The importance of the integrity of phytochromes for their biochemical and physiological function [Elektronische Ressource] / Rashmi Shah

Gp Nolorgues

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

The importance of the integrity of
phytochromes for their biochemical
and physiological function


Inaugural Dissertation
zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf



vorgelegt von

Rashmi Shah
aus Kathmandu (Nepal)



May 2011 angefertigt am Max Planck Institut für Bioanorganische Chemie
(Performed at Max Planck Institute for Bioinorganic Chemistry)













Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf












Referent: Prof. Dr. Wolfgang Gärtner (first referee)

Koreferent: Prof. Dr. Karl-Erich Jaeger (second referee)

Tag der mündlichen Prüfung: 30.05.2011 (date of the oral examination)

Acknowledgement

First of all I would like to express my sincere gratitude to Prof. Dr. Wolfgang Gärtner
for giving me the opportunity to work in his group and providing superb guidance
during the period of investigation. Furthermore, I am grateful to him for the critical
readings and corrections of the manuscript in spite of his busy schedules, after
which this thesis has come out in this form.

I would like to thank Prof. Dr. Karl-Erich Jaeger (Institute for Molecular Enzyme
Technology, University of Düsseldorf, Forschungszentrum Jülich) for kindly
accepting to be the second referee of this dissertation.

My sincere thanks go to Dr. Thomas Drepper (Institute for Molecular Enzyme
Technology, University of Düsseldorf, Forschungszentrum Jülich) for valuable
suggestions during the generation of the knockout mutants. Many thanks also go to
Prof. Jorge Casal (Institute for Agricultural Plant Physiology and Ecology, Buenos
Aires, Argentina) for helping with the infectivity experiments of knockout mutants.

I would like to thank Dr. Shivani Sharda for generously sharing her expertise and
labor in phytochrome expression and conducting the protein-purification
experiments. Special thanks go to colleagues Dr. Amrit Pal Kaur, Björn Zorn, Dr.
Cao Zhen, Dr. Christina Alessandra Hoppe, Jana Riethausen, Dr. Madina
Mansurova, Sarah Raffelberg and Sebastian Gandor for good working and the
inspiring atmosphere. It was a pleasant and unforgettable environment in the lab
because of cooperative and supportive attitude of all of the group members. I extend
my appreciation to Ms. Gülümse Koc-Weier and Mrs. Helene Steffen for maintaining
the order and providing support for keeping things functional in the laboratory.

I am deeply and forever indebted to my parents for their love, support and
encouragement throughout my entire life. My deepest gratitudes are to my sisters for
their love, belief and eternal support. A special thank to my son Garbit whose smile
always inspired me and coming home to him everyday made my life sweet. Finally, a
great thanks to my husband Gopal with whom I would like to share this work, for his
patience, support and above all for his love.
Publications and manuscripts

Sharda, S., Shah, R., and Gärtner, W., (2007) Domain interaction in cyanobacterial
phytochromes as a prerequisite for spectral integrity. Eur Biophys J.; 36(7) : 815-21.

Shah, R., Pathak, G., Drepper, T., and Gärtner, W. An Efficient, Linear DNA-based
in vivo Mutagenesis System for Pseudomonas syringae (Manuscript)

Shah, R., and Gärtner, W., Complex formation between heme oxygenase and
phytochrome during biosynthesis in Pseudomonas syringae pv tomato (Manuscript).
Summary
Summary

Phytochrome pigments were first characterized in higher plants in the 1950’s [1].
These red/far red sensing biological photoreceptors were later reported also for
cyanobacteria [2], and also in other non-photosynthetic bacteria and fungi [3].
Phytochromes interconvert between red light (664/704 nm, P states) and far-red R
light (707/750 nm, P states) absorbing forms. This photoconversion represents a FR
double bond photoisomerization of the covalently bound bilin chromophores

In this work, four different research projects from the area of biological
photoreceptors were followed:

