YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction

YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction Christiane Dahl, Jingjing Li, Fabienne Göbel, Hsun Hsu, Julian Koch, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4461547/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract Many sulfur-oxidizing prokaryotes oxidize sulfur compounds through a combination of initial extracytoplasmic and downstream cytoplasmic reactions. Facultative sulfur oxidizers adjust transcription to sulfur availability. While sulfur-oxidizing enzymes and transcriptional repressors have been extensively studied, sulfur import into the cytoplasm and how regulators sense external sulfur are poorly understood. Addressing this gap, we show that SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters and are encoded alongside sulfur oxidation and transcriptional regulation genes, fulfill these roles in the Alphaproteobacterium Hyphomicrobium denitrificans . SoxT1A mutants are sulfur oxidation-negative despite high transcription levels of sulfur oxidation genes, showing that SoxT1A delivers sulfur to the cytoplasm for its further oxidation. SoxT1B serves as a signal transduction unit for the transcriptional repressor SoxR, as SoxT1B mutants are sulfur oxidation-negative due to low transcription unless SoxR is also absent. Thus, SoxT1A and SoxT1B play essential but distinct roles in oxidative sulfur metabolism and its regulation. Hyphomicrobium denitrificans sulfur oxidation thiosulfate Sox pathway sulfur transport regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The biogeochemical cycle of sulfur is primarily driven by prokaryotes, which reduce sulfate or sulfite in an aerobic respiratory process to conserve energy 1 . Dissimilatory sulfur oxidizers maintain the cycle by oxidizing reduced sulfur compounds and using them as electron donors for energy conservation through respiration or photosynthesis 2,3 . Sulfide and thiosulfate (S 2 O 3 2 ) are common sulfur substrates in these organisms and in many cases their oxidation is initiated outside of the cytoplasm (if present in the bacterial periplasm). Further oxidative steps take place in the cytoplasm. This requires the import of sulfur into this cellular compartment 2,4–6 . In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of sulfur oxidation allows adaptation of metabolic flux to environmental conditions 7,8 . Sulfur transport across the cytoplasmic membrane is likely involved in the sensing and response to externally available reduced sulfur compounds. While intensive experimental work has been dedicated to elucidating the wide variety of redox reactions involved in prokaryotic sulfur oxidation 2,3 , less effort has been devoted to clarifying the mechanisms of sulfur transport required for its use as an electron source or in the course of signal transduction. Uptake of sulfur compounds for assimilatory purposes, i.e. for the biosynthesis of sulfur-containing cell constituents, has been much better investigated and provides starting points for answering the many open questions. Assimilation of sulfur is required for growth of all living beings and prokaryotes obtain it either from inorganic sulfate or from organosulfur compounds such as sulfonates, sulfate esters, or sulfur-containing amino acids 9–13 . Transporters mediating the import of such precursors include a variety of ABC-type systems with solute-binding proteins as the primary determinants of transporter specificity 11 . The Escherichia coli CysUWA complex is a prime example for this concept. It takes up sulfate and thiosulfate as a sulfur source and acts in combination with periplasmic Sbp and CysP, respectively 9,12 . Recent work has shown that E. coli has an additional transporter, TsuA, which imports thiosulfate as a source of sulfur 14–16 . The protein belongs to the YeeE/YedE family (COG2391; DUF395) and has nine transmembrane helices. The structurally characterized protein from Spirochaeta thermophila contains three conserved cysteine residues that play a role in transport, probably through transient hydrogen bond mediated interaction with thiosulfate ions 14 . In E. coli , the soluble cytoplasmic protein TsuB (YeeD), that is encoded immediately adjacent to tsuA , is essential for thiosulfate uptake via TsuA 15,16 . TsuB is similar to, but cannot replace, TusA 17 , which is a central sulfur hub in bacterial cells 18 . In the archaeon Methanococcus maripaludis a YedE-like protein is involved in transport of selenium, which is chemically similar to sulfur 19,20 . PmpA and PmpB from Serratia sp . ATCC39006 are other members of the YeeE/YedE family that have been predicted to transport sulfur-containing ions, albeit not for assimilatory purposes 21 . Similar proteins facilitate the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane, thereby increasing cellular sulfane sulfur levels in bacterial cells 22 . Genes encoding YeeE/YedE-like proteins also occur together with genes for sulfur-metabolizing enzymes in sulfur-oxidizing prokaryotes. In the Alphaproteobacteria Paracoccus pantotrophus GB17 T (DSM2944 T ), Pseudaminobacter salicylatoxidans KCT001, and Hyphomicrobium denitrificans X T (DSM 1869 T ), large sox gene clusters encoding the thiosulfate-oxidizing periplasmic Sox multienzyme system are accompanied by soxT genes encoding YeeE-like transporters 8,23–25 . In these organisms, soxT is located in a soxSRT arrangement. SoxR, a repressor protein, binds to the promoter-operator region of the sox operon and prevents transcription when sulfur compounds are absent 8,23,24 . This suggests a potential role in signal transduction for the membrane protein. In Paracoccus denitrificans PD1222 (DSM 104981), Cereibacter sphaeroides (formely Rhodobacter sphaeroides 26 ), and Roseovarius sp. 217, the sox genes are flanked by two soxT genes 27 . We denote the one in the soxRST arrangement soxT1 and term the other soxT2 . Two soxT genes are also found in H. denitrificans . This genetically tractable bacterium serves as a model for the elucidation of the cytoplasmic sulfur-oxidizing sHdr-LpbA pathway 5,7,28–31 . In H. denitrificans , thiosulfate oxidation starts in the periplasm, where the SoxXAB proteins work together to oxidatively conjugate thiosulfate to a conserved cysteine of the substrate-binding protein SoxYZ and release a sulfate molecule 2,7,32,33 . The second sulfur atom of the original thiosulfate molecule is by unknown means transferred to the cytoplasm, where it is oxidized to sulfite by the sHdr-LbpA system 5,6 . In H. denitrificans , the typical soxSRT arrangement resides immediately upstream of the genes for a TusA-like sulfur carrier protein and a putative cytochrome P450 7,8 . A second soxT gene is located downstream of the large set of genes that encode the enzymes for cytoplasmic sulfite formation and is transcribed divergently from them. Here, we set out to decipher the function of the two different potential SoxT transporters in H. denitrificans . To this end, we collected information on the distribution and phylogeny of related transporters in sulfur-oxidizing prokaryotes and constructed a set of informative mutant strains lacking the transporter genes, the genes for two different transcriptional regulators, soxR and shdrR , and combinations thereof. Phenotypic characterization of the mutants and comparative analysis of transcription levels for relevant sulfur-oxidizing proteins finally allow functional assignments. Results Occurrence and phylogeny of YeeE/YedE-like proteins. Members of the YeeE/YeeD family are found in organisms across a wide variety of metabolic pathways and prokaryotic phyla, both within the Archaea and the Bacteria 14,21,22,28 . As of March 2022, the Database of Clusters of Orthologous groups included complete genomes from 1187 bacteria and 122 archaea. Among the latter, YeeE-type proteins occur in Saccharolobus and Sulfolobus (Thermoproteota) and some representatives from the Thermoplasmatota. Among the bacteria, some YeeE-containing representatives are found in the phyla Actinobacteriota, Bacteroidota, Cyanobacteriota, Deinococcota, Bacillota, Spirochaetota, Verrucomicrobiota and Thermotogota, while there are many organisms with YeeE among the Pseudomonadota and the Desulfobacterota. Conspicuously, the proteins of the YeeE family vary greatly in length. The structurally characterized S. termophila TsuA and relatives, as well as the SoxT proteins, share lengths of 330 to 350 aa, nine transmembrane helices and three conserved cysteines. In contrast, PmpA and PmpB, as well as their relatives 21,22 , are much shorter, approximately 130 amino acids in length. They share four predicted transmembrane helices and one conserved cysteine residue. We re-evaluated the relationship between the long and short members of the family and found that PmpB and related proteins align perfectly with the N-terminal half of the full-length YeeE family members, while PmpA and relatives match with their carboxy-terminal half (Supplementary Fig. 1). PmpB contains one cysteines that is in the same position as the second conserved cysteine of S. termophila TsuA (Cys 91 ) and a PmpA cysteine matches the third conserved cysteine (Cys 293 ). Cys 91 and Cys 293 are indispensable for proper function of the S. termophila transporter 14 . The central transmembrane helix (H7 in S. thermophila TsuA) is not covered by the PmpAB sequences. We propose that PmpA and PmpB form a heterodimer and that together they perform functions similar to those of the YeeE proteins. The similarity of PmpA to PmpB suggests that they arose from a gene duplication. The two genes may then have fused