Exploration of Sulfur-Cycling Microorganisms from Anoxic Black Sea Waters and Sediment

Abstract

Marine microorganisms are drivers of elemental cycles, and thus have global biogeochemical importance. Many marine environments have dysoxic (<1 µM O2 ) or anoxic (<10 nM O₂) conditions. Here, sulfur-cycling microorganisms such as sulfuroxidizing and sulfate-reducing bacteria (SOB, SRB) play key roles. The largely anoxic Black Sea is a model ecosystem for studying sulfur-cycling microbes. Nonetheless, there is only rudimentary insight into the diversity and metabolism of SRB, sulfur-reducing/ disproportionating microorganisms, or microorganisms that degrade the sulfated polysaccharides ubiquitously available in marine environments. Therefore, we set out to advance our understanding of these sulfur-cycling microbes in the Black Sea.

We started off with a critical review of the literature on sulfur-cycling microbes in dysoxic marine waters, combined with genome-resolved metagenomics to obtain an improved view on the diversity of putative SOB and SRB in the Black Sea water column, both described in chapter 2. We chose to taxonomically name several of these uncultured putative SOB and SRB based on phylogenetic, metabolic and habitat profiling analyses, providing taxonomic ‘handles’ with specific criteria for future studies.

We sampled Black Sea sediment from 2,100 meters water depth, at the same site where metagenomics samples were taken, as inoculum for anaerobic cultivation. In chapter 3 we report enrichment of diverse bacteria in sulfate- and sulfur-reducing cultures, including Aegiribacteria spp. with unknown metabolism and novel sulfur-reducing Desulfuromonadales Sva1033 bacteria. Furthermore, we isolated and characterized Desulfopila canfieldii sp. nov., which showed to be capable of dissimilatory sulfate, sulfite, thiosulfate and manganese oxide reduction.Desulfopilaspp. encode the acetyl-CoA pathway, which is presumed bidirectional. However, they appear to use this pathway exclusively in the reductive direction from CO2 to acetyl-CoA, and not in the oxidative direction, as they are incomplete oxidizers. This is a fundamentally relevant avenue for future research on SRB.

We also enriched and isolated novel anaerobes that degrade sulfated polysaccharides, as described in chapter 4. We used commercially available Fucus vesiculosus fucoidan as substrate, which is a structurally complex sulfated polysaccharide with an α-1,3/α1,4-linked L-fucose backbone. Chapter 5 presents a genome-guided phenotypic and phylogenetic characterization of the isolates belonging to the Kiritimatiellaeota phylum, which we proposed to name Pontiella desulfatans F1T gen. nov., sp. nov. and Pontiella sulfatireligansF21^Tsp. nov. These bacteria grew only fermentatively on monosaccharides or the sulfated polysaccharides fucoidan, chondroitin sulfate, and iota-carrageenan. Further, they produced N-sulfated glycosaminoglycan-like exopolysaccharides during stationary phase, which are known to be produced by eukaryotes but not bacteria.

putative biosynthesis gene cluster and sulfotransferase genes were identified in P. sulfatireligans F21^T

The Pontiella spp. encoded unprecedented numbers of sulfatase genes (521 and 480, respectively) and glycoside hydrolase genes (422 and 388, respectively). We observed an increase in free sulfate during growth on iota-carrageenan in chapter 4 and on fucoidan in chapter 6, demonstrating sulfatase-mediated desulfation. In chapter 6 we looked deeper into the enzymatic mechanism of fucoidan and L-fucose degradation by P. desulfatans F1^Tby analysis of gene expression with transcriptomics, and prediction of the subcellular localization of proteins. This revealed high numbers of sulfatase and exo-acting glycoside hydrolase genes were induced by fucoidan.