Bacterial Transport Flashcards
1
Q
What are three qualities about the rate of uptake in the absence of a transporter?
A
- low
- proportional to concentration gradient
- no saturation
2
Q
What is an example of uptake without a transporter?
A
Simple diffusion of small molecules like ions
3
Q
What does the graph of uptake without a transporter look like?
A
Linear with no plateau even at high [S]
4
Q
What are three qualities about the rate of uptake with a transporter?
A
- Proportional to transporter activity
- Conform to michelas menten kinetics
- Can go against a concentration gradient
5
Q
What are two forms of energy driven transport?
A
Primary and secondary
6
Q
What drives primary transport?
A
energy-producing metabolic event
7
Q
What are four examples are primary transport?
A
- Oxidation-reduction reactions (ETC)
- ATP-dependent ion translocation
- Osmotic shock-sensitive transport systems
- Sugar uptake by the phosphotransferase (PTS) system
8
Q
What drives secondary transport?
A
Electrochemical gradients
9
Q
What kind of solutes are generally involved in secondary transport and where do they move?
A
Typically H+ and Na+
- move down electrochemical ion gradients
10
Q
What are the three types of secondary transport and how do they work?
A
- Uniport : solute translocation in the absence of a coupling ion, e.g. K+ uptake.
- Symport : solute uptake in which two solutes are carried in the same direction, e.g. lactose uptake.
- Antiport : coupled movement of two solutes in opposite directions.
11
Q
The LacY transporter for lactose is an example of a ____?
A
Symporter
-brings in lactose with a proton
12
Q
What is another example of a symport?
A
PO42- and proton
13
Q
Whats an example of an antiport system?
A
Na+ and H+
14
Q
Whats an example of uniport?
A
K+
15
Q
Explain the evidence of a H+/Lactose symport in E. coli?
A
As soon as you add lactose to the cell suspension, the pH of the media increases very quickly
- This indicates that protons must be being taken up by the cell
- You can only have so many protons coming in or the membrane potential will change too much
16
Q
What is homeostasis?
A
the ability of living organisms to maintain a constant internal environment despite changes in external environment
17
Q
All prokaryotes (and eukaryotes) maintain an intracellular pH
A
near neutral
18
Q
What are the 5 mechanisms of pH homeostasis?
A
- H+ efflux via proton pumps
- K+ influx
- Na+/H+ antiporter (either 3 OR 4)
- K+/H+ antiporter
- Na+ uptake via symport
19
Q
What is the effect of H+ efflux via proton pumps
A
Efflux will increase intracellular pH, H+ gradient develops and eventually efflux will slow down because the membrane potential that develops.
20
Q
What is the effect/purpose of K+ influx?
A
Bring in electro-positive charges to lower the membrane potential developed from H+ efflux.
21
Q
What is the effect/purpose of either the Na+/Na+ antiporter or the H+/K+ antiporter?
A
H+ brought in to lower the intracellular pH; Na+ or K+ are pumped out to prevent an inverted ΔΨ to develop.
22
Q
What is the purpose of Na+ intake via symport ?
A
To complete the Na+ circuit
23
Q
When does an inverted ΔΨ develop?
A
Inverted ΔΨ develops when inside is more +ve than outside.
24
Q
How do acidophiles maintain a constant pH?
A
maintain a cytoplasmic pH around neutral i.e. more alkaline than outside pH, H+ must be pumped out.
25
What kind of ΔΨ do acidophiles have?
An inverted one
26
What generates an inverted ΔΨ ?
greater influx of K+ and a smaller efflux of H+.
27
∆pH =
ΔpH = pHi-pHo
28
How do alkilophiles maintain a constant pH ?
to maintain a cytoplasmic pH around neutral i.e. more acidic than outside pH, H+ must be brought in
29
A large what must be generated in order to have a -∆pH? How is this done?
because of a -ve ΔpH, a large membrane potential must be generated and this is achieved by a Na+/H+ antiporter.
30
What maintains pH homeostasis for acidophiles?
Na+/H+ antiporter.
31
ΔΨ should be __ and __ for acidophiles?
large and negative
32
What are the 2 major classes of ATP-dependent ion pumps?
F type and P type ATPases
33
What are the components of the F type and P type?
```
F-type = F1F0
P-type = E1E2
```
34
What are the 4 common properties of the P-type ATPases?
1. A phosphorylated intermediate (DKTGT motif)
2. Two conformational forms of the phosphorylated intermediate (E1 and E2)
3. Contain 6 -8 membrane-spanning regions
4. TGES motif as the phosphatase site
35
At what amino acid residue are the P-types phosphorylated?
At the aspartic acid
| - start of the DKTGT motif
36
Name 2 (important ones of) of the 7 compounds the P-type can translocate
K+, Mg2+, Ca2+, Cd2+, Cu2+, mercury, arsenate
| -K+ and Cu2+ are the most important
37
How many classes of P-type ATPases are there?
3
38
Whats a morphological feature of type 1 P-type ATPases?
