Lecture 6: Nutrient Uptake

Question 1

Heterotrophs

Answer 1

Acquire carbon from organic compound (Other organism)

Question 2

Autotrophs

Answer 2

Organisms that acquire carbon from CO2

Question 3

Type of bacterial cell where the periplasmic space is visible

Answer 3

Gram-negative

Question 4

Simple/Passive Diffusion

Answer 4

-Small uncharged molecules
-No transport protein needed
-Down gradient

Question 5

Passive Transport/Facilitated diffusion

Answer 5

-Charged, hydrophilic, or large molecules
-Needs transport protein
-Down gradient

Question 6

Active Transport

Answer 6

-Charged, hydrophilic, or large molecules
-Needs transport protein
-Uses energy, typically against the gradient

Question 6

Coupled transport

Answer 6

-One molecule goes against the concentration gradient using the energy of a molecule going down its concentration gradient
-Sometimes a transport protein will transport something to open its channel and allow something to move in the opposite direction

Question 7

ATP binding Cassette (ABC) transporter

Answer 7

1. Solute binds to cognate periplasmic binding protein and the complex then binds to the membrane transporter
2. ATPase activity of one component powers the opening of the channel and movement of the solute into the cell

Question 7

Channel proteins

Answer 7

Forms a water-filled pre across the bilayer through which some solutes can diffuse

Question 8

Carrier protein

Answer 8

Alternates between 2 conformations so that the solute binding site is sequentially accessible on one side of the bilayer and then on the other

Question 9

4 steps in the transport cycle of a carrier

Answer 9

1. Bind solute
2. Conformational change
3. Release solute
4. Get ready for next round

Question 10

Carrier mediated transport characteristics (Rate of solute entry in relation to concentration of solute)

Answer 10

-Exhibits saturation kinetics
-Exhibits specificity

Question 11

Simple Diffusion characteristics (Rate of solute entry in relation to concentration of solute)

Answer 11

-Depends on concentration difference across membrane
-Linear dependence (No saturation kinetics)

Question 12

Porins

Answer 12

Protein channels that allow molecules <600-700 Da to pass

Question 13

TonB-dependent transporter

Answer 13

Transports scarce or weakly permeable substrates through outer membrane

Question 13

TonB-dependent transporter mechanism

Answer 13

-Vitamin B12 binds to outer membrane carrier (BtuB)
-TonB complex uses proton motive force to open BtuB allowing B12 into periplasm
-Intermembrane transporter imports B12 to cytoplasm

Question 14

Siderophore transporter process

Answer 14

1. Siderophore called enterochelin secreted and binds to Fe(3+)
2. Complex is transported to the periplasm through an outer membrane protein
3. A periplasmic binding protein escorts the complex to an ABC transporter
4. Enterocherlin-Fe(3+) complex enters the cell
5. In the cell the iron is released and reduced to Fe(2+)

Question 15

Group translocation in glucose

Answer 15

Import of glucose and other sugars is maintain by modification of glucose into a different molecule (G6P) which allows more glucose import as it would be down its gradient. Done using the phosphate of PEP

Question 16

Phosphotransferase System Steps

Answer 16

EI: Takes phosphate from PEP to place onto HPR
HPR: Carries phosphate to component EII
EIII: Complex scanning membrane protein that transports phosphate to different receptors on the membrane to phosphorylate different sugars.

Question 17

Endo-acting degradative enzymes

Answer 17

Random cuts along polymer

Question 18

Exo-acting degradative enzymes

Answer 18

Reducing end or Nonreducing end cuts unidirectionally

Question 19

Alpha-Amylase

Answer 19

Endo acting enzyme

Question 20

Beta-amylase

Answer 20

Exo-activity enzyme from non-reducing end

Question 21

Y-amylsae

Answer 21

Exo-acting enzyme from reducing end

Question 21

Shine-Dalgarno Sequence

Answer 21

Region on bacterial mRNA that contains a sequence complementary to a portion of ribosome that allows for binding to aid in translation

Question 22

Transcriptional Fusion Reporters

Answer 22

Reporter gene is held downstream of promoter so that when it is activated it will transcribe also. Reporter gene activity reflects transcriptional activity and tests the strength of promoter.

Question 23

Translational Fusion reporters

Answer 23

Reporter gene is fused to coding region so that when mRNA is translated the reporter gene product is also made. -The amount of protein made correlates with translational level -Can show how effective translation

Question 24

Fluorescence v. Luminescence

Answer 24

-Fluorescence: Electron excitation will result in light when electron returns to its original state (TYPE OF LUMINESCENCE) -Luciferase: Emission of light not resulting from heat but involves electron excitation

Question 25

Pros of reporters

Answer 25

-Convenient -High throughput -Used for microscopy imaging and flow cytometry -Study populations and single cells

Question 26

Cons of reporters

Answer 26

-Not monitoring the expression of native mRNA or protein -May cause artifacts -Can sometimes affect the expression of native genes

Question 27

Northern blot vs qRT-PCR

Answer 27

Northern Blot -Directly visualizes RNA bands -Detects splicing variant -Detect heavily modified RNAs qRT-PCR -More sensitive -Multiplex and high throughput

Question 28

Steps of RNA fluorescence in situ hybridization

Answer 28

1. Target hybridization 2. Fluorescence imaging 3. Photobleaching

Question 29

Method for the detection of individual proteins

Answer 29

Western blot

Question 30

Methods used in proteomics

Answer 30

-Mass spectrometry -Ribosome profiling