tema 7: solute transportacademic.uprm.edu/lrios/4368/tema7.pdftema 7: solute transport chapter 16...

Tema 7: Solute Transport

Chapter 16Pages 417 - 437

Water solublemolecules

Gram + bacteria

Permeabilitybarrier

Water solublemolecules

Gram - bacteria

Permeabilitybarrier

Permeabilitybarrier

This allows the bacterium to maintain an internal environment different from theexternal….

1- Metabolites could be maintained at an intracellular concentrationthat is orders of magnitude higher than the extracellularconcentration.

A- rapid enzymatic reactions.

B- retention of metabolic intermediates within the cell.

2- Minimizes the passive diffusion of ions.

A- maintain the electrochemical proton and sodiumion gradient.

1- ATP synthesis2- solute transport

This implies that:

All water soluble molecules must enter and leave the cellthrough…

permease

transporter

carrier

porter

in

out

How can we study transport?

Phospholipids+

water

sonicate buffer

Detergent-coated protein

liposomesproteoliposome

Cytochromeprotein

Lactose permease

Oxalate/formateantiporter Na+/H+

antiporter

Histidinepermease

Cytochromeoxidase

proteoliposome

V max

V max

2

K m [S]

v

Vmax S

Km + Sv =

Michaelis-Menten constant

liposome

proteoliposome

[S]

v

Energy-dependent transport

Primary transport systems: driven by an energy-producing metabolic event,include proton translocation drive by ATP, light, or OX-RED Rxn.

1- Na ion transporting decarboxylases2- uptake of organic or inorganic solutes3- uptake of sugars

Secondary transport systems: driven by electrochemical gradients.

ATP- binding cassette (ABC) transporters

2 subunits αβhydrophobic

2 subunits ααhydrophilic

in outGram - cell

porinOMCM

ABC transporter

cytoplasm periplasm

ATP

ADP + Pi

sugars

amino acids

Small peptides

ATP-drive K+ influx

Potassium is the principal cation in bacteria….. It play a role in:

Osmotic homeostasis, pH homeostasis, is a cofactor for many enzymes and ribosomes.

There are 2 transport systems:There are 2 transport systems:

1- TrK system (major route) is constitutively expressed and operatesat high rates but low affinity (Km ~ 2 mM).

2- Kdp system it depends on the salt-osmolarity of the medium, it has avery low Km ~ 2 µM

The phosphotransferase system

Accumulates carbohydrates as the phosphorylated derivative.

This system is lacking in aerobes for the most part and it does not existin archaea and eukaryotes

This is a group translocation system!!! And not active transport.

How to determine the source of energy for transport

Does it use ATP or ∆p??

We must do certain things before we ca determine this!!

1- inactivated the ATP synthase….How?

a- use ATP synthase mutants

b- DCCD (N,N’-diclohexylcorbodi-imide

2- perturbing the intracellular levels of ATP…..How?

a- starve the cell

b- add an inhibitor like arsenate (substrate level P)

c- to increase ATP, add glucose but limit respirationusing the inhibitor cyanide.

3- perturbing the ∆p……How?

a- using Ionophores

B- addition of substrates that feed electrons to the e- transport chain

Drug-export systemThey are an important way by which bacteria become resistant to

antimicrobial agents. (antibiotics, dyes, detergents, disinfectants,and antiseptics.)

Two types: 1) dedicated and 2) multidrug

In M. tuberculosis, the TAP system pumps out both:1) aminoglycosides (gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin and apramycin. 2) tetracycline

Antimicrobial Agents and Chemotherapy, December 2000, p. 3249-3256, Vol. 44, No. 12

Lakshmi P. K. et.al.

MDR system

Major classes: primary and secondary

The primary systems are energized by ATP hydrolysis.

They use an ABC transporter!

Membrane fusionProtein (AcrA)

The secondary systems are energized by proton antiport or sodium!

E. coli, N. gonorrhoea, N. meningitidis, V. cholerae,and V. parahaemolyticus.

Protein (AcrA)

porin

Efflux transpoter(AcrB)

OM channel(TolC)

AcrAB-TolCSystem pump