N-terminal additional pair of transmembrane helices
39
Whats a morphological feature of type 2 P-type ATPases?
Extended C-termimus with additional transmemb. helices
40
Whats a morphological feature of type 3 P-type ATPases?
PTT1 motif that forms a channel for ions to pass through
41
Heavy metals are associated with which type?
Type 1 transports them
42
K+ is transported by which type?
Type 3
43
What is the principle cation in bacteria?
K+
44
Why is K+ so important?
- acts as an intracellular osmolyte.
- helps to maintain a ΔpH.
- acts as a cofactor for ribosomes and many enzymes.
45
What are the two transport systems for ATP-driven K+ influx?
1. constitutive TrK system
| 2. de-repressed Kdp system
46
When is the TrK system active? What kind of affinity does it have?
works with Low affinity (Km = 2 mM)
non-P-type, “housekeeping” transport active under normal conditions
47
What does the TrK system use to take in K+?
∆P
48
What kind of affinity does the Kdp system have? When is it active?
Kdp system is activated by low K+ concentration and by high external salt concentration or internal ionic strength
49
What is the role of KdpD ? When is it active?
sensor protein, becomes activated (autophosphorylated) when external osmolarity and intracellular ionic strength are high
Phosphorylates KdpE
50
What is the role of KdpE ? When is it active?
KdpE, a responder protein, when it is phosphorylated by KpdD will activate kdpFABC transcriptio
51
Why is copper an essential element for cell growth? What is it also?
acts as a cofactor for enzymes
| **but is also toxic**
52
What are three ways in which intracellular copper levels are controlled?
1. decreasing uptake
2. Complexation by cell components
3. ATP-dependent efflux system
53
Which form of copper level control confers copper resistance?
ATP-dependent efflux system
54
Why do many baby creams have heavy metals in them ?
They can complex non specifically with proteins and act as an antimicrobial agent
55
The best characterized copper transport and resistance systems are that of :
Enterococcus faecalis and Streptococcus mutans.
56
S. mutans copYAZ operon codes for 3 proteins which are:
CopY, CopA, CopZ
57
What is CopY?
transcriptional repressor
58
What is CopA?
copper specific P-type ATPase
59
What is CopZ?
an antirepressor
60
CopYAZ system is responsible for :
copper resistance
61
5 morphological features of CopA: (is this important?)
1. N-terminal heavy metal binding motif (GMXCXXC)
2. eight transmembrane regions
3. a TGES motif as phosphatase site
4. a DKTGT motif as the phosphorylation site
5. a CPC motif as part of the ion channel
62
Explain the CopYAZ system (3 points)
1. Under physiological copper conc. conditions, CopY acts as a repressor.
2. When copper is limiting, CopY has no copper bound and repression does not occur.
- the gene will be transcribed and CopB produced
3. When copper is in excess, CopZ complexes with copper preventing CopY to function as a repressor.
63
What is the role of CopB?
To allow the cell to take in copper
64
Why is iron an essential nutrient for bacteria?
acts as a cofactor for many enzymes and redox centre in cytochromes and iron-sulfur proteins
65
What are 2 reasons that iron is inaccessible to bacteria in nature?
1. In the hosts, irons are sequestered by transferrin, lactoferrin, or ferritin.
2. Outside the hosts, Fe2+ is rapidly oxidized as Fe3+ and forms insoluble ferric hydroxide polymers.
66
What are siderophores?
small high-affinity iron-chelating molecules that are secreted by some bacteria
- bind soluble Fe3+
67
Why are siderophores potentially useful as drugs?
to use the iron transport abilities of siderophores to carry drugs into pathogens by preparation of conjugates between siderophores and antimicrobial agents.
-Because bacteria recognize and utilize only certain siderophores, such conjugates are anticipated to have selective antimicrobial activity.
68
what is enterobactin
A siderophore used by E-coli
69
What is FepA?
An outer membrane porin that allows the enterochelin-iron complex to pass through
70
What is the role of TonB?
interactions between TonB and FepA control the “opening” of the pore in FepA
71
What energy source is required for the interactions between FepA and TonB?
transducing energy from ∆P (PMF)
72
What is the role of FepB?
is a periplasmic binding protein that binds to the enterochelin-iron complex adnd transports it to the inner membrane ABC transporter
73
What is the inner membrane transporter in TonB-dependent iron transport?
FepCDG
74
What enzymes degrades the enterochelin?
Fes esterase
75
What happens to the Fe3+ once it is in the cell?
reduced to Fe2+ and incorporated into proteins such as cytochromes, iron-sulfur proteins, etc.
76
Is FepA a monomer, dimer, or trimer?
Monomer
77
How does TonB make contact with FepA?
Via an amino acid sequence on the FepA porin called the Ton Box
78
what is the structure of TonB? What is it associated with?
inner membrane protein and its transmembrane segment is associated with the ExbB and ExbD hexamer.