and acquired an element encoding an additional transmembrane helix, resulting in the full-length YeeE family proteins. As a first step towards a sequence-based grouping of YeeE-like proteins from dissimilatory sulfur-oxidizing bacteria, we created a phylogenetic tree including all YeeE-like transporters encoded in organisms containing the full set of soxXABYZ genes. The functionally characterized TsuA transporters from E. coli and S. thermophila were also included (Fig. 1 ). The tree reveals multiple paralogous groups with the TsuA proteins residing on a well separated branch. The most closely related group consists of SoxT2 proteins such as those encoded in close proximity to the sox genes in C. sphaeroides, P. denitrificans PD1222, and P. salicylatoxydans . SoxT1 proteins form another coherent clade, distant from the SoxT2 group. Both soxT genes from H. denitrificans are of the SoxT1 type and we term them SoxT1A (Hden_0681) and SoxT1B (Hden_0699). Further information was obtained by screening all sulfur-oxidizing prokaryotes containing shdr genes for the presence of soxT1 , soxT2, sox genes, genes for the sHdr-LbpA sulfur-oxidizing system and genes for the transcriptional repressors sHdrR 7 and SoxR 8 by HMSS2 37 . Clusters of genes encoding the sHdr-LbpA pathway for sulfane sulfur oxidation in the cytoplasm fall into two distinct categories. Type I and type II sHdr systems share the Fe/S flavoprotein sHdrA, the electron carrier protein sHdrC1 and the proposed catalytic subunit sHdrB1. The type I sHdrC2 and sHdrB2 polypeptides are encoded by a fused gene, shdrB3 in the type II-containing organisms 5,6 . As evident from Fig. 2 , SoxT transporters are only rarely present in genomes with the type I shdr genes and completely absent in genomes with type II sHdr, even though some of these organisms harbor the capacity for Sox-driven thiosulfate oxidation (see also Supplementary Table 1). In all genomes encoding the regulator SoxR, either SoxT1 or SoxT2 is present. The same is not true for the related repressor sHdrR. It does not always co-occur with SoxT1 or SoxT2. This is in line with a possible function for SoxT1 and/or SoxT2 in SoxR-dependent gene regulation, but contradicts a general role for the transporters in sulfur compound import. Nevertheless, sulfur import may be facilitated by either one of the transporters in a subset of sulfur oxidizers. Regulation of yeeE -like genes in Hyphomicrobium denitrificans . RT-qPCR provided initial evidence that SoxT1A and SoxT1B from H. denitrificans may be intricate components of the sulfur oxidation pathway and/or involved in its transcriptional regulation. The transcript abundance for soxT1A increased more than tenfold upon addition of thiosulfate in the H. denitrificans Δ tsdA reference strain, while soxT1B expression remained essentially unaffected and thus similar to the expression of the genes for the transcriptional repressors soxR and shdrR 8 . Here, we extend these analyses with genome-wide mRNA-Seq data for the reference strain, comparing transcription in the absence and presence of 2 mM thiosulfate. Of the 3529 predicted genes, 3379 mRNAs (95.7%) were identified. The availability of thiosulfate affected the abundance of a total of 136 (4.1%) of the detected mRNAs (Supplementary Fig. 2, Supplementary Tables 2 and 3). In the presence of thiosulfate, mRNA transcripts of 47 genes showed lower mRNA abdundance than in its presence (Supplementary Table 2), among them several genes for enzymes of fatty acid biosynthesis (acyl carrier protein (ACP), β-hydroxyacyl-ACP dehydratase, β-ketoacyl-ACP synthase) and assimilatory sulfate reduction (assimilatory sulfite reductase, sulfate adenylyltransferase). mRNA transcripts of 89 genes (18 genes for hypothetical proteins) were more abundant in the presence of thiosulfate (Supplementary Table 3). The most affected gene (Hden_0834, YeiH) with a log 2 -fold change of + 9.04 encodes a putative efflux pump belonging to the PSE (Putative Sulfate Exporter) family (entry 2.A.98, Transporter Classification Database). The classification as a putative sulfate exporter is based on a study in Paracoccus pantotrophus , where 3-sulfolactate is converted to pyruvate and sulfite during dissimilation of cysteate. It has been suggested that sulfite is oxidized to sulfate in the cytoplasm and then exported 39 . However, sulfite dehydrogenases of Paracococcus species are periplasmic enzymes and it is more likely that the transporter extrudes sulfite from the cytoplasm into the periplasm where it is then detoxified by oxidation to sulfate. A csimilar role may be played by YeiH in H. denitrificans . Strong increases, up to 20-fold, were also observed for the transcripts from the shdr-lbpA2-sox locus. Those for soxT1A were among the top three (Fig. 3 a). In full agreement with RT-qPCR analysis (Fig. 3 b), the transcription of only three genes in the genomic sulfur oxidation region shown in Fig. 3 a proved unaffected in the mRNA-Seq experiment, and these were the genes for the two transcriptional repressors, sHdrR and SoxR, and soxT1B . We state with confidence that soxT1A expression increases substantially during thiosulfate oxidation, while soxT1B expression, along with that of soxR and also shdrR , does not change significantly. These findings are corroborated by RT-qPCR analysis of H. denitrificans strains Δ tsdA Δ soxR 8 and Δ tsdA Δ shdrR 7 , which lack the individual repressor genes. In the repressor-negative strains, soxT1A expression is high even in the absence of thiosulfate, while soxT1B transcript abundance is hardly affected (Fig. 3 b). Our mRNA-Seq analyses also yielded insight into the transcription of further yeeE -like genes in H. denitrificans , i.e. pmpA and pmpB . In contrast to soxT1A , the expression of pmpA and pmpB is not affected by the availability of thiosulfate (Supplementary Fig. 3). These genes attracted our attention because they are located in close proximity to genes encoding proteins that may be related to sulfur metabolism, such as a Sox(YZ) fusion and SoxH 40 . However, close inspection revealed that the gene ensemble rather encodes a PQQ-dependent enzyme for alcohol catabolism, its electron acceptor and an associated transport system (Supplementary Fig. 3). Since there is no evidence that PmpAB are involved in transport processes relevant to oxidative sulfur metabolism in H. denitrificans , they were not analyzed further. Role of SoxT1B in H. denitrificans : gene inactivation, complementation and cysteine exchanges . To clarify the role of SoxT1B, an H. denitrificans strain carrying an in frame deletion of the gene was constructed and phenotypically characterized. In addition, a complemented strain was investigated. Both strains grew equally well on methanol in the absence of thiosulfate (Supplementary Fig. 4a). When the H. denitrificans Δ tsdA reference strain is grown with thiosulfate as an additional electron source, it excretes toxic sulfite 7 , which causes growth retardation (Fig. 4 b). Growth retardation was not observed for H. denitrificans strain Δ tsdA Δ soxT1B and returned upon complementation in cis of the Δ soxT1B deletion strain with an intact copy of the soxT1B gene (Fig. 4 b). Accordingly, the complemented strain H.denitrificans Δ tsdA soxT1Bcomp oxidized thiosulfate with the same rate as the reference strain, while the deletion mutant proved negative with regard to thiosulfate oxidation (Fig. 4 a). As a proof of principle, the three cysteine residues conserved in the YeeE and SoxT proteins (Supplementary Fig. 1) were individually replaced by serine through site directed mutagenesis of the chromosomal H. denitrificans soxT1B gene. All three strains encoding variants of SoxT1B with cysteine to serine substitutions showed no growth retardation in the presence of thiosulfate and were unable to oxidize the sulfur compound, confirming the essentiality of these residues (Fig. 4 ). Role of SoxT1B in H. denitrificans : Interaction with transcriptional regulators. In principle, the thiosulfate oxidation-negative phenotype of the H. denitrificans Δ tsdA Δ soxT1B strain can be explained by two fundamentally different functions of the membrane protein: ( 1 ) Either it is essential for import of oxidizable sulfur into the cytoplasm or ( 2 ) it is essential for signal transduction, informing one or both transcriptional repressors about the presence of external thiosulfate. In the latter case, simultaneous removal of the genes for the signal transducing membrane protein and the transcriptional repressor should allow thiosulfate oxidation, because transcription of the relevant genes would no longer be blocked. A signal-transducing unit would be dispensable in this case. On the other hand, if SoxT1B were responsible for import of oxidizable sulfur, it should be essential for thiosulfate oxidation even when the genes for other components of the sulfur-oxidizing machinery are constitutively expressed. To differentiate between these possibilities, the transcription of indicator genes was compared by RT-qPCR in the absence versus the presence of thiosulfate. We chose the genes soxXA and shdrA because they encode central components of thiosulfate oxidation in the periplasm and sulfane sulfur oxidation in the cytoplasm, respectively. The transcription of soxT1A and soxT1B was also followed. While thiosulfate increases transcript abundance for soxT1A , shdrA and soxXA in the H. denitrificans reference strain 8 , this is not the case for the strain lacking the soxT1B gene (Fig. 5 ). The thiosulfate oxidation-negative phenotype of this strain is therefore explained by a lack of enzymes required for the degradation of