport (e.g., melibiose permease) uses only Na+ gradient; does not directly use the PMF, but the Na+ gradient is often established by Na+ + H+ antiport.

Question 19

What is antiport?

Answer 19

Secondary transport. Simultaneous transport of two species in opposite directions by a single transporter, again driven by one flowing down a concentration gradient. Neutral molecule + H+ antiport uses both delta psi and delta pH. Types of secondary transport across the CM membrane. Positive molecule + H+ antiport (e.g., Na+/H+antiporter) driven by delta pH.

Question 20

What is uniport?

Answer 20

transport of a single species driven by delta psi; thus,either a positive species moved into cell or a negative species moved out.

Question 21

Describe signal transduction systems.

Answer 21

Components: sensors, transducers, regulators. Stimulus detection, signal processing, output.

Question 22

Describe histidine kinase-based signal transduction systems.

Answer 22

Most common system. Based on phosphotransfer rxns between histidine and aspartate residues. Two varieties: phosphorelay systems and and two-component systems. Depends on the number of phosphotransfer components.

Question 23

Describe two-component systems.

Answer 23

Signal transduction system. Components: sensor kinase and response regulator. Sensor kinase: contains a conserved His(H) residue that is phosphorylated in response to an environmental stimulus and an ATP-binding site. Response regulator: contains a conserved Asp(D) residue that is phosphorylated by the sensor kinase and that effects an output response.

Question 24

Describe the sensor kinase of two-component systems.

Answer 24

Consist of an N-terminal input (sensor) domain and a C-terminal transmitter domain. Function as homodimers, but do not always exist as such in the cell. Often membrane-bound. The input domain senses the external stimulus via a region that is usually on the external surface of the membrane. The input domain also contains 1 to 8 (usually 2) TM segments. The transmitter domain harbours two essential elements: the key His that is reversibly phosphorylated and a catalytic/ATP-binding site. The sensor kinase has two states: autokinase (K) and phosphatase (P). As an autokinase, the key His is phosphorylated at the expense of ATP. The phosphorylated transmitter domain transfers its phosphoryl group to a response regulator. The unphosphorylated transmitter domain can act as phosphatase, dephosphorylating the response regulator.

Question 25

Describe response regulators of two-component systems.

Answer 25

Soluble proteins that consist of an N-terminal receiver domain (REC) and a C-terminal output domain (effector). REC consists of ~125 residues and includes the conserved Asp residue whose phosphorylation state is mediated by the sensor kinase.The output domain has some kind of activity. Phosphorylation of the Asp residue induces a conformational change that leads to the activation of the output domain. The regulator is usually phosphorylated by a sensor kinase, but a phosphotransfer module may serve this function in more complex signalling pathways. REC domains are well conserved while the output domain varies significantly. Response regulators that bind DNA usually function as dimers. It is essential that a given sensor interacts with only its cognate regulator. This specificity is ensured by non-conserved sequences in the transmitter and receiver domains.

Question 26

What does the EnvZ/OmpR two-component system regulate?

Answer 26

The expression of numerous genes (OmpF and OmpC) in E. coli in response to the osmolarity and pH of the bacterium’s environment.

Question 27

Describe OmpF vs OmpC

Answer 27

Both allow passage of small hydrophilic molecules, OmpF ha a larger diameter. Osmolarity goes up: OmpC goes goes up, osmolarity goes down: OmpF goes up. Both belong to OmpR regulon.

Question 28

Describe the EnvZ/OmpR system.

Answer 28

EnvZ is the sensor kinase, and the cytoplasmic N region senses osmolytes that are made in response to increased extracellular osmolality. The C region contains the H and ATP-binding site. EnvZ~P phosphorylates OmpR regulator. C domain of OmpR binds to nucleotide sequence.

Question 29

Describe phosphorelay systems.

Answer 29

Phosphorelay systems have the sensor kinase and response regulator of two-component systems as well as two intermediary components: receiver (REC) and His-containing phosphotransfer (HPt) domains. The additional REC and HPt domains may be fused with the sensor kinase in a single polypeptide or can occur as independent proteins. In either case, the phosphoryl group is transferred between several His and Asp residues (H→D→H→D). The additional steps in phosphorelay systems may be functionally significant by providing additional sites for interaction with other systems.

Question 30

Describe ArcA and ArcB of the ArcAB system.

