Environmental Microbiology (2019) 21(1), 209–225 doi:10.1111/1462-2920.14442 Insight into the sulfur metabolism of Desulfurella amilsii by differential proteomics Anna P. Florentino,1 Inês A. C. Pereira ,2 processes and these proteins should be considered in Sjef Boeren,3 Michael van den Born,1 searches for sulfur metabolism in broader fields like Alfons J. M. Stams1,4 and Irene Sánchez-Andrea 1* meta-omics. 1Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE, Wageningen, The Netherlands. Introduction 2Instituto de Tecnologia Quimica e Biologica António Xavier, Universidade Nova de Lisboa, Elemental sulfur is one of the most ubiquitous sulfur spe- Av. da Republica-EAN, 2780-157, Oeiras, Portugal. cies in sediments and geological deposits, formed by bio- 3Laboratory of Biochemistry, Wageningen University, logical and chemical oxidation of H2S(Rabuset al., 2013). μ −1 Stippeneng 4, 6708 WE, Wageningen, The Netherlands. Elemental sulfur has a low solubility in water [5 gl at 4CEB-Centre of Biological Engineering, University of 20 C (Boulegue, 1978)] hampering fast growth of sulfur- Minho, Campus de Gualtar, 4710-057, Braga, Portugal. utilizing prokaryotes (Blumentals et al., 1990; Schauder and Müller, 1993). However, microorganisms able to utilize elemental sulfur are great in number and widespread over Summary the tree of life (Dahl et al., 2001; Friedrich et al., 2001; Many questions regarding proteins involved in micro- Rabus et al., 2013; Florentino et al., 2016a,b). bial sulfur metabolism remain unsolved. For sulfur Two mechanisms have been postulated in sulfur- respiration at low pH, the terminal electron acceptor is reducers to cope with the low solubility of elemental sulfur: 0 still unclear. Desulfurella amilsii is a sulfur-reducing (a) direct physical attachment of the cells to the solid S bacterium that respires elemental sulfur (S0) or thio- phase (Laska et al., 2003), or (b) a soluble intermediate sulfate, and grows by S0 disproportionation. Due to its form of sulfur terminally accepts electron. Regarding the versatility, comparative studies on D. amilsii may shed latter, when sulfide is available in the environment, it 0 light on microbial sulfur metabolism. Requirement of cleaves the S -ring of elemental sulfur by a nucleophilic physical contact between cells and S0 was analyzed. attack, which generates polysulfide (Eq. 1), enabling an Sulfide production decreased by around 50% when S0 enhancement of sulfur respiration (Blumentals et al., 1990, was trapped in dialysis membranes, suggesting Schauder and Müller, 1993, Hedderich et al., 1999). that contact between cells and S0 is beneficial, 0 − Ð 2− + ð Þ but not strictly needed. Proteome analysis was per- S8 +HS S8S +H 1 formed under the aforementioned conditions. A Mo- oxidoreductase suggested from genome analysis to The concentration and chain length of polysulfide act as sulfur reductase was not detected in any depend on several parameters, such as pH, elemental growth condition. Thiosulfate and sulfite reductases sulfur and sulfide concentrations, redox potential, and showed increased abundance in thiosulfate-reducing temperature. The maximum polysulfide concentration in cultures, while rhodanese-like sulfurtransferases were solution increases with increasing pH (Schauder and highly abundant in all conditions. DsrE and DsrL were Müller, 1993). Some binding proteins produced by sulfur- abundantly detected during thiosulfate reduction, sug- reducing microorganisms, such as the polysulfide sulfur gesting a modified mechanism of sulfite reduction. transferase showed high affinity for polysulfide at low Proteogenomics suggest a different disproportion- concentrations in Wolinella succinogenes (Klimmek ation pathway from what has been reported. This work et al., 1999, Lin et al., 2004). points to an important role of rhodaneses in sulfur At low pH, however, polysulfide is unstable and is readily converted to elemental sulfur (Steudel, 2003). While at 20C and pH 6.5, the maximum concentration of Received 25 March, 2018; revised 28 August, 2018; accepted 5 fi October, 2018. *For correspondence. E-mail irene.sanchezandrea@ polysul de in solution is around 0.1 mM, at pH 3 (in the wur.nl; Tel./Fax +31 317 483486/+31 317 483829. presence of elemental sulfur in excess and 1 mM of © 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 210 A.P. Florentino et al. − hydrogen sulfide) it is just around 10 12 Mat80C cell-sulfur interaction in sulfur respiration by trapping (Schauder and Müller, 1993). sulfur in dialysis membranes; and through comparative Therefore, depending on the environmental