the sulfur substrate. When the gene for the SoxR regulator was deleted together with Δ soxT1B from H. denitrificans Δ tsdA , this resulted in a thiosulfate oxidation-positive phenotype. Transcription was high for soxT1A, shdrA and soxXA irrespective of the presence of thiosulfate (Fig. 5 ). The constitutive expression of these genes is caused by the lack of the transcriptional repressor. In the next step, a strain was constructed that lacks genes soxT1B and shdrR . This strain behaves differently from H. denitrificans Δ tsdA Δ soxT1B Δ soxR . It cannot oxidize thiosulfate and the substrate does not induce substantial increase of transcript abundance of the tested sulfur oxidation genes (Fig. 5 ). SoxR is present in this strain and appears to be the major regulator that prevents transcription even in the presence of thiosulfate when the signal-transducing SoxT1B is not available. In conclusion the described experiments show that SoxT1B is dispensable for thiosulfate oxidation and that the import of sulfur for further oxidation is not its primary function. Instead, all results are consistent with a signal transduction function. Role of SoxT1A in H. denitrificans . Like SoxT1B, the related membrane protein SoxT1A could in principle act either as an importer of sulfur for further oxidation in the cytoplasm or as a means of transmitting the information that oxidizable sulfur is available externally. To decide between the two possibilities, the strain H. denitrificans Δ tsdA Δ soxT1A was constructed, phenotypically characterized and studied concerning shdr and sox gene transcription (Fig. 6 ). The strain proved to be thiosulfate oxidation negative, although transcript abundance for shdrA and soxXA increased significantly in the presence of thiosulfate. Further insights were obtained when H. denitrificans strains Δ tsdA Δ soxT1A Δ soxR and Δ tsdA Δ soxT1A Δ shdrR were studied. Both strains show very high transcript abundance for soxXA and shdrA in the absence as well as in the presence of thiosulfate, but are unable to oxidize thiosulfate, suggesting at an essential function of SoxT1A in the overall sulfur oxidation pathway. Discussion Here, we provide information on the distribution and phylogeny of YeeE-like transporters in sulfur-oxidizing prokaryotes and even more importantly, we assign fundamentally different functions to two of these proteins, SoxT1A and SoxT1B, that co-occur in the same Alphaproteobacterium, H. denitrificans . The completely different regulation of the respective genes upon exposure of the organism to thiosulfate is the first milestone for functional assignment. Expression of soxT1A is highly increased, while soxT1B expression is hardly affected at all by the presence of the reduced sulfur compound. All of our observations are consistent with a central role of SoxT1A in sulfur oxidation. The amount of SoxT1A molecules in the cells is increased to ensure efficient import of sulfur into the cytoplasm where it is further processed by the sHdr-LbpA system (Fig. 7 ). To the best of our knowledge, H. denitrificans SoxT1A is the only experimentally demonstrated sulfur importer in dissimilatory sulfur-oxidizing prokaryotes. However, it does not provide a general solution because it not even occurs in all sulfur oxidizers using the cytoplasmic sHdr pathway. SoxT transporters are completely absent genomes with type II sHdr, even though some of these organisms harbor the capacity for Sox-driven thiosulfate oxidation (Fig. 2 ). SoxT1B functions as a signal transducing module. The same function can be assumed for the SoxT proteins in Alphaproteobacteria with complete Sox systems. In these organisms, thiosulfate is completely oxidized to sulfate in the periplasm and accordingly they lack cytoplasmic sulfur-oxidizing enzymes. As a consequence, there is no need for mass import of sulfur as carried out by SoxT1A. In full agreement with these conclusions, a function of SoxT from Pseudaminobacter salicylatoxidans in the transport of an inducer to the cytosol to activate the transcriptional regulator SoxR has been suggested 42 . The genetic neighborhood of the soxT1A and soxT1B genes provides a basis for a model of how sulfur might be presented to the transporters, transported through them, and delivered to their final targets (Fig. 7 ). In immediate vicinity to and in the same direction of transcription with soxT1A , a gene (Hden_0679) is located that encodes a periplasmic DsbA-like thioredoxin with two thioredoxin-like cysteine motifs (Cys-X 2 -Cys), one of which resides at the very carboxy-terminal end of the protein. Thioredoxins serve as general protein disulfide oxidoreductases that interact with a broad range of proteins by a redox mechanism based on reversible oxidation of two cysteine thiol groups to a disulfide, accompanied by the transfer of two electrons and two protons (IPR013766). We consider the possibility that the H. denitrificans DsbA is involved in release of sulfane sulfur from the persulfidated periplasmic sulfur carrier SoxYZ and that the sulfur is then transferred into the cytoplasm through SoxT1A. In the cytoplasm, the sulfur is further handled by cytoplasmic Rhd442 (Hden_680), a protein that we recently characterized as a rhodanese-like sulfur transferase 6 . From there, the sulfur is delivered to the sulfur transferase DsrE3C and finally oxidized to sulfite by the sHdr-LbpA system, possibly involving TusA 43 . Hden_0678 encodes short 56 aa membrane protein, lacking cysteine residues and consisting of one central transmembrane helix (aa 12 to 27) with the N-terminus predicted to reside in the cytoplasm. Functional assignment is currently not possible. The genes in the vicinity of soxT1B appear to encode a second module dedicated to the transport of sulfur, albeit for a different purpose. As suggested earlier, it is conceivable that sulfur bound to the sulfur carrier protein SoxYZ is in this case presented to the transporter by the periplasmic thiol–disulfide oxidoreductase SoxS 44 . In fact, SoxS from P. denitrificans specifically binds SoxY 44 . Once in the cytoplasm, the sulfur transferase TusA 6,18 is a likely acceptor protein for the sulfur. This idea is corroborated by recent findings for the E. coli thiosulfate transporter TsuA 14,16 . TsuA belongs to same family as the SoxT transporters and the TusA-like TsuB protein was shown to be essential for TsuA mediated thiosulfate uptake in vivo. TsuB can cleave thiosulfate resulting in persulfidation of its conserved cysteine and the release of sulfite. In H. denitrificans , sulfur atoms could be passed on from TusA to either one or both or the transcriptional repressors encoded in the shr-lbpA-sox genomic region. For SoxR, we showed that it forms an intramolecular sulfur bridge between two conserved cysteines 8 . The formation of this bridge is the trigger to detach from its target DNA and thus to enable transcription. We assume that sHdrR, which closely resembles SoxR 8 , functions accordingly. Whether SoxR and/or sHdrR are indeed loaded with sulfur in a reaction mediated by TusA or rather directly by the sulfur species transported through SoxT1B, cannot be answered on the current data basis. The exact chemical nature of the sulfur species transported by SoxT1A and SoxT1B requires further investigation. The substrate for TsuA (YeeE) is thiosulfate. At present, we cannot rule out the possibility that in H. denitrificans a small fraction of the thiosulfate available for oxidation is itself used as a signal molecule, channeled through SoxT1B and then cleaved by TusA, as proposed for TsuA. However, thiosulfate is certainly not the substrate for SoxT1A. The periplasmic proteins of the truncated Sox system in H. denitrificans effectively oxidize this substrate and release sulfate and SoxYZ-bound sulfane sulfur from it, which then has to be further processed in the cytoplasm. In fact, a transport by passing sulfur from the SoxY cysteine along the three cysteines lining the central channel of the SoxT1 proteins is conceivable. On the other hand, Ikei and coworkers suggest that interaction of thiosulfate with the cysteine residues occurs via S─H─S hydrogen bonds 16 . The three cysteine residues in TsuA (YeeE) are linearly located at intervals of ~ 7 Å, while disulfide bonds are usually about 2.05 Å in length, and 3.0 Å is taken as the cutoff for disulfides in the PDB database. It is therefore questionable whether sulfur atoms can be directly transferred from one cysteine sulfur to the next. Free HS − ions or short polysulfides ( − S-S n -S − ), that are possibly formed by the action of the periplasmic protein disulfide oxidoreductases DsbA and SoxS, are alternatives and conceivable substrates for cytoplasmic sulfur transferases such as Rhd442 or TusA. Even a direct reaction of polysulfides with the transcriptional repressors, as occurs in vitro 8 , is conceivable. Methods Bacterial strains, plasmids, primers, and growth conditions. Supplementary Table 4 lists the bacterial strains, and plasmids that were used for this study. Escherichia coli strains were grown on complex lysogeny broth (LB) medium 45 . E. coli 10β was used for molecular cloning. H. denitrificans strains were cultured in minimal medium kept at pH 7.2 with 100 mM 3-( N -Morpholino)propanesulfonic acid (MOPS) buffer as previously described 28 . Media contained 24.4 mM methanol. Antibiotics for E. coli and H. denitrificans were used at the following concentrations (in µg ml − 1 ): ampicillin, 100; kanamycin, 50; streptomycin, 200; chloramphenicol, 25. Recombinant DNA techniques. Standard techniques for DNA manipulation and cloning were used unless otherwise indicated 46 . Restriction enzymes, T4 ligase and Q5 polymerase were obtained from New England Biolabs (Ipswich, UK) and used according to the manusfacturer’s instructions. Oligonucleotides were obtained from Eurofins Genomics Germany GmbH (Ebersberg, Germany). Plasmid DNA from E. coli was purified using the GenJET Plasmid Miniprep kit (Thermo Scientific, Waltham, USA). Chromosomal DNA from H. denitrificans strains was prepared using the Simplex Easy DNA Extract Kit (GEN-IAL GmbH, Troisdorf, Germany). DNA fragments were extracted from agarose gels using the GeneJET Gel Extraction Kit (Thermo Scientific, Waltham, USA). Construction of H. denitrificans mutant strains. Plasmids for reverse genetics in H. denitrificans were constructed using the suicide plasmid pk18 mobsacB 47 and the tetracycline cassette from pHP45Ω-Tc 48 on the basis of previously published procedures 28,29 . For markerless in frame deletion of the individual H. denitrificans soxT1A and soxT1B genes by splicing overlap extension (SOE) 49 , PCR fragments were constructed using the primers listed in Supplementary Table 4. The soxT1A or soxT1B fragments were inserted into pk18 mobsacB using XbaI and SaII or XbaI and PstI restriction sites, respectively. The SmaI-excised tetracycline cassette from pHP45Ω-Tc 48 was inserted into the SmaI site, resulting in plasmids pK18 mobsacB- Δ soxT1A- Tc and pK18 mobsacB- Δ soxT1B- Tc. Another plasmid was constructed for concomitant deletion of soxR and soxT1B by SOE PCR with primers P1 fwd up hden_0700, P5 fwd down hden_soxR/soxT1B, P6 rev down hden_ soxR/soxT1B and P7 rev up hden_ soxR/soxT1B (Supplementary Table 4). The PCR fragment was cloned into the XbaI and PstI sites of pk18 mobsacB -Tc 7 . For chromosomal complementation of the H. denitrificans Δ tsdA Δ soxT1B strain, the s oxT1B gene was amplified together with upstream and downstream regions using primers SoxT1B_Del_Up_Fw and SoxT1B_Del_Down_Rev and cloned into the XbaI/PstI sites of pk18 mobsacB -Tc. For chromosomal integration of the genes encoding SoxT1B Cys 24 Ser, SoxT1B Cys 98 Ser and SoxT1B Cys 304 Ser, the modified genes and upstream and downstream sequences were amplified by SOE PCR using the appropriate primers listed in Supplementary Table 4. All final constructs were electroporated into the desired H. denitrificans strains and transformants were selected using previously published procedures 28,29 . H. denitrificans Δ tsdA served as acceptor for plasmids pK18 mobsacB- Δ soxT1B- Tc, pK18 mobsacB- Δ soxT1A- Tc and pk18 mobsacB _Tc_Δ soxR/soxT1B . H. denitrificans Δ tsdA Δ shdrR and H. denitrificans Δ tsdA Δ soxR served as strain backgrounds for deletion of soxT1A . The soxT1B deletion was also established in the H. denitrificans Δ tsdA Δ shdrR strain. The plasmids for complementation and cysteine exchanges of SoxT1B were transferred into H. denitrificans Δ tsdA Δ soxT1B in all cases, single crossover recombinants were Cm r and Tc r . Double crossover recombinants were Tc s and survived in the presence of sucrose due to loss of both, the vector-encoded levansucrase (SacB) and the tetracyclin resistance gene. The genotype of the H. denitrificans strains generated in this study were confirmed by PCR. Characterization of phenotypes, quantification of sulfur compounds and biomass content. Growth experiments with H. denitrificans were run in medium with 24.4 mM methanol in Erlenmeyer flasks or in 96-well microtiter plates as described earlier 7 . 2 mM thiosulfate were added when needed. Biomass content, thiosulfate and sulfite concentrations were determined by previously described methods 7,50 . All growth experiments were repeated three to five times. Representative experiments with two biological replicates for each strain are shown. All quantifications are based on at least three technical replicates. Expression studies based on RT-qPCR . Total RNA of the relevant H. denitrificans strains was isolated from cells harvested in mid-log phase according to an established procedure 8 . RNA samples of 100 ng were used for RT-qPCR analysis which was performed with the primers listed in Supplementary Table 4 following the method described in Li et al 2023 8 . Genome-wide transcriptomic analysis of H. denitrificans Δ tsdA in the absence and presence of thiosulfate. For transcriptome sequencing (RNA-Seq), H. denitrificans Δ tsdA was cultured in 50 ml minimal medium containing either 24.4 mM methanol or 24.4 mM methanol plus 2 mM thiosulfate in 200 ml Erlenmeyer flasks at 30°C with shaking at 200 rpm to early log phase. Cells from 20 ml culture were harvested and flash frozen in liquid N 2 and stored at -70°C. From the frozen pellets, the RNA was purified with the FastGene RNA Premium Kit (NIPPON Genetics EUROPE, Düren, Germany) according to the manufacturer’s instructions. A modification was introduced regarding the cell lysis step. After addition of the lysis buffer that contained 1% (v/v) 2-mercaptoethanol, cells were disrupted by bead beating (Bead Ruptor 12 Bead Mill Homogenizer, Omni International, Kennesaw, GA, USA) for three cycles of 30 s at maximum speed and incubation on ice for 1 min. RNA quality was checked on 1% agarose gels and its concentration was measured using NanoPhotometer NP80 (IMPLEN, Munich, Germany). The RNA was shipped on dry ice to Eurofins Genomics GmbH (Ebersberg, Germany). The subsequent analysis pipeline included rRNA depletion, library preparation (mRNA fragmentation, strand specific cDNA synthesis), Illumina paired end sequencing (2 x 150 bp, minimum 10 MB reads)), and bioinformatic analysis (mapping against the reference genome, identification and quantification of transcripts, pairwise comparison of expression levels and determination of significant fold differences) and was conducted by the company. Generation of datasets for phylogenetic analyses. Archaeal and bacterial genomes were downloaded from Genome Taxonomy Database (GTDB, release R207). In GTDB, all genomes are sorted according to validly published taxonomies, they are pre-validated and have high quality (completeness minus 5*contamination must be higher than 50%). One representative of each of the current 65,703 species clusters was analyzed. Open reading frames were determined using Prodigal 51 and subsequently annotated for sulfur related proteins via HMSS2 37 . Annotation was extended by HMMs from TIGRFAMs 52 and Pfam 53 databases representing the 16 syntenic ribosomal proteins RpL2, 3, 4, 5, 6, 14, 15, 16, 18, 22, and 24, and RpS3, 8, 10, 17, and 19. A type I sHdr system was considered to be present if the core genes shdrC1B1AHC2B2 were present in a syntenic gene cluster. For a type II sHdr system gene cluster shdrC1B1AHB3 and etfAB had to be present in a single syntenic gene cluster 29,54 . Phylogenetic tree inference. For species tree inference, results for each ribosomal protein were individually aligned, trimmed and subsequently concatenated before they were used for phylogenetic tree construction. Proteins were aligned using MAFFT 55 and trimmed with BMGE 56 (entropy threshold = 0.95, minimum length = 1, matrix = BLOSUM30). Alignments were then used for maximum likelihood phylogeny inference using IQ-TREE v1.6.12 57 implemented on the “bonna” high performance clusters of the University of Bonn. The best-fitting model of sequence evolution was selected using ModelFinder 58 . Branch support was then calculated by SH-aLRT (2000 replicates), 59 aBayes (2000 replicates) 60 and ultrafast bootstrap (2000 replicates) 34 . Finally, trees were displayed using iTol 61 . Statistics and reproducibility. Experimental data are expressed as the mean ± standard deviation of the mean (SEM) of the number of tests stated for each experiment. All analysis was reproduced in at least three independent experiments. The significant difference between the two groups was analyzed using an independent student’s t-test; the p-value < 0.05 indicated statistical significance. Reporting summary. Further information on research design will be available in the Nature Portfolio Reporting Summary linked to this article. Data availability The authors declare that the data supporting the findings of this study are available within the article (and its supplementary information files). Declarations Acknowledgements This work was funded by the Deutsche Forschungsgemeinschaft (Grants Da351/8-2, Da 351/13-1 and Da 351/14-1). Jingjing Li was financed by a Scholarship of the Chinese Scholarship Council and Tomohisa Sebastian Tanabe received a scholarship from the Studienstiftung des Deutschen Volkes. We thank Stefania de Benedetti for help with RNA isolation. Author contributions All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at Correspondence and requests for material should be addressed to Christiane Dahl. References Rabus, R. et al. 