Environmental genomics and proteomics of plant-associated microbial dimethylsulfide degradation in a coastal salt marsh

Abstract

The methylated sulfur compound dimethylsulfide (DMS) plays a major role in the biogeochemical sulfur cycle and atmospheric chemistry. Bacteria are a main sink for DMS in the global sulfur cycle and can utilise DMS as a sole carbon and energy source. This study investigated the diversity and activity of bacteria capable of DMS degradation and associated with the salt marsh plant Spartina anglica known to be a producer of the DMS precursor dimethylsulfoniopropionate (DMSP). Initially, it was shown that S. anglica is rich in DMSP throughout the entire seasonal cycle in the Stiffkey salt marsh providing a likely hotspot for DMSP- and DMS-degrading bacteria. DMS uptake experiments demonstrated that DMS degradation takes place in the phyllosphere and rhizosphere of S. anglica and denaturing gradient gel electrophoresis (DGGE) and high-throughput amplicon sequencing of the 16S rRNA revealed the dominance of bacteria related to α - and γ- Proteobacteria, as well as Flavobacteria in the phyllosphere of S. anglica, whereas the rhizosphere was mainly colonised by members of the classes γ-, δ-, α-, and ε-Proteobacteria and Bacteroidia. The diversity of DMS-degrading bacteria associated with S. anglica was first assessed by enrichment culture. DGGE analysis and high-throughput sequencing diversity of DMS enriched samples using the 16S rRNA gene as a marker suggested the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like bacteria in DMS-enrichments of phyllosphere and rhizosphere samples of S. anglica. A functional gene marker analyses was carried out using the gene encoding methanethiol oxidase (mtoX), a key enzyme in DMS degradation and the gene encoding a DMSP lyase (dddP) to determine the diversity of bacteria degrading DMS and DMSP, respectively, in the phyllosphere and rhizosphere of S. anglica. The analysis for mtoX showed a great diversity of this gene in phyllosphere and rhizosphere of S. anglica and that major clades of mtoX clustered together with Sedimenticola, Methylohalobius, Methylophaga and other mtoX clones previously detected in surface sediments of the same salt marsh. The results for the functional marker gene analysis for the dddP gene suggested the dominance of Ruegeria-like species and Roseobacter-like bacteria but also of unidentified Ddd+ bacteria in the phyllosphere and rhizosphere of S. anglica. In order to identify the active DMS degraders in the phyllosphere and rhizosphere of S. anglica stable-isotope probing (SIP) combined with DGGE and highthroughput sequencing of the 16S rRNA gene was carried out. The SIP experiments revealed the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like microorganisms in the rhizosphere of S. anglica. However, the DMS-degrading microbial community in the phyllosphere seemed more diverse than in the rhizosphere and microorganisms like Halothiobacillus, Xanthomonadaceae, Rhodanobacter but also Piscirickettsiaceae seemed to be involved. A comparative proteomic and transcriptomic experiment of Methylophaga thiooxydans, a microorganism found in phyllosphere and rhizosphere of S. anglica, revealed the general pathways involved in methanol but especially DMS degradation. During DMS cycling the protein and the protein-encoding gene for the methanethiol oxidase (MtoX/mtoX) was highly expressed. A metaproteogenomic experiment provided an insight into the taxonomy and functional diversity of the microbial community associated with the Spartina anglica phyllosphere. Analysis of the metagenome provided evidence that the microbial community associated with S. anglica is dominated by γ-Proteobacteria such as Halomonadales, Alteromonadales, Oceanospirillales, and Thiotrichales and the alphaproteobacterial order Rhodobacterales and showed therefore a major difference to the bacterial community composition in the phyllosphere of for instance A. thaliana, clover, soybean and rice. The detection of DMSP lyase encoding genes and genes encoding proteins for DMS degradation confirmed the genetic potential for the observed DMSP and DMS degradation activity previously measured in the phyllosphere of S. anglica. The metaproteomic experiment allowed a first insight into the proteins expressed in the phyllosphere of S. anglica which also suggested that mainly γ-Proteobacteria and α-Proteobacteria are dominant populations occurring in this habitat. New insights were gained into the activity and diversity of DMS-degrading microbial communities associated with a salt marsh plant that represents a significant component of salt marsh plant communities world wide. Not only was the taxonomic and functional diversity of DMS-degrading microorganisms associated with S. anglica greater then previously realised, the observation of considerable potential of above-ground plant-associated DMS degradation in the phyllosphere demonstrates a previously unrealised sink in the DMS cycle in coastal ecosystems, which is clearly more complex than previously appreciated