TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Mikrobiologie




Elucidation of the Cell Division Mechanism and
Characterization of Tubulins
in the Bacterial Phylum Verrucomicrobia


Martin Pilhofer



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen
Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. W. Höll

Prüfer der Dissertation: 1. Univ.-Prof. (i. R.) Dr. K.-H. Schleifer
2. Univ.-Prof. Dr. W. Liebl
3. Priv.-Doz. Dr. G. Petroni
(Università di Pisa, Italy)





Die Dissertation wurde am 15.04.2008 bei der Technischen Universität
München eingereicht und durch die Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt am 29.05.2008
angenommen.












































meiner Familie

Contents

CONTENTS


Abbreviations............................................................................................................ 7

Original Publications................................................................................................ 9

A General Introduction.................................................................................... 11
A.1 The Cytoskeleton: Eukaryotes versus Bacteria.......................................... 13
A.2 Bacterial Cell Division................................................................................. 21
A.3 The Bacterial Phylum Verrucomicrobia ...................................................... 25
A.4 Aims of This Study ..................................................................................... 33

B Results and Discussion............................................................................... 35
B.1 Development of the Two-Step Gene Walking Method................................ 37
B.2 23S rRNA Phylogeny of Verrucomicrobia: Inter- and Intraphylum
Relationships ............................................................................................. 39
B.3 Detection of Tubulin Genes in Verrucomicrobia ......................................... 41
B.4 Cell Division in the PVC Superphylum ....................................................... 42
B.5 Characterization of Bacterial Tubulins........................................................ 50
B.6 Origin of Bacterial Tubulins ........................................................................ 71

C Summary....................................................................................................... 75
C.1 Summary.................................................................................................... 77
C.2 Zusammenfassung..................................................................................... 79

D References.................................................................................................... 81

Appendices ............................................................................................................. 97
Appendix A............................................................................................................ 99
Appendix B.......................................................................................................... 119
Appendix C ......................................................................................................... 131
Appendix D ......................................................................................................... 149
Appendix E.......................................................................................................... 177

Acknowledgments................................................................................................ 187

Curriculum Vitae................................................................................................... 191
Figures
FIGURES


Figure 1. The structure of a eukaryotic microtubule and its subunit [Alberts et al.
(2002), modified]...................................................................................... 14
Figure 2. Structure comparison of tubulin homologs (Michie and Lowe 2006) ......... 16
Figure 3. Model of the septum and order of protein recruitment (Margolin 2005)..... 22
Figure 4. dcw gene cluster in different bacteria (Mingorance and Tamames 2004). 24
Figure 5. Epixenosomes and their host (Petroni et al. 2000).................................... 26
Figure 6. Stalked bacteria from Lake Alexander (Henrici and Johnson 1935).......... 28
Figure 7. Prosthecobacter debontii........................................................................... 29
Figure 8. 16S rRNA tree showing the sister-phyla Verrucomicrobia and
Lentisphaerae.......................................................................................... 31
Figure 9. SDS gel showing cultures overexpressing different btub-operon genes.... 54
Figure 10. Western hybridizations using antibodies specific for btub-operon gene
products................................................................................................... 57
Figure 11. Determination of specificity for immunofluorescence staining ................. 59
Figure 12. Immunofluorescence staining of recombinant E. coli expressing btubA-
btubB-bklc................................................................................................ 61
Figure 13. Intracellular rod-like structure in recombinant E. coli expressing btubA-
btubB-bklc................................................................................................ 62
Figure 14. TEM micrographs of recombinant E. coli cells expressing the btub-operon
................................................................................................................. 64
Figure 15. TEM micrographs of P. debontii .............................................................. 66
Figure 16. Immunogoldstaining of Prosthecobacter cells ......................................... 68


Abbreviations

ABBREVIATIONS

AMP adenosine monophosphate
ATP adenosine triphosphate
bklc bacterial kinesin light chain
bp basepair
BSA bovine serum albumin
btub bacterial tubulin
Ca Candidatus
cDNA complementary DNA
CODEHOP consensus degenerate hybrid oligonucleotide primer
Da dalton
dcw division and cell wall
ddl d-alanine d-alanine ligase
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
°C degree Celsius
DNA deoxyribonucleic acid
DTT dithiothreitol
FRAP fluorescence recovery after photo bleaching
fts filamentous temperature sensitive
GDP guanosine diphosphate
GFP green fluorescent protein
GTP guanosine triphosphate
h hour
His histidine
IF intermediate filament
IgG immunoglobulin G
IMG Integrated Microbial Genomes
IPTG isopropyl-beta-D-thiogalactopyranosid
kb kilobases
kDa kilodaton
klc kinesin light chain
kV kilovolt
7
Abbreviations
M molar
µF microfarad
µg microgram
µm micrometer
µM micromolar
MT microtubule
MTOC microtubule organizing center
mg milligram
ml milliliter
min minute
ng nanogram
Ni nickel
nm nanometer
OD optical density
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
Pde Prosthecobacter debontii
Pdj Prosthecobacter dejongeii
rpm rounds per minute
Pva Prosthecobacter vanneervenii
PVC Planctomycetes-Verrucomicrobia-Chlamydiae
PVDF polyvinylidene fluoride
RNA ribonucleic acid
rRNA ribosomal RNA
RT reverse transcription / real time
SDS sodiumdodecylsulphate
TBS tris buffered saline
TEM transmission electron microscopy
TPR tetratricopeptide repeat
V volt
v/v volume/volume
Vsp Verrucomicrobium spinosum
w/v weight/volume
8
List of Original Publications

ORIGINAL PUBLICATIONS

Some main results of this study and the corresponding discussion, conclusions and materials
and methods are described in detail in the publications listed below. The original articles and
the corresponding author contributions can be found in the section Appendix A-D. Also, the
study contains unpublished data for which additional materials and methods are specified in
section Appendix E.
The symbol at the beginning of a chapter designates that the chapter content was
published as part of the indicated appendix / publication.



Appendix A Pilhofer M, Rosati G, Ludwig W, Schleifer KH, Petroni G.
Coexistence of tubulins and ftsZ in different Prosthecobacter species.
Molecular Biology and Evolution 2007 Jul;24(7):1439-42.

Appendix B Pilhofer M, Bauer AP, Schrallhammer M, Richter L, Ludwig W,
Schleifer KH, Petroni G.
Characterization of bacterial operons consisting of two tubulins and a
kinesin-like gene by the novel Two-Step Gene Walking method.
Nucleic Acids Research 2007 Nov;35(20):e135.

Appendix C Pilhofer M, Rappl K, Eckl C, Bauer AP, Ludwig W, Schleifer KH,
Petroni G.
Characterization and evolution of cell division and cell wall synthesis
genes in the bacterial phyla Verrucomicrobia, Lentisphaerae,
Chlamydiae and Planctomycetes and phylogenetic comparison with
rRNA genes.
Journal of Bacteriology 2008 doi:10.1128/JB.01797-07.

Appendix D Pilhofer M, Ludwig W, Schleifer KH, Petroni G.
Phylogenetic relationships of bacterial tubulins within the Tubulin/FtsZ
superfamily: Implications on their evolutionary origin
Prepared for submission.

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