Identification of the biotin transporter in Escherichia coli, biotinylation of histones in Saccharomyces cerevisiae and analysis of biotin sensing in Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Stefan Ludwig Ringlstetter

Stefan Löfving

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Identification of the biotin transporter in
Escherichia coli, biotinylation of histones in
Saccharomyces cerevisiae and analysis of biotin
sensing in Saccharomyces cerevisiae
Dissertation
Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der
naturwissenschaftlichen Fakultät III - Biologie und Vorklinische Medizin -
der Universität Regensburg
vorgelegt von
Stefan Ludwig Ringlstetter
aus Straubing
Regensburg im Februar 2010Promotionsgesuch eingereicht am: 23.02.2010
Tag der mündlichen Prüfung: 29.04.2010
Die Arbeit wurde angeleitet von: PD Dr. Jürgen Stolz
Prüfunfgsausschuss: Vorsitzender: Prof. Dr. Gernot Längst
1. Prüfer: PD Dr. Jürgen Stolz
2. Prüfer: Prof. Dr. Ludwig Lehle
3. Prüfer: Prof. Dr. Reinhard Sterner
IIContents
1 Introduction 1
1.1 History of vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.1 Structure and chemistry . . . . . . . . . . . . . . . . . . . . . 3
1.2.2 Physiological role . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Biotin metabolism in Escherichia coli . . . . . . . . . . . . . . . . . . 8
1.3.1 Biosynthesis in E. coli . . . . . . . . . . . . . . . . . . . . . . 8
1.3.2 Biotin transport in gram-positive bacteria and E. coli . . . . . 10
1.3.3 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.4 Biotin metabolism in Saccharomyces cerevisiae . . . . . . . . . . . . . 15
1.4.1 Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4.2 Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4.3 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5 Biotin in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.5.1 Biotin as a vitamin . . . . . . . . . . . . . . . . . . . . . . . . 21
1.5.2 Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.5.3 Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1.5.4 Biotinylation of histones . . . . . . . . . . . . . . . . . . . . . 24
1.6 Aims of this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2 Material and methods 26
2.1 Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.1.1 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.1.2 Databases, websites and software . . . . . . . . . . . . . . . . 27
2.1.3 Chemicals and enzymes . . . . . . . . . . . . . . . . . . . . . 28
2.1.4 Buffers and solutions . . . . . . . . . . . . . . . . . . . . . . . 29
2.1.5 Culture media . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.1.6 Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.7 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.1.8 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.2.1 Cell maintainence . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.2.2 Molecular biology methods . . . . . . . . . . . . . . . . . . . . 44
2.2.3 Methods with DNA . . . . . . . . . . . . . . . . . . . . . . . . 46
III2.2.4 Methods with proteins . . . . . . . . . . . . . . . . . . . . . . 48
2.2.5 Reporter-genes . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.2.6 Electrophoretic mobility shift assays (EMSA) . . . . . . . . . 53
2.2.7 Pyruvate carboxylase activity measurements . . . . . . . . . . 53
2.2.8 Biotin uptake experiments . . . . . . . . . . . . . . . . . . . . 54
2.2.9 Isolation of membrane fractions of E. coli and reconstitution
in membrane vesicles . . . . . . . . . . . . . . . . . . . . . . . 54
2.2.10 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3 Results 56
3.1 Biotin uptake in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.1.1 Candidate genes. . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.1.2 In silico analysis of YigM . . . . . . . . . . . . . . . . . . . . 59
3.1.3 Immunological detection . . . . . . . . . . . . . . . . . . . . . 62
3.1.4 Uptake experiments . . . . . . . . . . . . . . . . . . . . . . . . 62
3.1.5 Expression of a codon-adapted yigM from pET24 . . . . . . . 64
3.1.6 K -value of YigM for biotin uptake . . . . . . . . . . . . . . . 64M
3.1.7 Energetization of biotin transport . . . . . . . . . . . . . . . . 65
3.1.8 Uptake experiments in membrane vesicles . . . . . . . . . . . 68
3.1.9 Sequence of YigM from the E. coli biotin transport mutant
S1039 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.1.10 C-terminal truncation of yigM . . . . . . . . . . . . . . . . . . 71
