Lecture 1: Solute Transport - Exam 3

Question 1

What is the difference between Primary Transport and Secondary Transport?

Answer 1

Primary: Export driven by an exergonic reaction - proton transport.
H+in + energy -> H+out
Secondary: Is indirectly coupled to primary transport by an energy-yielding chemical reaction by ion currents (proton or sodium gradient)
H+out + Soluteout -> H+in + Solutein

Question 2

What does the phospholipid bilayer allow the cell to do? Why is it important for the cell?

Answer 2

The phospholipid bilayer of the bacterial cell membrane acts as a barrier that blocks the diffusion of water-soluble molecules into and out of the cell. It allows bacteria to maintain an internal environment different from the external environment.

Question 3

Metabolites can be maintained at intracellular concentration that is orders of magnitude _______ than extracellular concentrations.
What does this promote and allow?

Answer 3

Higher
This promotes more rapid enzymatic reactions and allows for retention of metabolic intermediates within the cell.

Question 4

How does the phospholipid bilayer affect solute transport?

Answer 4

The phospholipid bilayer minimizes the passive diffusion of ion, including protons, allowing the membrane to maintain electrochemical proton and sodium ion gradients.

Question 5

The membrane is able to maintain electrochemical proton and sodium ion gradients. The gradients do what?

Answer 5

The gradients drive ATP synthesis, solute transport, and other membrane activities.

Question 6

The phospholipid bilayer is a permeability barrier, anything that is not _________ soluble must enter the cell through _________. Including:

Answer 6

Lipid ;
Integral membrane proteins
Including transporters, carriers, permeases, and porters.

Question 7

Bacterial cell membranes consist in large part of a phospholipid matrix that acts as a permeable barrier blocking the diffusion of water-soluble molecules into and out of cells. Why do we care?

Answer 7

1) Metabolism - need to bring nutrients inside before eating them (catabolism), and remove waste products.
2) Multidrug resistance - preventing uptake, or pumping drugs out of the cell can confer resistance to antimicrobial compounds.

Question 8

Describe solute transport in G- bacteria.

Answer 8

Solutes must pass through outer membrane and inner membrane (cell membrane).

Question 9

Describe the transport of SMALL molecules through the OUTER membrane.

Answer 9

Transport of small molecules through the outer membrane occurs through Porins.
Porins are outer membrane proteins that form narrow channels for free diffusion of small hydrophilic molecules (< 600-700 Da) into the periplasm.

Question 10

What kind of molecules are transported through porins?

Answer 10

Sugars, amino acids, ions etc are transported to the periplasm through porin channels.

Question 11

Describe the transport of LARGER solutes in gram negative bacteria.

Answer 11

Transport of larger solutes require specific transport proteins such as TonB-dependent proteins.

Question 12

What kinds of molecules require specific transport proteins in gram-negative bacteria?

Answer 12

Cobalamin, siderophores, oligosaccharides require specific transport proteins.

Question 13

Describe solute transport in Gram Positive bacteria.

Answer 13

Solutes must pass through the cell membrane.

Question 14

Biological membranes are _________ (hydrophobic/hydrophilic) making them impermeable to most water-soluble molecules. What is the major advantage and disadvantage of this.

Answer 14

Hydrophobic
Major advantage: separation of cytoplasm from environment, enables membrane potential
Major disadvantage: need specific mechanisms for solute transport.

Question 15

Solute transport is _______ (with/against) solute gradient. Meaning what?

Answer 15

Against, this means energy is required.

Question 16

The rate of solute uptake is directly proportional to…?

Answer 16

The extent of saturation of the transporters.

Question 17

What is the affinity constant (Km)?

Answer 17

The concentration of solute that produces 1/2 the maximum initial rate of transport (1/2 Vm).
It is called the affinity constant because the Km is considered to be a measure of the affinity of the solute for transporter.

Question 18

Is Km the same for evert transporter?

Answer 18

NO!

Question 19

Km is a characteristic of each specific transporter. If the Km is high, what does this mean?
If the Km is low, what does this mean?

