Lecture 1: Solute Transport - Exam 3 Flashcards

1
Q

What is the difference between Primary Transport and Secondary Transport?

A

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

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2
Q

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

A

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.

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3
Q

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

A

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

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4
Q

How does the phospholipid bilayer affect solute transport?

A

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

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5
Q

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

A

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

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6
Q

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

A

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

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7
Q

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?

A

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.

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8
Q

Describe solute transport in G- bacteria.

A

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

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9
Q

Describe the transport of SMALL molecules through the OUTER membrane.

A

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.

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10
Q

What kind of molecules are transported through porins?

A

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

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11
Q

Describe the transport of LARGER solutes in gram negative bacteria.

A

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

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12
Q

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

A

Cobalamin, siderophores, oligosaccharides require specific transport proteins.

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13
Q

Describe solute transport in Gram Positive bacteria.

A

Solutes must pass through the cell membrane.

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14
Q

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

A

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

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15
Q

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

A

Against, this means energy is required.

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16
Q

The rate of solute uptake is directly proportional to…?

A

The extent of saturation of the transporters.

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17
Q

What is the affinity constant (Km)?

A

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.

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18
Q

Is Km the same for evert transporter?

A

NO!

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19
Q

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?

A

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.

20
Q

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

A

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

21
Q

Do all solutes require transport systems?

A

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.

22
Q

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

A

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).

23
Q

What are the three major primary solute transport systems?

A
  1. ABC (ATP Binding Cassette) transporter systems (active transport)
  2. ATP-linked Ion Motive Pump (active transporter)
  3. Group Translocation system
24
Q

What is active transport?

A

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

25
Q

What is Group Translocation?

A

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)

26
Q

Describe the ABC transporter system process.

A
  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.
27
Q

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

A

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

28
Q

What domains of life are ABC Transporter systems present in?

A

All three domains of life

29
Q

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

A

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.

30
Q

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

A

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

31
Q

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

A

higher

32
Q

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

A

TrK system
Kdp system

33
Q

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

A

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.

34
Q

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:

A
  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.
35
Q

How does group translocation differ from other transport systems?

A

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.

36
Q

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

A

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

37
Q

Describe the glucose PTS system in E. coli.

A

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.

38
Q

Go through the steps of the PTS.

A
  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.
39
Q

What is catabolite repression?

A

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.

40
Q

Catabolite repression assures…?

A

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

41
Q

What are the two ways that catabolite repression occurs?

A

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

42
Q

What is inducer exclusion of catabolite repression?

A

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

43
Q

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

A

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.

44
Q

What is the secondary transport system? What is it?

A

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

45
Q

What is uniport, symport, and antiport?

A

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).

46
Q

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

A

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.