3.1.11 Gene regulation of yigM . . . . . . . . . . . . . . . . . . . . . 73
3.1.12 Luciferase-reporter constructs . . . . . . . . . . . . . . . . . . 73
3.1.13 Electrophoretic mobility-shift assays . . . . . . . . . . . . . . 75
3.2 Biotin sensing in S. cerevisiae . . . . . . . . . . . . . . . . . . . . . . 78
3.2.1 VHR1 and biotin sensing . . . . . . . . . . . . . . . . . . . . 79
3.2.2 The function of pyruvate carboxylases in biotin sensing . . . . 83
3.2.3 Single and double knockouts of PYC1 and PYC2 . . . . . . . 85
3.2.4 Complementationofpyc1Δpyc2Δwithpyr1+fromSchizosac-
charomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . 86
3.2.5 Truncation of Pyc2p C-terminus . . . . . . . . . . . . . . . . . 88
3.2.6 Co-immunoprecipitation of Pyc2p . . . . . . . . . . . . . . . . 95
3.3 Histone biotinylation in S. cerevisiae . . . . . . . . . . . . . . . . . . 97
4 Discussion 101
4.1 Biotin transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.1.1 The E. coli biotin transporter represents a new class of bacte-
rial biotin transporters . . . . . . . . . . . . . . . . . . . . . . 101
4.1.2 yigM encodes the E. coli biotin transport protein . . . . . . . 103
4.1.3 Transport mechanism of YigM . . . . . . . . . . . . . . . . . . 104
4.1.4 Homologues of yigM might represent biotin transporters of
other gram negative bacteria . . . . . . . . . . . . . . . . . . . 107
4.1.5 Expression of yigM is regulated by biotin . . . . . . . . . . . . 109
IV4.2 Biotin in S. cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
4.2.1 VHR1 and biotin sensing . . . . . . . . . . . . . . . . . . . . . 112
4.2.2 Pyruvate carboxylases and biotin sensing . . . . . . . . . . . . 113
4.2.3 Histone biotinylation . . . . . . . . . . . . . . . . . . . . . . . 116
5 Summary 120
Literature 120
6 Appendix 143
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Danksagung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Erklärung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
V1 Introduction
1.1 History of vitamins
More than 100 years ago Sir Frederick Gowland Hopkins realized from the results
of his experiments with young rats that the animals could not survive from being
fed with only a mixture of pure protein, fat and carbohydrates [87]. He claimed
there must be some other so called "minor" or "accessory factors" that are essential
for normal growth and development. The term "vitamin" was invented in 1912 by
Casimir Funk. He tried to isolate a substance to heal the Beriberi disease which is
caused by a lack of thiamine. As Funk found out that the substance contained an
amino group he called it vitamin (vita lat. = life, amin from amino-group). Several
at that time uncharacterized growth factors, although not all of them contained an
amino-group were then also designated as vitamins. Today vitamins are defined
as organic substances that are essentiall in small amounts because they can not
be synthesized at all or not in the required quantity. Vitamins are not needed
as an energy-source but fulfill functions as cofactors, antioxidants or hormone-like
substances. Although only very low doses in the mg org range of these substances
are necessary, the symptoms of a lack of vitamins can lead to metabolic defects and
severe illness. For humans 13 fat- and water-soluble vitamins are known today. An
overview is shown in table 1.1.
Not all of the listed vitamins are essential for all organisms. Every organism has
its own special set of vitamins. Several plants and microorganisms are still able to
synthesizesomeorevenallofthesesubstances,butmosthigherorganismsdependend
on the uptake of a certain amount from food or in part from microbial synthesis in
theintestine. Therelativecontributionofintestinal synthesis tovitaminsupplymay
be different for individual vitamins and in different species and can in many cases
not be precisely quantified.
11 Introduction 2
Vitamin (class) active substance function in metabolsim
fat-soluble:
vitamin A retinol, retinal light perception, antioxidants
vitamin D calciferol regulation of calcium- and
phosphate-metabolism, hormon-
like
vitamin E tocopherol, antioxidants for unsaturated
tocotrienol membrane-lipids
vitamin K phyllochinone, γ-carboxylation of glutamate
menachinone (blood clotting)
water-soluble:
vitamin B thiamine aldehyde-transfer1
vitamin B riboflavin oxidation and reduction2
vitamin