Answer 19

If the Km is high, this means that this is a low affinity transporter because it takes a lot of solute to produce 1/2 Vmax.
If the Km is low, this means that this is a high affinity transporter (because it takes very little solute to produce 1/2 Vmax.

Question 20

Describe the kinetics of solute (S) transport (on the graph).

Answer 20

The rate (V) of solute transport approaches a maximum (Vm) as the fraction of transporters bound to solute reaches a maximum.

Question 21

Do all solutes require transport systems?

Answer 21

No, some small molecules can enter via simple diffusion.
In simple diffusion, the rates of solute uptake are low and proportional to the concentration gradient.

Question 22

What are the two classes of energy-requiring solute transport systems? How are they different?

Answer 22

Primary: Requires energy-generating metabolic reaction such as hydrolysis of molecules with high group transfer potential (e.g. ATP)
Secondary: Driven by electrochemical gradient (e.g. PMF, a proton gradient across the cell membrane that powers the ATPase to generate ATP).

Question 23

What are the three major primary solute transport systems?

Answer 23

1. ABC (ATP Binding Cassette) transporter systems (active transport)
2. ATP-linked Ion Motive Pump (active transporter)
3. Group Translocation system

Question 24

What is active transport?

Answer 24

A subset of primary solute transport where the solute is not chemically altered.
- Energy dependent process (usually ATP hydrolysis)
- Moves molecules against the gradient
- Involves carrier proteins
- Concentrates molecules inside the cell
Ex: ABC transporter systems and ATP-linked ion motive pump system

Question 25

What is Group Translocation?

Answer 25

A type of primary solute transport that chemically modifies molecules as they are brought into the cell.
Phosphoenolpyruvate (PEP) group translocation (or Phosphotransferase system (PTS)) transports a variety of sugars while phosphorylating them using PEP as the phosphate donor.
Group translocation is NOT considered active transport (due to the chemical modification)

Question 26

Describe the ABC transporter system process.

Answer 26

1. The solute enters the periplasm through the porin pore.
2. Inside the periplasm, the solute binds to a binding protein, which is induced to have a conformational change.
3. The binding protein carries the solute to the transport proteins (a, b, and c) located in the cell membrane.
4. Energized by ATP hydrolysis, the transporter (permease) goes through a conformational change that opens the pore.
5. The solute is passed from one binding site of the permease to another, and diffuses into the cytoplasm.
6. The pore closes after solute transport.

Question 27

What is different between G- and G+ bacterial cells when it comes to the ABC transporter system?

Answer 27

G- bacteria have the binding protein located in the periplasm.
G+ bacteria have the binding proteins tethered to the cell membrane.

Question 28

What domains of life are ABC Transporter systems present in?

Answer 28

All three domains of life

Question 29

Who has the Shock-sensitive ABC transport system? What does it do?

Answer 29

Gram negative bacteria.
It is active when the cell is under osmotic shock. Can uptake a wide range of solutes (sugars, amino acids, ions). Have very high efficiencies for solute uptake. This allows accumulation of high internal concentrations of solute in order to survive osmotic shock when in a high concentration of external solutes.

Question 30

Potassium (K+) is the ____________ within bacterial cells. What role does it play?

Answer 30

Principal cation. It plays an important role in osmotic and pH homeostasis.

Question 31

Bacteria accumulate K+ to a level much _______ than external concentrations.

Answer 31

higher

Question 32

K+ transport in E. coli is through two transport systems. What are they?

Answer 32

TrK system
Kdp system

Question 33

What do the TrK and Kdp systems do? When are they expressed? How does expression work?

Answer 33

TrK: A low affinity K+ transporter but is constitutively expressed (continuously transcribed). Always present and binding potassium here and there. This system has a high Km (~ 2 mM).
Kdp: High affinity K+ transporter system in many bacteria with a low Km (~2 uM). This system is a two component signal transduction system.

Question 34

The Kdp System is a two component signal transduction system. When E. coli cells are grown in a medium with very low K+ and high osmolarity:

Answer 34

1. The KdpD protein in the cytoplasmic membrane phosphorylates KdpE, a transcription factor that then increases transcription of the kdpFABC operon.
2. The KdpA protein binds K+ and forms a channel through which K+ translocates across the cell membrane.
3. The KdpB protein is an integral membrane protein that has an ATPase domain (i.e. “nucleotide-binding domain) that provides the energy necessary for K+ uptake.