(i) The phytochromes CphA and CphB from the cyanobacterium Calothrix PCC7601
were investigated for the essential protein domains required to maintain the spectral
integrity. Both proteins fold into PAS-, GAF-, PHY-, and Histidine-kinase (HK)
domains. CphA binds phycocyanobilin (PCB) as chromophore and CphB binds
biliverdin (BV) IXα. Removal of the HK domains had no effect on the absorbance
maxima of the resulting PAS–GAF–PHY constructs (CphA: 663/ 707 nm, CphB:
704/750 nm, P /P , respectively); these values are similar to the full length protein R FR
CphA and CphB. Further deletion of the “PHY” domains caused a blue-shift of the
P and P absorption of CphA (λ : 658/698 nm) and increased the amount of R FR max
unproperly folded apoprotein, seen as a reduced capability to bind the chromophore
in a photoconvertible manner. In CphB, however, this deletion practically impaired
the formation of P The intermediate that can be generated shows an absorption FR.
band with very low oscillator strength, whereas the spectral features of the P form R
remain unchanged.

(ii) In an approach to generate non-naturally existing hybrid proteins, the blue-light
sensor kinase module (PAS motif) of P. syringae pv tomato was fused with the
red/far red photoreceptor HK from the cyanobacterial phytochrome CphA to study
the kinase activity of such a reprogrammed (swapped) sensor kinase. This
i Summary
reprogrammed sensor LOV-kinase retained the typical photochemical properties of
PstLOV.

(iii) Recent genome sequence data of the model plant pathogen Pseudomonas
syringae pv tomato DC3000 have revealed the presence of two red/far red sensing
putative phytochrome photoreceptors (PstBphP1 and PstBphP2) and one blue-light
photoreceptor (PstLOV). The bacterial phytochromes from P. syringae pv tomato
(PstBphPs) were recombinantly expressed in E. coli and characterized in vitro. Both
phytochromes show the general modular architecture of a three domain
chromophore-binding region (PAS-GAF-PHY) that is followed by a histidine kinase
domain at the C-terminal part. PstbphP1 is arranged in an operon with a preceding
gene encoding a heme oxygenase (PstbphO), whereas PstbphP2 is followed by a
response regulator. Heterologous expression of the heme oxygenase yielded a
green protein (λ = 650 nm), indicative for bound biliverdin. Heterologous max
expression of PstbphP1 and PstbphP2 yielded the apoproteins for both
phytochromes, however, only in case of PstBphP1 a holoprotein was formed upon
addition of biliverdin. The two phytochromes were also co-expressed with PstbphO
as a two-plasmid approach yielding a fully assembled holoprotein for PstBphP1,
whereas again for PstBphP2 no chromophore absorbance could be detected. An
even further increased yield for PstBphP1 was obtained when the operon PstbphO:
PstbphP1 was expressed in E. coli. A construct placing the gene for PstBphP2
exactly at the position of PstbphP1 in this operon again gave the negative result
such that no phytochrome-2 chromoprotein was formed. The reason for the
improved yield for the operon expression PstbphO: PstbphP1 is the formation of a
complex formed between both proteins during biosynthesis.

(iv) The regulatory functions of these red/ far red genes and of the also present blue
light photoreceptor gene were studied by generating insertional knockout mutants.
As the commonly applied protocol of gene transfer by conjugation/homologous
recombination is a time-consuming process of low-efficiency, an alternative method
was developed in this study to create interposon- as well as point mutations of the
ii Summary
corresponding photoreceptors genes by employing linear DNA constructs. Four
single mutant strains, bphP1∆, bphP2∆, bphO∆, pspto_2896 (LOV∆) and one
double mutant strain, bphP1∆bphP2∆ were generated using the new method. The
bphO- and the pspto_2896 (LOV) genes encode the heme oxygenase and the blue
light-sensitive photoreceptor, described in sub-project (iii). The pspto_2896 mutant
strain was tested for the motility in blue light (447 nm), and all the other mutants
were tested under red/ far red (625, 660, 720, 740 nm) light for changes in their
motility. All mutant strains showed photokinesis response to the blue/red/ far red
light. Further, the effect of mutations on infectivity on plants was studied for the
phytochrome mutant strain (bphP1∆), and the blue-light photoreceptor (LOV∆)
mutant strain. Our preliminary experiments of plant-mutant interaction indicate that
light perceived by the LOV-domain photoreceptor reduces bacterial growth (this
behavior is not observed for the mutant strain), whereas no such effect is observed
for the phytochrome photoreceptor.



iii Zusammenfassung
Zusammenfassung