The ISME Journal (2020) 14:2261–2274 https://doi.org/10.1038/s41396-020-0684-5 ARTICLE A novel bacterial thiosulfate oxidation pathway provides a new clue about the formation of zero-valent sulfur in deep sea 1,2,3,4 1,2,4 3,4,5 1,2,3,4 4,5 1,2,4 Jing Zhang ● Rui Liu ● Shichuan Xi ● Ruining Cai ● Xin Zhang ● Chaomin Sun Received: 18 December 2019 / Revised: 6 May 2020 / Accepted: 12 May 2020 / Published online: 26 May 2020 © The Author(s) 2020. This article is published with open access Abstract Zero-valent sulfur (ZVS) has been shown to be a major sulfur intermediate in the deep-sea cold seep of the South China Sea based on our previous work, however, the microbial contribution to the formation of ZVS in cold seep has remained unclear. Here, we describe a novel thiosulfate oxidation pathway discovered in the deep-sea cold seep bacterium Erythrobacter flavus 21–3, which provides a new clue about the formation of ZVS. Electronic microscopy, energy-dispersive, and Raman spectra were used to confirm that E. flavus 21–3 effectively converts thiosulfate to ZVS. We next used a combined proteomic and genetic method to identify thiosulfate dehydrogenase (TsdA) and thiosulfohydrolase (SoxB) playing key roles in the conversion of thiosulfate to ZVS. Stoichiometric results of different sulfur intermediates further clarify the function of TsdA − – – – − 1234567890();,: 1234567890();,: in converting thiosulfate to tetrathionate ( O3S S S SO3 ), SoxB in liberating sulfone from tetrathionate to form ZVS and sulfur dioxygenases (SdoA/SdoB) in oxidizing ZVS to sulfite under some conditions. Notably, homologs of TsdA, SoxB, and SdoA/SdoB widely exist across the bacteria including in Erythrobacter species derived from different environments. This strongly indicates that this novel thiosulfate oxidation pathway might be frequently used by microbes and plays an important role in the biogeochemical sulfur cycle in nature. Introduction system [2–4]. The production of ZVS is an important energy-conserving strategy for prokaryotes inhabiting these Zero-valent sulfur (ZVS) is widespread under the sea floor areas. Intimately associated with microbial cells, ZVS is [1], particularly in the cold seep and the hydrothermal regarded as a bio-signature of the activity of sulfur- oxidizing microorganisms [5–7]. The process of ZVS pro- duction begins with the formation of polysulfide, which has Supplementary information The online version of this article (https:// been implicated in a number of important geochemical doi.org/10.1038/s41396-020-0684-5) contains supplementary reactions, such as pyritization [8]. Cyclooctasulfur S8 and 2− material, which is available to authorized users. inorganic polysulfide (Sn ) are important forms of ZVS, which is a critical intermediate in the biogeochemical sulfur * Chaomin Sun [email protected] cycle [9, 10]. As a common substrate oxidized by almost all sulfur 1 CAS Key Laboratory of Experimental Marine Biology & Center lithotrophs, thiosulfate has been suggested to fulfill a key of Deep Sea Research, Institute of Oceanology, Chinese Academy role in the sulfur cycle [11]. At least three thiosulfate oxi- of Sciences, Qingdao, China dation pathways are postulated to exist in sulfur oxidizing 2 Laboratory for Marine Biology and Biotechnology, Qingdao bacteria [12, 13], and each has the possibility to form ZVS National Laboratory for Marine Science and Technology, fi Qingdao, China either extra- or intracellularly. The rst is the Sox pathway, which is governed by the conserved operon SoxXYZABCD 3 College of Earth Science, University of Chinese Academy of Sciences, Beijing, China and operates in photo- and chemo-lithotrophic alphapro- teobacteria [14, 15]. The oxidation of thiosulfate is per- 4 Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China formed via a multienzyme complex consisting of the thiosulfate-induced periplasmic proteins SoxAX, SoxYZ, 5 CAS Key Laboratory of Marine Geology and Environment & Center of Deep Sea Research, Institute of Oceanology, Chinese SoxB, and SoxCD. The complete Sox pathway results in Academy of Sciences, Qingdao, China sulfate as the sole product, whereas the incomplete pathway 2262 J. Zhang et al. (with SoxCD absent) results in either intra- [16–19]or Erythrobacteraceae offer a valuable source for further stu- extracellular [20] accumulation of ZVS. However, the col- dies focused on aerobic anoxygenic phototrophic metabo- orless sulfur bacteria Thiomicrospira thermophile, which lism [34, 35], carbon cycling in the ocean [36], and have contains a complete Sox pathway, exhibits pH-dependent high potential for applications in biotechnology because thiosulfate oxidation to either extracellular ZVS and sulfate they contain important enzymes, such as epoxide hydrolase at low pH (incomplete) or sulfate only at alkaline pH [37, 38] and manganese oxydase [39]. The genus Ery- (complete) [21]. throbacter is abundant in different marine environments The second thiosulfate oxidation pathway is the tetra- [40–42] including the deep-sea cold seep where the relative thionate intermediate (S4I) pathway, which involves pro- abundance of Erythrobacter could reach 4% of the total duction and consumption of polythionate [22]. S4I is widely bacterial community [43], which strongly suggests that found in Beta- and Gammaproteobacteria, particularly the Erythrobacter plays an important ecological role in the obligate chemolithotrophic genera including Acid- deep-sea cold seep. However, to the best of our knowledge, ithiobacillus, Thiobacillus, Halothiobacillus, Thermithioba- the involvement of Erythrobacter in the oxidation of thio- cillus, and Advenella [23, 24]. The production of sulfate and other reductive sulfur compounds has not been tetrathionate is likely coupled with enzymes that subse- elucidated. quently split tetrathionate to thiosulfate, sulfate, or elemental In our previous study, a large amount of ZVS was found sulfur [25, 26]. In addition, the Betaproteobacteria Advenella in the chemosynthetic communities of the Formosa Ridge kashmirensis either oxidizes tetrathionate to sulfate or uses it cold seep in the South China Sea, suggesting that ZVS may as an intermediate during thiosulfate oxidation [27]. be a key intermediate in the sulfur cycle in the deep-sea cold The third thiosulfate oxidation pathway is branched seep [4]. However, the microbial contribution to the for- thiosulfate oxidation, comprehensively characterized in the mation of ZVS was unknown. Here, we report a novel purple sulfur bacterium Allochromatium vinosum, and this thiosulfate oxidation pathway in Erythrobacter (and other pathway involves the interaction of Sox- and S4I-related bacteria), that provides a new clue about the formation of enzyme systems [16, 28]. Under neutral or slightly acidic ZVS in the deep-sea cold seep. The key genes responsible growth conditions thiosulfate dehydrogenase (TsdA) cata- for thiosulfate oxidation and ZVS production in E. flavus lyzes some of the thiosulfate to tetrathionate, while thio- 21–3 were determined by a combination of proteomic, sulfate oxidizes to sulfate with the formation of sulfur genetic, and stoichiometric methods. The broad distribution globules through the Sox system. In conjunction with the of these genes was investigated and discussed. dissimilatory sulfite reductase system, sulfur globules are further oxidized to sulfate [29]. Sulfur oxidizing bacteria are defined as microbes that use Materials and methods reductive sulfur compounds as an energy source for growth. They are highly diverse, and include the green and purple Bacterial strains, plasmids, primers, and growth sulfur bacteria, purple non-sulfur bacteria and colorless conditions sulfur bacteria. All green sulfur bacteria belong to the phylum Chlorobi and most oxidize sulfide and ZVS to E. flavus 21–3 was isolated from sediment samples col- sulfate [20]. Purple sulfur bacteria include the families lected by RV KEXUE from the cold seep in the South China Chromatiaceae and Ectothiorhodospiraceae and produce Sea (119°17′04.956′′ E, 22°06′58.384′′N) at a depth of intra- and extracellular sulfur when grown in the presence of ~1143 m in September 2017 (Supplementary Table S1). sulfide and thiosulfate [29]. Purple non-sulfur bacteria are a The ~0.5 g sediment sample was added to 100 mL of sulfur highly diverse and heterogeneous group of bacteria [30] that producing medium (SPM). The SPM contained: 10 g prefer photoheterotrophic growth under anaerobic condi- Na2S2O·5H2O, 0.2 g K2HPO4, 1 g NaHCO3, 0.4 g NH4Cl, tions, and include many species that can grow photo- 0.6 g MgSO4, 0.001 g FeCl3, and 0.001 g MnCl2 in 1 L of autotrophically with hydrogen or sulfide as the electron sea water, and 15 g/L agar was added to make the corre- donor. The colorless sulfur bacteria are often identified as sponding solid medium. Samples were incubated at 28 °C chemolithoautotrophic, chemolihoheterotrophic, or che- with shaking at a speed of 150 rpm for 2 days. For isolation moorganoheterotrophic [31]. The famous genera in this of single colony, gradient dilutions were made before group include Thiobacillus, Acidithiobacillus, Halothioba- spread plating. Genomic DNA was extracted (water-boil- cillus, and Beggiatoa [10, 32]. ing) from the isolates, and PCR was performed to amplify The genus Erythrobacter belongs to the family Ery- the 16S rRNA gene sequence as described previously [44]. throbacteraceae, members of which have been isolated To determine the phylogenetic position of E. flavus 21–3, from seawater, tidal flats, marine sediment,