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The hidden sulfur cycle in freshwater wetlands: an eco-systems biology approach to identify and characterize major microbial players

Final Report Summary - WETLAND-ECOSYSBIOL (The hidden sulfur cycle in freshwater wetlands: an eco-systems biology approach to identify and characterize major microbial players)

Freshwater wetlands are a major source of the greenhouse gas methane but can also act as carbon sink, storing currently more than one third of the terrestrial organic carbon. Understanding their intertwined biogeochemistry and microbiology is therefore indispensable to foresee their influence to positive and negative climate feedback cycles. The aim of this project was to elucidate the identity and ecophysiology of sulfate reducing microorganisms (SRM) driving a highly active but hidden sulfur cycle in wetlands, which is not apparent from the low standing pools of sulfate and thus has been severely understudied. The project was subdivided into three major focus areas dealing with (i) the ecophysiology of active SRM in peatlands, (ii) analyzing the hidden sulfur cycle in rice paddies using an eco-systems approach, and (iii) performing a comparative genome analysis of Desulfosporosinus species as important wetland SRM. All of the anticipated goals in the individual focus areas were achieved.

In focus area 1, a large scale incubation of peat soil under various substrate and sulfate scenarios was set up as replicated microcosm experiment. Microbial activities in these incubations were monitored using biogeochemical methods and total DNA and RNA was extracted from four selected time points. These DNA and RNA extracts were subjected to 16S rRNA (gene) amplicon sequencing and obtained results were amended to bioinformatics and statistical analysis. Natively abundant (≥0.1% estimated genome abundance) species-level operational taxonomic units (OTUs) showed no significant response to sulfate. In contrast, low-abundance OTUs responded significantly to sulfate in incubations with propionate, lactate and butyrate. These OTUs included members of recognized sulfate-reducing taxa (Desulfosporosinus, Desulfopila, Desulfomonile, Desulfovibrio) and also members of taxa that are either yet unknown sulfate reducers or metabolic interaction partners thereof. Most responsive OTUs markedly increased their ribosome content but only weakly increased in abundance. Responsive Desulfosporosinus OTUs even maintained a constantly low population size throughout 50 days, which suggests a novel strategy of rare biosphere members to display activity (Hausmann et al., 2016, ISME Journal – Nature Publishing Group). In parallel, the obtained DNA and RNA extracts were subjected to metagenome and metatranscriptome analysis. Here, draft genomes of seven novel Acidobacteria species with the potential for dissimilatory sulfite or sulfate respiration were recovered. Surprisingly, the genomes also encoded DsrL, which so far was only found in sulfur-oxidizing microorganisms. Metabolic potentials included polysaccharide hydrolysis and sugar utilization, aerobic respiration, several fermentative capabilities, and hydrogen oxidation. These findings extend both, the known physiological and genetic properties of Acidobacteria and the known taxonomic diversity of microorganisms with a DsrAB-based sulfur metabolism (Hausmann et al., 2018a ISME Journal – Nature Publishing Group).

In focus area 2, a greenhouse experiments with rice plant microcosms in the presence and absence of the sulfate-containing soil additive gypsum was conducted. Biogeochemical parameters were monitored from the rhizosphere, the bulk soil, as well as from the headspace of the plant microcosms during the whole incubation period. The obtained results demonstrated that gypsum as a typical sulfate-containing amendment to rice paddy soil decreased methane emissions by up to 99% from this habitat but had no major impact on the general phylogenetic composition of the bacterial community. It rather selectively stimulated or repressed a small number of microorganisms in the rhizosphere and bulk soil. Gypsum-stimulated OTUs were affiliated with several potential sulfate-reducing (Syntrophobacter, Desulfovibrio, unclassified Desulfobulbaceae, unclassified Desulfobacteraceae) and sulfur-oxidizing taxa (Thiobacillus, unclassified Rhodocyclaceae). Abundance correlation networks suggested that two abundant (>1%) OTUs (Desulfobulbaceae, Rhodocyclaceae) were central to the reductive and oxidative parts of the sulfur cycle (Wörner et al., 2016, Environ Microbiol Rep). In parallel, the obtained DNA extracts were amended to a metagenomics analysis, which was flanked by a metaproteomics analysis. Here, we assembled the genome of Nitrospirae bacterium Nbg-4 as a representative of Nitrospirae species regularly observed in environmental surveys of anoxic marine and freshwater habitats. Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression thereof in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia (DNRA) with expression of its first step being detected in bulk soil without gypsum amendment. Comparison to publicly available Nitrospirae genome bins revealed the pathway for dissimilatory sulfate reduction also in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we proposed for Nbg 4 the name Candidatus Sulfobium mesophilum, gen. nov., spec. nov. (Zecchin et al., 2018, Appl Environ Microbiol).

In focus area 3, we used environmental systems biology to follow a sulfate-reducing Desulfosporosinus species in peatland soil. This cosmopolitan rare biosphere member was identified to be active in cryptic sulfur cycling in focus area 1. Using differential metagenomics, we obtained a 98%-complete genome of this rare biosphere member directly from environmental samples. The transcriptional response of the peatland Desulfosporosinus species was followed by metatranscriptomics in the incubations outlined in focus area 1. Under sulfate-amended conditions a 56- to 188-fold increase of its transcriptional activity was evident in acetate, propionate, lactate, and butyrate treatments as opposed to the no-substrate control, revealing a generalist lifestyle. This correlated with a significant overexpression of genes encoding ribosomal proteins but not of genes encoding cell replication or sporulation. Our results provided proof for the first time that rare biosphere members transcribe metabolic pathways relevant for carbon and sulfur cycling over prolonged time periods while being growth-arrested in their lag phase (Hausmann et al., 2018b, in submission).

The studied research area could be advanced beyond the state-of-the-art in three aspects. Results obtained in focus area 1 and 3 added a new concept to microbial ecology. We could clearly show that the so called rare biosphere plays an important role in the hidden sulfur cycle of peatlands. In addition, the obtained results clearly showed that rare biosphere microorganisms can be growth-arrested in their lag phase while being metabolically active. Results obtained in focus area 2 were important from an agricultural point of view. Surprisingly, gypsum as a typical sulfate-containing soil additive had only a minor impact on the overall bacterial community and rather selectively selected microorganisms active in sulfur cycling. However, the ecosystem service of this small sulfur community was dramatic by decreasing emission of the otherwise produced greenhouse gas methane by 99%. Last but not least, our results expanded the known diversity of SRM to the phylum Acidobacteria and mesophilic Nitrospirae with the proposal of 3 novel Candidatus genera. Additional work comprised a publications on the phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases as functional marker genes for SRM (Müller et al., 2015), the establishment of a high throughput amplicon sequencing method for the dsrA and dsrB genes (Pelikan et al., 2015), and the discovery of the capacity for sulfate/sulfite reduction in the genomes of organisms from thirteen bacterial and archaeal phyla (Anantharaman et al.. 2018, ISME Journal). Furthermore, I contributed as first or co-author to seven further publications (Pester et al., 2014, Gruber-Dorninger et al., 2014, Herbold et al., 2017, Schlotz et al., 2014, Müller et al., 2015, Patil et al., 2015, Dawson et al., 2017). In summary, my research achievements enabled me to obtain a position as full professor at the Technical University of Braunschweig and the Leibniz Institute DSMZ in 2017.