79
How is the power of the PMF harnessed in the TonB transport mechanism?
the passage of protons through the ExbBD hexamer. This somehow energizes TonB to interact with FepA, triggering a conformational change in FepA allowing the enterochelin-iron complex to enter.
80
What are the 2 classes of multidrug resistance transport systems? what is this distinction based on?
primary and secondary
based on the energy source used
81
How do primary MDRs get their energy and what do they used to pump drugs out?
ATP hydrolysis, use ABC transporter to pump out drugs
82
How do secondary MDRs get their energy and what do they used to pump drugs out?
H+ antiport (efflux of drugs coupled to influx of H+)
83
what system in E-coli is an example of an MDR?
AcrAB-TolC system
84
What does the AcrAB-TolC system accomplish?
Pumps out many toxic substances (dyes, detergents ,bile salts, tetracycline, penicillins, erythromycin, etc) once they enter the periplasm
-dont enter the cytoplasm
85
Which transport system is characteristic of gram negative bacteria?
Osmotic shock-sensitive transport systems
86
What is osmotic shock?
a treatment that makes the outer membrane permeable and releases periplasmic proteins
87
Are Osmotic shock-sensitive transport systems effective or not?
Yes they are highly effective
88
What are osmotic shock-sensitive transport systems responsible for? What is an important item?
the transport of a wide variety of sugars, peptides and amino acids, anions, and vitamins.
** vitamin B12 **
89
Describe the 4 steps in the osmotic shock process
1. Resuspend cells in 20% sucrose, 1 mM EDTA, 20 mM Tris buffer (pH 8.0) --> causes plasmolysis
2. Centrifuge and remove supernatant.
3. Quickly resuspend cells in ice-cold distilled water.
4. Centrifuge and supernatant contains periplasmic contents.
- Remaining cells have lost their periplasmic contents.
90
What is plasmolysis?
Plasmolysis is the process in cells where the cell membrane pulls away from the cell wall due to the loss of water through osmosis
91
what is the role of EDTA in the osmotic shock process?
EDTA is a chelator for divalent cations like magnesium. This destabilizes the outer membrane because magnesium is often found on the LPS.
92
Describe the 5 steps of
1. Solute enters the periplasm through the outer membrane porin.
2. Solute binds to a specific periplasmic binding protein.
3. Binding protein undergoes a conformational change that allows it to bind to an ABC transporter (ATP-Binding Cassette transporter) in the cell membrane.
4. Solute is delivered to the ABC transporter.
5. Solute is translocated across the membrane by the hydrolysis of ATP. Binding protein and transporter regenerated.
93
What is the phosphotransferase system responsible for?
The uptake of sugars and alcohols
94
The PTS is present in which types of bacteria?
common in anaerobes and facultative anaerobes
95
What is the phosphoryl donor in the PTS?
PEP
96
What is the overall reaction of the PTS?
PEP + (CH2O)[out] --> pyruvic acid + (CH2O)-P[in]
97
Describe the 4 steps of the PTS
1. Transfer of phosphate from PEP to Enzyme I (EI).
2. Enzyme I transfers the phosphate group to a small cytoplasmic protein HPr.
* *Enzyme I and HPr are common to all PTS systems.
3. HPr transfers the phosphate group to a carbohydrate specific permease complex, Enzyme II (EII).
4. EII transfers the phosphate group to the carbohydrate.
98
Which enzymes is different among PTS systems?
enzyme II
99
What is the EII set up for glucose uptake in E. coli? How does each part work?
consists of two proteins IIA and IIBC.
IIAGlc is cytoplasmic, which accepts P from HPr.
IIBCGlc is membrane bound. IIB domain accepts P from IIA and then transfers P to IIC
100
What is the EII set up for mannose uptake in E. coli?
consists of three proteins; IIAB as a single cytoplasmic protein and IIC and IID are membrane bound.
101
What is the setup/parts for mannotil uptake in E. coli?
consists of a single membrane protein with 3 domains A, B,C. Domain C is in the membrane and domains A and B project into the cytoplasm.
102
what does Glucose or catabolite repression mean for enteric bacteria
They preferrentially use glucose as a carbon source over other sugars
103
what is Inducer exclusion
the inhibition by glucose of the transport and metabolism of non-PTS carbohydrates (e.g. lactose).
104
Which protein of enzyme II in glucose uptake by PTS can exist in the phosphorylated and dephosphorylated form? When is it in each form?
IIA (Glucose)
Dephosphorylated when glucose levels are high during PTS glucose transport
Phosphorylated form in glucose limiting cases
105
What does the phosphorylated IIA stimulate?
stimulates adenylate cyclase which leads to cyclic AMP production.
cAMP complexes with its receptor (CRP) and stimulates the expression many catabolic enzymes (including LacY, LacZ coded by the lac operon).
106
What is a diauxic growth curve? what causes it?
double growth, is caused by the presence of two sugars on a culture growth media, one of which is easier for the target bacterium to metabolize
-results from catabolite repression
107
Who discovered the lac operon?
Jacques Monod