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Supplementary Files SoxTsupplementarydata.pdf Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4461547","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":310729672,"identity":"ea89c8bb-6679-4654-8717-2695ccc7dd67","order_by":0,"name":"Christiane Dahl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBAC/hn8B5gZCmwYGJgZ2CBCBwhokbjBk8DMYJBGghaDCB4DoJbDIDaxWqR7DD8XGJxP3M7OwPbg4447DHzHGwhokTljLD3D4HbizmYGdsOZZ54xSJ4hYI2BRI6BNA9Qy4bD/N+kedsOMxjcSCCoxfg3j8E5oBYGNoiW+w8IaInIMQPacgBJyw38OoCBnJNmzWOQbAzUAvRL22EeyTMEHMY/I//wbZ4KO9kN5w8AQ6ztsBzf8QMErEEHPCSqHwWjYBSMglGADQAAkdVDJMnWCkkAAAAASUVORK5CYII=","orcid":"","institution":"University of Bonn","correspondingAuthor":true,"prefix":"","firstName":"Christiane","middleName":"","lastName":"Dahl","suffix":""},{"id":310729673,"identity":"353c5d20-cbac-4e06-b121-578fbc75c174","order_by":1,"name":"Jingjing Li","email":"","orcid":"https://orcid.org/0000-0003-1799-4374","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Li","suffix":""},{"id":310729674,"identity":"6b5451cd-0d12-42c3-94b6-748884fd1f1c","order_by":2,"name":"Fabienne Göbel","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Fabienne","middleName":"","lastName":"Göbel","suffix":""},{"id":310729675,"identity":"8fa2b886-4516-4859-bd2a-1f36f52e645e","order_by":3,"name":"Hsun Hsu","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Hsun","middleName":"","lastName":"Hsu","suffix":""},{"id":310729676,"identity":"a6bad8ee-c51a-4852-bfe3-b04b4411209d","order_by":4,"name":"Julian Koch","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Koch","suffix":""},{"id":310729677,"identity":"d8b19a00-d88b-4785-bf65-2bbbf4be01be","order_by":5,"name":"Natalie Hager","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"","lastName":"Hager","suffix":""},{"id":310729678,"identity":"a1b883e1-b3a0-43a0-affd-c637b8255319","order_by":6,"name":"Wanda Flegler","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Wanda","middleName":"","lastName":"Flegler","suffix":""},{"id":310729679,"identity":"9a7797d9-98fc-4cc3-8f96-c1cfb316d134","order_by":7,"name":"Tomohisa Tanabe","email":"","orcid":"https://orcid.org/0000-0003-2154-7980","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Tomohisa","middleName":"","lastName":"Tanabe","suffix":""}],"badges":[],"createdAt":"2024-05-22 14:21:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4461547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4461547/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-024-07270-7","type":"published","date":"2024-11-21T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58596496,"identity":"3ad9bc0d-0d58-4951-9155-d64f42ef7edb","added_by":"auto","created_at":"2024-06-18 16:51:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUnrooted phylogenetic tree for YeeE-like proteins in Sox-containing sulfur oxidizers. \u003c/strong\u003eGroups of proteins encoded in or immediately adjacent to \u003cem\u003esox\u003c/em\u003e clusters (SoxT1 and SoxT2) are highlighted. SoxT1 includes both studied transporters from \u003cem\u003eH. denitrificans. \u003c/em\u003eThe TsuA group includes the thiosulfate uptake proteins from \u003cem\u003eE. coli \u003c/em\u003eand \u003cem\u003eS. thermophila\u003c/em\u003e\u003csup\u003e14,16\u003c/sup\u003e. The tree was calculated with 1000 bootstrap resamplings using Ultrafast Bootstrap\u003csup\u003e34\u003c/sup\u003e and IQ-Tree\u003csup\u003e35,36\u003c/sup\u003e. Bootstrap values between 50% and 100% are displayed as scaled circles at the branching points. Protein accession numbers and species names are available at \u003ca href=\"https://github.com/WandaFlegler/Masterarbeit/blob/main/Galaxy3-%5BBMGE_Cleaned_sequences_\"\u003ehttps://github.com/WandaFlegler/Masterarbeit/blob/main/Galaxy3-%5BBMGE_Cleaned_sequences_\u003c/a\u003e Fasta%5D.fasta.treefile.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/178a582433b2c2d17e3ffd3a.png"},{"id":58594965,"identity":"6074cffd-a31c-40c5-bb24-6914717b27ce","added_by":"auto","created_at":"2024-06-18 16:35:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of SoxT transporters in sulfur oxidizers with sHdr systems. \u003c/strong\u003eThe distribution of genes for type I and type II sHdr systems, full Sox systems enabling complete oxidation of thiosulfate in the periplasm (SoxAXBYZCD), truncated Sox systems requiring formation of sulfite in the cytoplasm (SoxXABYZ), SoxT and two different transcriptional repressors, SoxR and sHdrR, is shown. In order to be classified as present, at least the proteins SoxA, SoxB SoxY and SoxZ had to be encoded in a syntenic gene cluster. The \u003cem\u003esox\u003c/em\u003e genes of species of the order Ectothiorhodospirales are not syntenic and were therefore assigned manually as described before\u003csup\u003e38\u003c/sup\u003e. A type I sHdr system was marked positive when the core genes \u003cem\u003eshdrC1B1AHC2B2\u003c/em\u003e were present in a syntenic gene cluster. For assignment of a type II sHdr system, \u003cem\u003eshdrC1B1AHB3\u003c/em\u003e and \u003cem\u003eetfAB\u003c/em\u003e had to be present in a single syntenic gene cluster\u003csup\u003e6\u003c/sup\u003e The gene for the regulator sHdrR was only considered positive when located in or immediately next to a \u003cem\u003eshdr\u003c/em\u003e gene cluster. The species tree was calculated as described before\u003csup\u003e6\u003c/sup\u003e. The data underlying the figure is provided in Supplementary Table 1.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/4814ae97c28375af96c20a34.png"},{"id":58595896,"identity":"75154668-253d-4df8-919b-f4e614ce4b0a","added_by":"auto","created_at":"2024-06-18 16:43:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":129287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThiosulfate-dependent regulation of gene expression in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003elip-shdr-lbpA-sox\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e locus in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. denitrificans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Δ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etsdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reference strain.\u003c/strong\u003e \u003cstrong\u003ea. \u003c/strong\u003eThiosulfate-dependent regulation of gene expression as assessed by mRNA-Seq analysis. Fold changes for transcript abundancies are relative to a culture grown on 2 mM thiosulfate (white columns) versus cells grown in the absence of thiosulfate (gray columns). Transcript abundance changes of genes encoding enzymes involved in thiosulfate oxidation in \u003cem\u003eH. denitrificans\u003c/em\u003e. Columns in gray show the reference values for cells grown in the absence of thiosulfate, white columns apply to cells grown with 2 mM thiosulfate. The experiment was conducted in duplicate, each time using mRNA preparations from two different cultures. Adjusted p values were all below 0.05 (Supplementary Table 3). \u003cstrong\u003eb. \u003c/strong\u003eRelative mRNA levels of four indicative genes/combinations of genes located in the \u003cem\u003eshdr\u003c/em\u003e-\u003cem\u003esox\u003c/em\u003e genetic region (depicted in panel \u003cstrong\u003ea\u003c/strong\u003e) from \u003cem\u003eH. denitrificans\u003c/em\u003e for the Δ\u003cem\u003etsdA\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ereference\u003cstrong\u003e \u003c/strong\u003estrain in the presence of thiosulfate as assessed by RT-qPCR. Results for \u003cem\u003eH. denitrificans\u003c/em\u003e strains\u003cem\u003e \u003c/em\u003eΔ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxR \u003c/em\u003eand Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003eshdrR\u003c/em\u003e grown in the absence of thiosulfate are also shown. All changes are compared to \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ein the absence of thiosulfate. Results were adjusted using \u003cem\u003eH. denitrificans rpoB\u003c/em\u003e, which encodes the b-subunit of RNA polymerase, according to\u003csup\u003e41\u003c/sup\u003e. TS oxidation, thiosulfate oxidation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/8d72377060afb6e3af9b0e32.png"},{"id":58595895,"identity":"3efd4a16-cd54-441d-aa30-1850c819268e","added_by":"auto","created_at":"2024-06-18 16:43:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":128573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and thiosulfate consumption for the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. denitrificans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eΔ\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etsdA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reference strain compared with strains lacking \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esoxT1B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e or producing SoxT1B with cysteine to serine exchanges.\u003c/strong\u003e Thiosulfate consumption (\u003cstrong\u003ea\u003c/strong\u003e) and growth (\u003cstrong\u003eb\u003c/strong\u003e) of \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e (black), Δ\u003cem\u003etsdA \u003c/em\u003eΔ\u003cem\u003esoxT1B\u003c/em\u003e (bright red), Δ\u003cem\u003etsdA soxT1Bcomp\u003c/em\u003e (gray), Δ\u003cem\u003etsdA soxT1B C\u003c/em\u003e\u003csup\u003e\u003cem\u003e24\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eS\u003c/em\u003e (orange), Δ\u003cem\u003etsdA soxT1B C\u003c/em\u003e\u003csup\u003e\u003cem\u003e98\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eS\u003c/em\u003e (pink) and Δ\u003cem\u003etsdA soxT1B C\u003c/em\u003e\u003csup\u003e\u003cem\u003e304\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eS\u003c/em\u003e (dark red). All strains were grown in 48-well microtiter plates as described in \u003csup\u003e7\u003c/sup\u003e in medium containing 24.4\u0026nbsp;mM methanol and 2 mM thiosulfate. Precultures contained 2\u0026nbsp;mM thiosulfate.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/2ef1e8047a0d69a9e3d548f6.png"},{"id":58594963,"identity":"66b76839-bfe5-4d97-9cf4-623ce8ba80cc","added_by":"auto","created_at":"2024-06-18 16:35:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscription of sulfur oxidation genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH.denitrificans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains lacking \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esoxT1B.