A novel type of pmoA [Elektronische Ressource] : presence and distribution among methanotrophs ; expression in methylocystis strain SC2 / by Tchawa Yimga Merlin

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A Novel type of pmoA: presence and distribution among methanotrophs - expression in Methylocystis strain SC2 Doctoral thesis for the fulfilment of the grade of Doctor (Dr. rer. nat.) of the Philipps University of Marburg Submitted to the faculty of Biology of the Philipps University of Marburg/Lahn by Tchawa Yimga Merlin from Bangoua, Cameroon Marburg/Lahn 2002 Pledge Pledge I certify that the present thesis entitled: “A Novel type of pmoA: presence and distribution among methanotrophs- expression in Methylocystis strain SC2” was carried out without any unlawful devices. I did not use any other than the described literature sources or technical devices. This work has never been submitted before in this or similar form to any other university and has not been used before any examination. Marburg, 11.09.2002 Tchawa Yimga Merlin Dedication I dedicate this work to: - my family, - my friends, - and those who will find it valuable.

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A Novel type ofpmoA:
presence and distribution among
methanotrophs - expression in
Methylocystisstrain SC2
Doctoral thesis for the fulfilment of the grade of Doctor (Dr. rer. nat.) of the Philipps University of Marburg Submitted to the faculty of Biology of the Philipps University of Marburg/Lahn
by Tchawa Yimga Merlin from Bangoua, Cameroon
Marburg/Lahn 2002
an
e
efor
has not been used b
Marburg, 11.09.2002
Tchawa Yimga Merlin
g
d
e
l
 P
e
ex
y
een submitted before i
amination.
This
work has never b
his o
n t
ilar form t
r sim
y oth
o an
niv
er u
y a
ersit
n
d
ources or tech
literature s
nical d
evices.
was carried out without any unlawful devices. I did
not
use any other than
the
described
Methylocystisstrain SC2
A Novel type ofpmoA: presenc
e and distribution among methanotrophs- ex
pression in
tled:
resent thesis enti
e p
I certify that th
Pledge
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my family, my friends, and those who
I d
ed
i
.
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___________________________________________________________________________
The present work was carried out between April 1999 and September 2002 at the Max-
Planck-Institute for Terrestrial Microbiology, Marburg, G
Dr. Werner Liesack.
ermany under the supervision of PD
By the Biology department of the Philipps University, Marburg as doctoral thesis accepted
on: 04 November 2002.
Date of oral examination: 04 November 2002.
First reviewer: PD Dr. Werner Liesack
Second reviewer: Prof. Dr. Wolfgang Buckel
 Publications
The following papers were published or in preparation by the date of submission of the
present thesis:
1)- Horz H. P., M. Tchawa Yimga and W. Liesack. 2001. Molecular analysis of
methanotrophs associated with roots of submerged rice plants by targeting functional and
phylogenetic genes, includingpmoA-based T-RFLP profiling. Appl. Environ. Microbiol.
67:4177-4185.
2)- Dunfield P. F., M. Tchawa Yimga, S. N. Dedysh; U. Berger, W. Liesack and J. Heyer.
2002. Isolation of aMethylocystisstrain containing a novelpmoA-like gene. FEMS Microbiol.
Ecol.41:17-26.
3)- Tchawa Yimga, M.; J. Heyer; P. F. Dunfield, and W. Liesack. 2002. A Novel type of
pmoA: presence and distribution among methanotrophs  expression inMycolyhtesits strain
SC2 (in preparation).
 Table of contents
Table of contents List of abbreviations1...I. Summary3.
II. Introduction5
1. Balancing the global methane budget.5
2. Methanotrophic bateria...6
3. Physiology, biochemistry and molecular biology of methane oxidation...7
4. Molecular ecology of methanotrophs...10
5. Functional gene probes for methanotrophs...11
6. Anaerobic methane oxidation...13
7. Aim of work..13
III. Materials and Methods..611. Materials...16
1.1 Environmental samples and DNA extracts ....16
1.2 Microorganisms..16
1.3 Cloning vectors...16
1.4 Enzymes and Kits...18
1.5 Nucleic acid standards19
1.6 Oligonucleotide primers and probes...19
1.7 Chemicals and reagents..19
1.8 Gases...19
1.9 Buffers and solutions..19
1.10 Culture media....22
2. Methods.22
2.1 DNA extraction from environmental samples...22
2.2 Nucleic acids extraction from pure cultures..24
2.3 Agarose gel electrophoresis..24
2.4 Quantification of nucleic acids.25
2.5 PCR amplification.25
2.5.1 Amplification of 16S rDNA of type I and type II methanotrophs..25
 Table of contents
2.5.2 Development of specific PCR assays for the retrieval of sequence types belonging to a
 novelpmoAnelieag.26
2.5.3 PCR amplification ofpmoAfrom methanotrophic pure cultures.26
2.6 Amplification of 16S rDNA from methanotrophic pure cultures..27
2.7 Cloning of PCR products...27
2.8 Screening of clone libraries....28
2.9 Long-term storage of clones...28
2.10 T-RFLP analysis....29
2.11 Southern hybridisation..30
2.12 Northern hybridisation..31
2.13 Reverse transcription-PCR (RT-PCR)..32
2.14 Dideoxynucleotide DNA sequencing33
2.15 Blast searches and phylogenetic analyses.33
IV. Results36.1. 16S rDNA-based diversity of type I and type II methanotrophs on rice roots..36
1.1 Type I MOB clone library...36
1.2 Type II MOB clone library..36
2. Development of PCR assays for the specific retrieval of novelpmoA-like sequences.38
2.1 Specific primers for novelpmoA38
2.2 Specific PCR assays andpmoAsequences of the novel lineage..40
2.3 T-RFLP for assessingpmoAdiversity in selected environmental DNA extracts.42
3.Methylocsyitsstrain SC2 harbours a novelpmoA-like gene...43
4. Screening of pure cultures for the presence of the novelpmoAgene type.46
4.1 Establishment of a T-RFLP-based screening method...46
4.2 Screening of pure cultures.47
5. Expression of the novelpmoA-like gene in strain SC2...51
V. Discussion4.5.
1. 16S rRNA-based diversity of type I and type II methanotrophs on rice roots....54
2. The novelpmoAgene copy.56
2.1 Development of molecular tools for the detection of the novel gene copy...56
2.2 Evidence for the presence of a novelpmoAgene inMethylocystisstrain SC2.57
2.3 Expression of the novel gene in strain SC2...58
 Table of contents
2.4 Screening of methanotrophic pure cultures for the presence of the novelpmoA58
2.5 Southern hybridisation.60
2.6 Functional significance of the novelpmoA..60
VI. References62..
Curriculum vitae Acknowledgement