conditions, proteomics (ii) elucidate enzymes involved in sulfur/thio- the terminal electron acceptor for sulfur-reducing sulfate respiration and sulfur disproportionation, and enzymes might differ. The enzymes possibly involved in (iii) compare autotrophic and organotrophic growth to the respiration of those compounds were only isolated identify the enzymes involved. This combined strategy and characterized from a few microorganisms, such as allowed us to obtain information about energy and carbon Wolinella succinogenes (Klimmek et al., 1991), Pyrococ- metabolism of D. amilsii, as well as to reveal the impor- cus furiosus (Ma and Adams, 1994) and Acidianus tance of some enzymes – rhodanese-like sulfurtrans- ambivalens (Laska et al., 2003). Unfortunately, a well- ferases – in sulfur metabolism. This study is the first defined activity of those enzymes in the metabolism of proteomic study performed under different sulfur metabo- sulfur utilizers is not yet available. lizing conditions in a comparative way, helping to eluci- Sulfur disproportionation, a process involving oxidative date some aspects of this important metabolism. and reductive routes, is also a poorly characterized part of the sulfur metabolism. Although those reactions have been described for several sulfur reducers from the Pro- Material and methods teobacteria, Firmicutes and Thermodesulfobacteria phyla Growth medium and cultivation (Finster et al., 1998, Finster, 2008, Hardisty et al., 2013, Florentino et al., 2016a,b), the formation and oxidation of D. amilsii cells were grown in 1 L bottles containing sulfite as a key intermediate is not yet understood 500-ml anoxic medium (Florentino et al., 2015). To adjust (Finster, 2008, Hardisty et al., 2013). In a similar manner, the pH of the medium, bicarbonate-buffer was omitted as sulfite is postulated to be an intermediate in thiosulfate described by Sánchez-Andrea et al. (2013) and pH was reduction (Surkov et al., 2000, Stoffels et al., 2012), but adjusted to 3.5 or 6.5 with HCl. In the set analyzed for the complete mechanism of thiosulfate respiration leading disproportionation of elemental sulfur, elemental sulfur to sulfide is still unclear. was added at a concentration of 25 mM with dissolved Desulfurella amilsii is an acidotolerant sulfur-reducing carbon dioxide or bicarbonate likely used as carbon bacterium isolated from sediments of the acidic Tinto source. For the cultures incubated under sulfur-respiring river in Spain. It can grow in a broad range of pH (from conditions, either elemental sulfur or thiosulfate (25 mM) 3 to 7) and it is able to grow by respiring elemental sulfur were added as electron acceptors; in the chemolitho- or thiosulfate, and by disproportionating elemental sulfur trophic cultures subset H2/CO2 (1.5 atm, 80:20, v/v) was (Florentino et al., 2016a,b). Based on its genome added to provide H2 as electron donor and CO2 as car- sequence, D. amilsii encodes an NADH-dependent ferre- bon source, while the heterotrophic cultures were sup- doxin:NADP+ oxidoreductase (NfnAB; DESAMIL20_1852– plied with acetate (5 mM) as electron donor and carbon 1853) that in the past was reported to act as sulfide source. Bottles with medium were sealed with butyl rub- dehydrogenase (Ma and Adams, 1994); and a Mo- ber stoppers (Rubber BV, Hilversum, The Netherlands) oxidorreductase with no confident functional assignment and autoclaved for 30 min at 105 C. Except otherwise (DESAMIL20_1358–1361), but likely involved in sulfur indicated, cultures were incubated statically at 50 C and metabolism, possibly playing a role as sulfur reductase in the dark. (Laska et al., 2003) or tetrathionate reductase. The multi- Chemically synthesized elemental sulfur was the only ple enzymes open possibilities for both polysulfide and form of elemental sulfur utilized for all experiments in this fi elemental sulfur serving as terminal electron acceptors study. Sample identi cation stands for electron donor, − − (Florentino et al., 2017). The reduction of thiosulfate is electron acceptor and pH of cultivation: e donor_e likely catalyzed by another Mo-oxidoreductase serving as acceptor_pH. When there was no electron donor added, thiosulfate reductase (PhsAB; DESAMIL20_9–10) and the S_ stands for disproportionation. sulfite reductase (DsrAB; DESAMIL20_1434–1435) encoded in the genome of D. amilsii. A number of rhodanese-like Dialysis membranes experiment sulfurtransferase genes are also encoded in the genome of D. amilsii without a clear function. Sulfur- and heavy metals-free Spectra/Por (Spectrum, Performing important conversions of the reductive sul- Rancho Dominguez, CA)