Answer 30

ArcB is a membrane-bound sensor kinase with fused REC and HPt domains and ArcA is a response regulator with DNA-binding activity. Both are dimers.

Question 31

What does ArcAB regulate?

Answer 31

Preferential use of O2 as a TEA in E. coli.

Question 32

Describe cyt d and cyt o the ArcAB system.

Answer 32

In high aeration, cyt o is most abundant; cyt d is most abundant under microaerobic conditions. Cyt o has a relatively low affinity for O2 but consumes cytoplasmic H+ and acts as a H+ pump. Overall, it contributes 2H+/e-to the proton motive force. By contrast, cyt d has a relatively high affinity for O2 but does not NOT pump H+, so only contributes 1H+/e- to the PMF.

Question 33

WHat is the signal detected by ArcB?

Answer 33

Oxidation state of quinones. The relative concentration of oxidized quinones increases when the flow of electrons into Complex I is slowed (less NADH is being made) or when the flow of electrons out of Complex IV chain is accelerated (high O2 tension). The sensing mechanism in ArcB involves two pairs of cysteine residues that are oxidized to form disulphide bonds by oxidized quinones, Q. In this state, ArcB is K-P+.

Question 34

Describes the steps of the ArcAB system.

Answer 34

Two component system.
High O2 conc: Q. ArcB is oxidized, K-P+. Not much ArcA~P. Cyo expressed, cyd repressed.
Low O2 conc: QH2. ArcB is reduced, K+P-. More ArcA~P. Cyo repressed by ArcA~P, cyd expressed by Arc~P.

Question 35

Describe the components of the Spo phosphorelay system.

Answer 35

Separate proteins. The KinA sensor kinase functions as phosphodonor for Spo0F, an intermediate receiver domain. Spo0F~P is the phosphodonor of Spo0B, anHPt domain. Spo0B~P is the phosphodonor of Spo0A, a DNA-binding response regulator. Spo0A~P activates genes necessary for sporulation and represses genes involved in competence.

Question 36

What are the sensor kinases of the Spo system?

Answer 36

KinA (cytoplasmic), KinB (membrane-bound), KinC, KinD and KinE. Among the signals sensed are nutritional stress, cell density, Krebs cycle, DNA damage and presence of extracellular matrix in biofilms.

Question 37

Describe the phosphatases of the Spo system.

Answer 37

Multiple phosphatases for different steps. RapA, RapB, RapE dephosphorylate Spo0F~P while the Spo0E phosphatase targets Spo0A~P.

Question 38

Describe the flagella of E. coli.

Answer 38

3 parts: basal body, hook, filament. CCW = run, CW = tumble.

Question 39

What are the sensors of chemotaxis?

Answer 39

Chemoreceptors, long, needle-like integral membrane proteins. They function as heterotrimers of dimers arranged in “rafts”or “patches” that typically occur at eachpole of the cell. Also called methyl-accepting chemotaxis proteins (MCPs).

Question 40

What are the 4 types of chemoreceptors?

Answer 40

Tar –Taxis to aspartate, glutamate and maltose and from repellents (nickel and cobalt)
Tsr –Taxis to serine and from repellents(leucine, indole and weak acids)
Trg–Taxis to ribose and galactose
Tap –Taxis to to dipeptides

Question 41

What are the transducers of chemotaxis?

Answer 41

CheA, CheB,CheR, CheW, CheY and CheZ

Question 42

Describe the steps of chemotaxis.

Answer 42

Unstimulated: CheW -> CheA autophosphorylates -> CheA~P phosphorylates CheY -> CheY~P binds to flagellar motor switch and causes it to spin CW, tumble.
CheZ acts as phosphatase and dephosphorylates CheY
Binding of chemoattractant -> decrease in autophosphorylation of CheA -> CCW, run

Question 43

Describe the mechanism of adaptation of the chemotaxis system.

Answer 43

An MCP can be methylated at up to 4 or 5 sites (glutamate residues) of its cytoplasmic domain. Methylation is catalyzed by CheR (a methyltransferase) and demethylation is catalyzed by the phosphorylated form of CheB (CheB has no activity, but CheB-P is a methylesterase). Methylated forms of MCPs have a lower binding affinity for chemoattractant. The methylated, desensitized MCP can sense only an increased concentration of chemoattractant, triggering another cycle of response,extended run and further adaptation. CheA phosphorylates CheB and CheB autodephosphorylates.