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003eRT-qPCR analysis is shown for four indicative genes in three different \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e strains in the absence (\u003cstrong\u003ea\u003c/strong\u003e) and in the presence of 2 mM thiosulfate (\u003cstrong\u003eb\u003c/strong\u003e). The ability of the strains to oxidize thiosulfate is indicated. The growth experiments are shown in full in Supplementary Fig. 5. All strains grew equally well on methanol in the absence of thiosulfate (Supplementary Fig. 4a). TS, thiosulfate.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/ae64027c3e4e5289875dc38a.png"},{"id":58594969,"identity":"6ac57e16-5d77-4b52-91fa-6f95b7b3a7ba","added_by":"auto","created_at":"2024-06-18 16:35:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscription of sulfur oxidation genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH.denitrificans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains lacking \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esoxT1A.\u003c/strong\u003e\u003c/em\u003eRT-qPCR analysis for four indicative genes in three different \u003cem\u003eH. denitrificans\u003c/em\u003e strains in the absence (\u003cstrong\u003ea\u003c/strong\u003e) and in the presence of 2 mM thiosulfate (\u003cstrong\u003eb\u003c/strong\u003e). Note that the fold change for \u003cem\u003eshdrA\u003c/em\u003e transcript abundance was 201.7 ± 35.5\u003cem\u003e \u003c/em\u003eand thus far exceeds the y-axis range appropriate for all other cases. The ability of the strains to oxidize thiosulfate is indicated. The growth experiments are shown in full in Supplementary Fig. 6. All strains grew equally well on methanol in the absence of thiosulfate (Supplementary Fig. 4b). TS, thiosulfate.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/b9c2e9438a100751207de07f.png"},{"id":58594968,"identity":"712f8c8c-aa79-438d-b217-edf67897f763","added_by":"auto","created_at":"2024-06-18 16:35:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of thiosulfate oxidation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH.denitrificans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e integrating the sulfur transport and signal transduction functions of SoxT1A and SoxT1B, respectively\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e TauE, putative sulfite exporter encoded by Hden_0720\u003csup\u003e28\u003c/sup\u003e. Transcripts are 2.6-fold more abundant when thiosulfate is present (Supplementary Table 3). YeiH (Hden_0834) is another candidate for sulfite export, with increase transcript abundance in the presence of oxidizable sulfur. The \u003cem\u003elipS1,lipT, lipS2, slpl(AB)\u003c/em\u003e and \u003cem\u003elipX\u003c/em\u003e genes encode enzymes that assemble the cofactor on the lipoate-binding protein LbpA2\u003csup\u003e30\u003c/sup\u003e. RNAP, RNA polymerase. Sulfur atoms printed in red stem from the sulfane sufur atom of thiosulfate, the oxidation of which is initiated in the periplasm. sHdrH and sHdrI are soluble, cytoplamsic proteins of unknown function. \u003cem\u003ehyp1\u003c/em\u003e, encodes a56-aa transmembrane protein; \u003cem\u003ehyp2\u003c/em\u003e encodes a putative cytochrome P450.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/b2c5d1a013acca7a9345d731.png"},{"id":69610659,"identity":"8575db5f-63c8-44e1-b01c-33b024c299e7","added_by":"auto","created_at":"2024-11-22 08:09:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1717837,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/0ce9b8ef-7d2a-4950-aea3-3683da6be5ca.pdf"},{"id":58683093,"identity":"4faee12f-b6cd-43a1-be12-0cd15a888513","added_by":"auto","created_at":"2024-06-19 18:41:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1920692,"visible":true,"origin":"","legend":"","description":"","filename":"SoxTsupplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4461547/v1/2de8506e5d649df22e768021.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe biogeochemical cycle of sulfur is primarily driven by prokaryotes, which reduce sulfate or sulfite in an aerobic respiratory process to conserve energy\u003csup\u003e1\u003c/sup\u003e. Dissimilatory sulfur oxidizers maintain the cycle by oxidizing reduced sulfur compounds and using them as electron donors for energy conservation through respiration or photosynthesis\u003csup\u003e2,3\u003c/sup\u003e. Sulfide and thiosulfate (S\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e) are common sulfur substrates in these organisms and in many cases their oxidation is initiated outside of the cytoplasm (if present in the bacterial periplasm). Further oxidative steps take place in the cytoplasm. This requires the import of sulfur into this cellular compartment\u003csup\u003e2,4\u0026ndash;6\u003c/sup\u003e. In organisms that use reduced sulfur compounds as alternative or additional electron donors to organic compounds, transcriptional regulation of sulfur oxidation allows adaptation of metabolic flux to environmental conditions\u003csup\u003e7,8\u003c/sup\u003e. Sulfur transport across the cytoplasmic membrane is likely involved in the sensing and response to externally available reduced sulfur compounds. While intensive experimental work has been dedicated to elucidating the wide variety of redox reactions involved in prokaryotic sulfur oxidation\u003csup\u003e2,3\u003c/sup\u003e, less effort has been devoted to clarifying the mechanisms of sulfur transport required for its use as an electron source or in the course of signal transduction. Uptake of sulfur compounds for assimilatory purposes, i.e. for the biosynthesis of sulfur-containing cell constituents, has been much better investigated and provides starting points for answering the many open questions.\u003c/p\u003e \u003cp\u003eAssimilation of sulfur is required for growth of all living beings and prokaryotes obtain it either from inorganic sulfate or from organosulfur compounds such as sulfonates, sulfate esters, or sulfur-containing amino acids\u003csup\u003e9\u0026ndash;13\u003c/sup\u003e. Transporters mediating the import of such precursors include a variety of ABC-type systems with solute-binding proteins as the primary determinants of transporter specificity\u003csup\u003e11\u003c/sup\u003e. The \u003cem\u003eEscherichia coli\u003c/em\u003e CysUWA complex is a prime example for this concept. It takes up sulfate and thiosulfate as a sulfur source and acts in combination with periplasmic Sbp and CysP, respectively \u003csup\u003e9,12\u003c/sup\u003e. Recent work has shown that \u003cem\u003eE. coli\u003c/em\u003e has an additional transporter, TsuA, which imports thiosulfate as a source of sulfur\u003csup\u003e14\u0026ndash;16\u003c/sup\u003e. The protein belongs to the YeeE/YedE family (COG2391; DUF395) and has nine transmembrane helices. The structurally characterized protein from \u003cem\u003eSpirochaeta thermophila\u003c/em\u003e contains three conserved cysteine residues that play a role in transport, probably through transient hydrogen bond mediated interaction with thiosulfate ions\u003csup\u003e14\u003c/sup\u003e. In \u003cem\u003eE. coli\u003c/em\u003e, the soluble cytoplasmic protein TsuB (YeeD), that is encoded immediately adjacent to \u003cem\u003etsuA\u003c/em\u003e, is essential for thiosulfate uptake via TsuA\u003csup\u003e15,16\u003c/sup\u003e. TsuB is similar to, but cannot replace, TusA \u003csup\u003e17\u003c/sup\u003e, which is a central sulfur hub in bacterial cells\u003csup\u003e18\u003c/sup\u003e. In the archaeon \u003cem\u003eMethanococcus maripaludis\u003c/em\u003e a YedE-like protein is involved in transport of selenium, which is chemically similar to sulfur\u003csup\u003e19,20\u003c/sup\u003e. PmpA and PmpB from \u003cem\u003eSerratia sp\u003c/em\u003e. ATCC39006 are other members of the YeeE/YedE family that have been predicted to transport sulfur-containing ions, albeit not for assimilatory purposes\u003csup\u003e21\u003c/sup\u003e. Similar proteins facilitate the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane, thereby increasing cellular sulfane sulfur levels in bacterial cells\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenes encoding YeeE/YedE-like proteins also occur together with genes for sulfur-metabolizing enzymes in sulfur-oxidizing prokaryotes. In the Alphaproteobacteria \u003cem\u003eParacoccus pantotrophus\u003c/em\u003e GB17\u003csup\u003eT\u003c/sup\u003e (DSM2944\u003csup\u003eT\u003c/sup\u003e), \u003cem\u003ePseudaminobacter salicylatoxidans\u003c/em\u003e KCT001, and \u003cem\u003eHyphomicrobium denitrificans\u003c/em\u003e X\u003csup\u003eT\u003c/sup\u003e (DSM 1869\u003csup\u003eT\u003c/sup\u003e), large \u003cem\u003esox\u003c/em\u003e gene clusters encoding the thiosulfate-oxidizing periplasmic Sox multienzyme system are accompanied by \u003cem\u003esoxT\u003c/em\u003e genes encoding YeeE-like transporters\u003csup\u003e8,23\u0026ndash;25\u003c/sup\u003e. In these organisms, \u003cem\u003esoxT\u003c/em\u003e is located in a \u003cem\u003esoxSRT\u003c/em\u003e arrangement. SoxR, a repressor protein, binds to the promoter-operator region of the \u003cem\u003esox\u003c/em\u003e operon and prevents transcription when sulfur compounds are absent\u003csup\u003e8,23,24\u003c/sup\u003e. This suggests a potential role in signal transduction for the membrane protein. In \u003cem\u003eParacoccus denitrificans\u003c/em\u003e PD1222 (DSM 104981), \u003cem\u003eCereibacter sphaeroides\u003c/em\u003e (formely \u003cem\u003eRhodobacter sphaeroides\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e), and \u003cem\u003eRoseovarius\u003c/em\u003e sp. 217, the \u003cem\u003esox\u003c/em\u003e genes are flanked by two \u003cem\u003esoxT\u003c/em\u003e