Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.
| Identifer | oai:union.ndltd.org:CCSD/oai:tel.archives-ouvertes.fr:tel-01017178 |
| Date | 30 May 2013 |
| Creators | Farhan Ul Haque, Muhammad |
| Publisher | Université de Strasbourg |
| Source Sets | CCSD theses-EN-ligne, France |
| Language | English |
| Detected Language | English |
| Type | PhD thesis |
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