Molekularbiologische Untersuchungen zu Funktion und Phylogenie methanotropher Bakterien

This work focuses on the characterisation of methanotrophic bacteria (MB) by the aid of moleculare and microbiological methods as well as by bioinformatical methods. The results obtain in the course of this study are reported cumulative. The following topics will be reported: 1) Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 is present in other methane-oxidizing bacteria (MOB), and if it is expressed. We identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme. 2) Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2).A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5_ rapid amplification of cDNA ends we identified transcriptional start sites for sites both, pmoCAB1 and pmoCAB2. Immediately upstream of the transcriptional start, sequence motifs similar to Escherichia coli _70 promoters were identified. 3) MOB of the "upland soil cluster a" (USCa) are assumed to be responsible for methane oxidation at atmospheric methane concentrations but have as yet escaped all cultivation attempts. A metagenomic fosmide library consisting of 250.000 clones was constructed from total DNA of an acidic forest soil previously characterized as a sink for atmospheric methane. Using pmoA-targeted PCR assays, two clones were identified to carry genomic fragments of USCa representatives. Shotgun-based sequencing revealed that both inserts belonged to the same strain or two highy related USCa genotypes. Thus sequences were assembled resulting in a 42-kb contig. The next cultured relative of USCa is, based on partial pmoA phylogenies, the acidophilic M. acidiphila. Consequently, a 100-kb fragment of the M. acidiphila genome containing the pmo operon was sequenced. Annotation and comparative analysis of both genomic fragments against publicly available completely sequenced genomes revealed highest similarities to members of the Bradyrhizobiaceae. The affiliation of USCa to the Alphaproteobacteria was also confirmed by a SOM-based analysis of di-, tri- and tetra-nucleotid patterns of the USCa genomic fragment. A detailed comparative analysis of the pmo operons of USCa and M. acidiphila showed that both the pmo operon structure and the predicted secondary structure of pMMO are highly conserved among all MB. Together with the recently reported pmoCAB1 and pmoCAB2 of Methylocystis strain SC2, this study doubled the number of completely analysed pMMO operons. It enabled a phylogenetic analysis of concatenated pmoCAB sequences rather than only of partial pmoA, thereby roughly sextupling the amount of underlying genetic information. Next to the studies characterised above, two additional projects, focussing on the NifH and NifD phylogenies of methanotrophic bacteria and, respectively, on a new in silico tRFLP prediction tool implemented in the software ARB, which was tested on the field of MOB, will be reported