Sugar influx sensing by the
phosphotransferase system of
Escherichia coli
Referees : Prof. Dr. Victor Sourjik
Prof. Dr. Micheal Knop
Contents
I Introduction
1
1 The PTS of E. coli
2
1.1
General PTS components El and H p r ....................................................
3
1.2
Enzyme II com plexes................................................................................
4
1.3
Sugar preference in E. c o l i ......................................................................
6
1.3.1
Catabolite repression and growth r a te ........................................
6
1.3.2
EIIAGlc activates adenylate c y c la s e ...........................................
8
1.3.3
Growth rate and cAMP concentration
.....................................
8
1.3.4
cAMP receptor protein (G R P )....................................................
9
1.3.5
Inducer exclusion..........................................................................
10
Phosphoenolpyruvate: Phosphate donor and
metabolic n o d e .........................................................................................
12
Catabolite repression by non-PTS
compounds ...............................................................................................
13
Other functions of P T S .............................................................................
14
1.4
1.5
1.6
2 Chemotaxis in E. coli
15
2.1
Molecular machinery of chem otaxis.......................................................
16
2.2
Chemoreceptors - sensing and stru ctu re.................................................
17
2.3
Trimers of dimers and higher order complex formation
...............
18
2.4 Localization and architecture of chemosensory com plexes..................
20
2.5
Signal amplification by the chemotaxis p a th w a y ..................................
21
2.6 Taxis to sugars in E. c o li.........................................................................
22
2.7
23
.
Chemotactic signaling by the phosphotransferase s y s t e m ..................
3 Other sugar transporters in E. coli
3.1
ATP binding cassette (ABC) transporters
25
...........................................
25
3.1.1
The nucleotide bindingdomains (N B D )......................................
26
3.1.2
The transmembrane domains (T M D )........................................
26
3.2
The catalytic cycle of ABC transporters
..............................................
27
3.3
The major facilitator superfamily (MFS) of tr a n s p o rte r s ..................
29
4 Aim s of the current work
31
II
33
Materials andMethods
5 M aterials
34
5.1
Media and P l a t e s ......................................................................................
34
5.2
Buffers and Solutions................................................................................
35
5.2.1
Agarose gel electrophoresis..........................................................
35
5.2.2
SDS P A G E ...................................................................................
36
5.3
B uffers.........................................................................................................
37
5.4
A ntibiotics..................................................................................................
37
5.5
In d u c e rs.....................................................................................................
37
5.6
Stock s o lu tio n s .........................................................................................
38
5.7
Reaction kits
38
............................................................................................
6 M ethods
6.1
6.2
39
Molecular cloning......................................................................................
39
6.1.1
Polymerase chain reaction ( P C R ) ..............................................
39
6.1.2
Restriction enzymes
40
6.1.3
L ig a tio n ....................................................
40
6.1.4
Competent c e lls.............................................................................
40
6.1.5
One-step preparation of competent cells
..................................
41
6.1.6
Transformation of chemical competent cells...............................
41
Strains and p la s m id s................................................................................
41
6.2.1
42
....................................................................
Glycerol stock forstorageof bacterial stra in s..............................
6.3
6.2.2
Pl-transduction . . .......................................................................
42
6.2.3
Soft agar plates . ..........................................................................
42
6.2.4
Growth and preparation of c e lls .................................................
43
Fluorescence m icroscopy.............................................................................
43
6.3.1
Im a g in g .........................................................................................
43
6.3.2
Stimulus-dependent F R E T ..........................................................
44
6.3.3
Flow cytom etry.............................................................................
45
6.3.4
Sugar uptake assays
46
....................................................................
III Results and Discussion
47
7 Results
48
7.1
In vivo protein-protein interaction analysis by FRET assay...............
48
7.2
Cytoplasmic PTS components are recruited to transporters on stimu­
lation ...........................................................................................................
49
7.3 PTS network senses the overall influx of sugars
..................................
51
7.4 Inducer exclusion is a general phenom enon...........................................
54
7.5
Kinetics of reactions
................................................................................
57
7.6
PTS signals propagate linearly to the chemotaxis p a th w a y ...............
59
7.7 PTS integrates and transmits additional metabolic s i g n a ls ...............
62
7.8
7.9
Default (basal) uptake of carbon sources correlates with their metabolic
efficiency........................................................................................................
PTS mediated regulation of cAMP sy n th esis........................................
7.10 Metabolic efficiency and chemotacticeffectiveness of s u g a r ....................
8 Discussion
69
70
73
77
8.1
Interactions among the PTS com ponents..............................................
78
8.2
PTS senses influx of PTS sugars and integrates metabolic signals
. .
79
8.3
Chemotactic processing of PTS s ig n a ls .................................................
82
8.4
Inducer exclusion is a general phenom enon...........................................
83
8.5
PTS, chemotaxis, and g r o w th ................................................................
84
9 Outlook
86
IV Appendix
88
A Tables
89
B Bibliography
96