genes\u003csup\u003e27\u003c/sup\u003e. We denote the one in the \u003cem\u003esoxRST\u003c/em\u003e arrangement \u003cem\u003esoxT1\u003c/em\u003e and term the other \u003cem\u003esoxT2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTwo \u003cem\u003esoxT\u003c/em\u003e genes are also found in \u003cem\u003eH. denitrificans\u003c/em\u003e. This genetically tractable bacterium serves as a model for the elucidation of the cytoplasmic sulfur-oxidizing sHdr-LpbA pathway\u003csup\u003e5,7,28\u0026ndash;31\u003c/sup\u003e. In \u003cem\u003eH. denitrificans\u003c/em\u003e, thiosulfate oxidation starts in the periplasm, where the SoxXAB proteins work together to oxidatively conjugate thiosulfate to a conserved cysteine of the substrate-binding protein SoxYZ and release a sulfate molecule\u003csup\u003e2,7,32,33\u003c/sup\u003e. The second sulfur atom of the original thiosulfate molecule is by unknown means transferred to the cytoplasm, where it is oxidized to sulfite by the sHdr-LbpA system\u003csup\u003e5,6\u003c/sup\u003e. In \u003cem\u003eH. denitrificans\u003c/em\u003e, the typical \u003cem\u003esoxSRT\u003c/em\u003e arrangement resides immediately upstream of the genes for a TusA-like sulfur carrier protein and a putative cytochrome P450\u003csup\u003e7,8\u003c/sup\u003e. A second \u003cem\u003esoxT\u003c/em\u003e gene is located downstream of the large set of genes that encode the enzymes for cytoplasmic sulfite formation and is transcribed divergently from them.\u003c/p\u003e \u003cp\u003eHere, we set out to decipher the function of the two different potential SoxT transporters in \u003cem\u003eH. denitrificans\u003c/em\u003e. To this end, we collected information on the distribution and phylogeny of related transporters in sulfur-oxidizing prokaryotes and constructed a set of informative mutant strains lacking the transporter genes, the genes for two different transcriptional regulators, \u003cem\u003esoxR\u003c/em\u003e and \u003cem\u003eshdrR\u003c/em\u003e, and combinations thereof. Phenotypic characterization of the mutants and comparative analysis of transcription levels for relevant sulfur-oxidizing proteins finally allow functional assignments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eOccurrence and phylogeny of YeeE/YedE-like proteins.\u003c/b\u003e Members of the YeeE/YeeD family are found in organisms across a wide variety of metabolic pathways and prokaryotic phyla, both within the Archaea and the Bacteria\u003csup\u003e14,21,22,28\u003c/sup\u003e. As of March 2022, the Database of Clusters of Orthologous groups included complete genomes from 1187 bacteria and 122 archaea. Among the latter, YeeE-type proteins occur in \u003cem\u003eSaccharolobus\u003c/em\u003e and \u003cem\u003eSulfolobus\u003c/em\u003e (Thermoproteota) and some representatives from the Thermoplasmatota. Among the bacteria, some YeeE-containing representatives are found in the phyla Actinobacteriota, Bacteroidota, Cyanobacteriota, Deinococcota, Bacillota, Spirochaetota, Verrucomicrobiota and Thermotogota, while there are many organisms with YeeE among the Pseudomonadota and the Desulfobacterota.\u003c/p\u003e \u003cp\u003eConspicuously, the proteins of the YeeE family vary greatly in length. The structurally characterized \u003cem\u003eS. termophila\u003c/em\u003e TsuA and relatives, as well as the SoxT proteins, share lengths of 330 to 350 aa, nine transmembrane helices and three conserved cysteines. In contrast, PmpA and PmpB, as well as their relatives\u003csup\u003e21,22\u003c/sup\u003e, are much shorter, approximately 130 amino acids in length. They share four predicted transmembrane helices and one conserved cysteine residue. We re-evaluated the relationship between the long and short members of the family and found that PmpB and related proteins align perfectly with the N-terminal half of the full-length YeeE family members, while PmpA and relatives match with their carboxy-terminal half (Supplementary Fig.\u0026nbsp;1). PmpB contains one cysteines that is in the same position as the second conserved cysteine of \u003cem\u003eS. termophila\u003c/em\u003e TsuA (Cys\u003csup\u003e91\u003c/sup\u003e) and a PmpA cysteine matches the third conserved cysteine (Cys\u003csup\u003e293\u003c/sup\u003e). Cys\u003csup\u003e91\u003c/sup\u003e and Cys\u003csup\u003e293\u003c/sup\u003e are indispensable for proper function of the \u003cem\u003eS. termophila\u003c/em\u003e transporter\u003csup\u003e14\u003c/sup\u003e. The central transmembrane helix (H7 in \u003cem\u003eS. thermophila\u003c/em\u003e TsuA) is not covered by the PmpAB sequences. We propose that PmpA and PmpB form a heterodimer and that together they perform functions similar to those of the YeeE proteins. The similarity of PmpA to PmpB suggests that they arose from a gene duplication. The two genes may then have fused and acquired an element encoding an additional transmembrane helix, resulting in the full-length YeeE family proteins.\u003c/p\u003e \u003cp\u003eAs a first step towards a sequence-based grouping of YeeE-like proteins from dissimilatory sulfur-oxidizing bacteria, we created a phylogenetic tree including all YeeE-like transporters encoded in organisms containing the full set of \u003cem\u003esoxXABYZ\u003c/em\u003e genes. The functionally characterized TsuA transporters from \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. thermophila\u003c/em\u003e were also included (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tree reveals multiple paralogous groups with the TsuA proteins residing on a well separated branch. The most closely related group consists of SoxT2 proteins such as those encoded in close proximity to the \u003cem\u003esox\u003c/em\u003e genes in \u003cem\u003eC. sphaeroides, P. denitrificans\u003c/em\u003e PD1222, and \u003cem\u003eP. salicylatoxydans\u003c/em\u003e. SoxT1 proteins form another coherent clade, distant from the SoxT2 group. Both \u003cem\u003esoxT\u003c/em\u003e genes from \u003cem\u003eH. denitrificans\u003c/em\u003e are of the SoxT1 type and we term them SoxT1A (Hden_0681) and SoxT1B (Hden_0699).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther information was obtained by screening all sulfur-oxidizing prokaryotes containing \u003cem\u003eshdr\u003c/em\u003e genes for the presence of \u003cem\u003esoxT1\u003c/em\u003e, \u003cem\u003esoxT2, sox\u003c/em\u003e genes, genes for the sHdr-LbpA sulfur-oxidizing system and genes for the transcriptional repressors sHdrR\u003csup\u003e7\u003c/sup\u003e and SoxR\u003csup\u003e8\u003c/sup\u003e by HMSS2\u003csup\u003e37\u003c/sup\u003e. Clusters of genes encoding the sHdr-LbpA pathway for sulfane sulfur oxidation in the cytoplasm fall into two distinct categories. Type I and type II sHdr systems share the Fe/S flavoprotein sHdrA, the electron carrier protein sHdrC1 and the proposed catalytic subunit sHdrB1. The type I sHdrC2 and sHdrB2 polypeptides are encoded by a fused gene, \u003cem\u003eshdrB3\u003c/em\u003e in the type II-containing organisms\u003csup\u003e5,6\u003c/sup\u003e. As evident from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, SoxT transporters are only rarely present in genomes with the type I \u003cem\u003eshdr\u003c/em\u003e genes and completely absent in genomes with type II sHdr, even though some of these organisms harbor the capacity for Sox-driven thiosulfate oxidation (see also Supplementary Table\u0026nbsp;1). In all genomes encoding the regulator SoxR, either SoxT1 or SoxT2 is present. The same is not true for the related repressor sHdrR. It does not always co-occur with SoxT1 or SoxT2. This is in line with a possible function for SoxT1 and/or SoxT2 in SoxR-dependent gene regulation, but contradicts a general role for the transporters in sulfur compound import. Nevertheless, sulfur import may be facilitated by either one of the transporters in a subset of sulfur oxidizers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulation of\u003c/b\u003e \u003cb\u003eyeeE\u003c/b\u003e\u003cb\u003e-like genes in\u003c/b\u003e \u003cb\u003eHyphomicrobium denitrificans\u003c/b\u003e. RT-qPCR provided initial evidence that SoxT1A and SoxT1B from \u003cem\u003eH. denitrificans\u003c/em\u003e may be intricate components of the sulfur oxidation pathway and/or involved in its transcriptional regulation. The transcript abundance for \u003cem\u003esoxT1A\u003c/em\u003e increased more than tenfold upon addition of thiosulfate in the \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e reference strain, while \u003cem\u003esoxT1B\u003c/em\u003e expression remained essentially unaffected and thus similar to the expression of the genes for the transcriptional repressors \u003cem\u003esoxR\u003c/em\u003e and \u003cem\u003eshdrR\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e. Here, we extend these analyses with genome-wide mRNA-Seq data for the reference strain, comparing transcription in the absence and presence of 2 mM thiosulfate. Of the 3529 predicted genes, 3379 mRNAs (95.7%) were identified. The availability of thiosulfate affected the abundance of a total of 136 (4.1%) of the detected mRNAs (Supplementary Fig.\u0026nbsp;2, Supplementary Tables\u0026nbsp;2 and 3). In the presence of thiosulfate, mRNA transcripts of 47 genes showed lower mRNA abdundance than in its presence (Supplementary Table\u0026nbsp;2), among them several genes for enzymes of fatty acid biosynthesis (acyl carrier protein (ACP), β-hydroxyacyl-ACP dehydratase, β-ketoacyl-ACP synthase) and assimilatory sulfate reduction (assimilatory sulfite reductase, sulfate adenylyltransferase).