Question 35

How does group translocation differ from other transport systems?

Answer 35

Does not use active transport. Catalyzes the accumulation of carbohydrates (e.g. sugars) as the phosphorylated derivative instead of the free carbohydrate. This occurs most commonly in anaerobic and facultatively anaerobic bacteria.

Question 36

Phosphotransfer system (PTS) (a group translocation system) has what kind of energy source? What is the PTS used for?

Answer 36

The energy source and phosphoryl donor is phosphoenolpyruvate (PEP). PEP is an intermediate generated during glycolysis.
PTS is used for catabolite repression.

Question 37

Describe the glucose PTS system in E. coli.

Answer 37

Glucose enters the cells as glucose-6-phosphate. The phosphorylated EIIA^Glc binds to the adenylate cyclase (CyaA) and activates CyaA activity. The dephosphorylated EIIA^Glc binds to non-PTS permeases and inhibits their transport. So, maltose, lactose and melibiose will be blocked from entering the cell.

Question 38

Go through the steps of the PTS.

Answer 38

1. The phosphoryl group (P+O32-) is transferred from PEP to Enzyme I (EI).
2. Enzyme I transfers the phosphoryl group to a small cytoplasmic protein, HPr.
3. HPr transfers the phosphoryl group to a carbohydrate-specific* permease called Enzyme II (EII).
   -EII has three domains (A, B, &C) which can be part of a receptor protein for a singe sugar (mannitol) or separate sugars (mannose or glucose)
4. The phosphoryl group is transferred from EIIA -> EIIB -> EIIC
   -EIIC is an integral membrane protein that catalyzes the uptake and phosphorylation of the carbohydrate.

Question 39

What is catabolite repression?

Answer 39

When E. coli are provided both glucose and another carbon source, the bacteria will preferentially use glucose and delay the use of the other carbon source. This is catabolite repression and is utilized during diauxic growth.

Question 40

Catabolite repression assures…?

Answer 40

That the preferred carbon source (usually glucose) is used first by repression expression of genes involved in nonpreferred carbon source utilization.

Question 41

What are the two ways that catabolite repression occurs?

Answer 41

Inducer exclusion and repression of cyclic AMP (cAMP) accumulation.

Question 42

What is inducer exclusion of catabolite repression?

Answer 42

Represses the uptake of less-preferred C sources.
-EIIA^glu inhibits activity of non-PTS carbohydrate transport proteins (S1 and S2)
-melibiose or lactose uptake

Question 43

What is repression of cyclic AMP (cAMP) accumulation of catabolite repression?

Answer 43

EIIA^glu stimulates cAMP production, which stimulates transcription of genes involved in C metabolism.
-Glucose uptake results in cAMP dephosphorylation, which reduces the transcription of cAMP related genes involved in C source utilization.

Question 44

What is the secondary transport system? What is it?

Answer 44

Chemiosomotic-driven transport system: Transport of solutes across the membrane along its electrochemical gradient, such as the PMF or Na+ gradient.

Question 45

What is uniport, symport, and antiport?

Answer 45

Uniport: movement of single substrate
Symport: simultaneously transport of two substrates in the same direction
Antiport: simultaneously transport of two-like charged substrates in opposite direction (ex: proton:sodium antiporter used for pumping Na+ ions out of the cell).

Question 46

What is the AcrAB-TolC of E. coli used for?

Answer 46

AcrAB-TolC of E. coli is an example of a multi-drug resistance (MDR) transport system.
It is a PMF powered efflux system and pumps out dyes, detergents and many different antibiotics. The drug will come through a porin into the periplasm of a G- cell.
In the case of an amphiphilic drug, the lipophilic region will partition into the cell membrane.
AcrB is a cell membrane protein (cell membrane transporter) that transports the drug with the help of AcrA to TolC, an outer membrane channel which will pump the drug out of the cell.