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003emRNA transcripts of 89 genes (18 genes for hypothetical proteins) were more abundant in the presence of thiosulfate (Supplementary Table\u0026nbsp;3). The most affected gene (Hden_0834, YeiH) with a log\u003csub\u003e2\u003c/sub\u003e-fold change of +\u0026thinsp;9.04 encodes a putative efflux pump belonging to the PSE (Putative Sulfate Exporter) family (entry 2.A.98, Transporter Classification Database). The classification as a putative sulfate exporter is based on a study in \u003cem\u003eParacoccus pantotrophus\u003c/em\u003e, where 3-sulfolactate is converted to pyruvate and sulfite during dissimilation of cysteate. It has been suggested that sulfite is oxidized to sulfate in the cytoplasm and then exported\u003csup\u003e39\u003c/sup\u003e. However, sulfite dehydrogenases of \u003cem\u003eParacococcus\u003c/em\u003e species are periplasmic enzymes and it is more likely that the transporter extrudes sulfite from the cytoplasm into the periplasm where it is then detoxified by oxidation to sulfate. A csimilar role may be played by YeiH in \u003cem\u003eH. denitrificans\u003c/em\u003e. Strong increases, up to 20-fold, were also observed for the transcripts from the \u003cem\u003eshdr-lbpA2-sox\u003c/em\u003e locus. Those for \u003cem\u003esoxT1A\u003c/em\u003e were among the top three (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In full agreement with RT-qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), the transcription of only three genes in the genomic sulfur oxidation region shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea proved unaffected in the mRNA-Seq experiment, and these were the genes for the two transcriptional repressors, sHdrR and SoxR, and \u003cem\u003esoxT1B\u003c/em\u003e. We state with confidence that \u003cem\u003esoxT1A\u003c/em\u003e expression increases substantially during thiosulfate oxidation, while \u003cem\u003esoxT1B\u003c/em\u003e expression, along with that of \u003cem\u003esoxR\u003c/em\u003e and also \u003cem\u003eshdrR\u003c/em\u003e, does not change significantly. These findings are corroborated by RT-qPCR analysis of \u003cem\u003eH. denitrificans\u003c/em\u003e strains Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxR\u003c/em\u003e\u003csup\u003e8\u003c/sup\u003e and Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003eshdrR\u003c/em\u003e\u003csup\u003e7\u003c/sup\u003e, which lack the individual repressor genes. In the repressor-negative strains, \u003cem\u003esoxT1A\u003c/em\u003e expression is high even in the absence of thiosulfate, while \u003cem\u003esoxT1B\u003c/em\u003e transcript abundance is hardly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eOur mRNA-Seq analyses also yielded insight into the transcription of further \u003cem\u003eyeeE\u003c/em\u003e-like genes in \u003cem\u003eH. denitrificans\u003c/em\u003e, i.e. \u003cem\u003epmpA\u003c/em\u003e and \u003cem\u003epmpB\u003c/em\u003e. In contrast to \u003cem\u003esoxT1A\u003c/em\u003e, the expression of \u003cem\u003epmpA\u003c/em\u003e and \u003cem\u003epmpB\u003c/em\u003e is not affected by the availability of thiosulfate (Supplementary Fig.\u0026nbsp;3). These genes attracted our attention because they are located in close proximity to genes encoding proteins that may be related to sulfur metabolism, such as a Sox(YZ) fusion and SoxH\u003csup\u003e40\u003c/sup\u003e. However, close inspection revealed that the gene ensemble rather encodes a PQQ-dependent enzyme for alcohol catabolism, its electron acceptor and an associated transport system (Supplementary Fig.\u0026nbsp;3). Since there is no evidence that PmpAB are involved in transport processes relevant to oxidative sulfur metabolism in \u003cem\u003eH. denitrificans\u003c/em\u003e, they were not analyzed further.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRole of SoxT1B in\u003c/b\u003e \u003cb\u003eH. denitrificans\u003c/b\u003e: \u003cb\u003egene inactivation, complementation and cysteine exchanges\u003c/b\u003e. To clarify the role of SoxT1B, an \u003cem\u003eH. denitrificans\u003c/em\u003e strain carrying an \u003cem\u003ein frame\u003c/em\u003e deletion of the gene was constructed and phenotypically characterized. In addition, a complemented strain was investigated. Both strains grew equally well on methanol in the absence of thiosulfate (Supplementary Fig.\u0026nbsp;4a). When the \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e reference strain is grown with thiosulfate as an additional electron source, it excretes toxic sulfite\u003csup\u003e7\u003c/sup\u003e, which causes growth retardation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Growth retardation was not observed for \u003cem\u003eH. denitrificans\u003c/em\u003e strain Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e and returned upon complementation \u003cem\u003ein cis\u003c/em\u003e of the Δ\u003cem\u003esoxT1B\u003c/em\u003e deletion strain with an intact copy of the \u003cem\u003esoxT1B\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Accordingly, the complemented strain \u003cem\u003eH.denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA soxT1Bcomp\u003c/em\u003e oxidized thiosulfate with the same rate as the reference strain, while the deletion mutant proved negative with regard to thiosulfate oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAs a proof of principle, the three cysteine residues conserved in the YeeE and SoxT proteins (Supplementary Fig.\u0026nbsp;1) were individually replaced by serine through site directed mutagenesis of the chromosomal \u003cem\u003eH. denitrificans soxT1B\u003c/em\u003e gene. All three strains encoding variants of SoxT1B with cysteine to serine substitutions showed no growth retardation in the presence of thiosulfate and were unable to oxidize the sulfur compound, confirming the essentiality of these residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRole of SoxT1B in\u003c/b\u003e \u003cb\u003eH. denitrificans\u003c/b\u003e: \u003cb\u003eInteraction with transcriptional regulators.\u003c/b\u003e In principle, the thiosulfate oxidation-negative phenotype of the \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e strain can be explained by two fundamentally different functions of the membrane protein: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Either it is essential for import of oxidizable sulfur into the cytoplasm or (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) it is essential for signal transduction, informing one or both transcriptional repressors about the presence of external thiosulfate. In the latter case, simultaneous removal of the genes for the signal transducing membrane protein and the transcriptional repressor should allow thiosulfate oxidation, because transcription of the relevant genes would no longer be blocked. A signal-transducing unit would be dispensable in this case. On the other hand, if SoxT1B were responsible for import of oxidizable sulfur, it should be essential for thiosulfate oxidation even when the genes for other components of the sulfur-oxidizing machinery are constitutively expressed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo differentiate between these possibilities, the transcription of indicator genes was compared by RT-qPCR in the absence versus the presence of thiosulfate. We chose the genes \u003cem\u003esoxXA\u003c/em\u003e and \u003cem\u003eshdrA\u003c/em\u003e because they encode central components of thiosulfate oxidation in the periplasm and sulfane sulfur oxidation in the cytoplasm, respectively. The transcription of \u003cem\u003esoxT1A\u003c/em\u003e and \u003cem\u003esoxT1B\u003c/em\u003e was also followed. While thiosulfate increases transcript abundance for \u003cem\u003esoxT1A\u003c/em\u003e, \u003cem\u003eshdrA\u003c/em\u003e and \u003cem\u003esoxXA\u003c/em\u003e in the \u003cem\u003eH. denitrificans\u003c/em\u003e reference strain\u003csup\u003e8\u003c/sup\u003e, this is not the case for the strain lacking the \u003cem\u003esoxT1B\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The thiosulfate oxidation-negative phenotype of this strain is therefore explained by a lack of enzymes required for the degradation of the sulfur substrate. When the gene for the SoxR regulator was deleted together with Δ\u003cem\u003esoxT1B\u003c/em\u003e from \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e, this resulted in a thiosulfate oxidation-positive phenotype. Transcription was high for \u003cem\u003esoxT1A, shdrA\u003c/em\u003e and \u003cem\u003esoxXA\u003c/em\u003e irrespective of the presence of thiosulfate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The constitutive expression of these genes is caused by the lack of the transcriptional repressor. In the next step, a strain was constructed that lacks genes \u003cem\u003esoxT1B\u003c/em\u003e and \u003cem\u003eshdrR\u003c/em\u003e. This strain behaves differently from \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e Δ\u003cem\u003esoxR\u003c/em\u003e. It cannot oxidize thiosulfate and the substrate does not induce substantial increase of transcript abundance of the tested sulfur oxidation genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). SoxR is present in this strain and appears to be the major regulator that prevents transcription even in the presence of thiosulfate when the signal-transducing SoxT1B is not available. In conclusion the described experiments show that SoxT1B is dispensable for thiosulfate oxidation and that the import of sulfur for further oxidation is not its primary function. Instead, all results are consistent with a signal transduction function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRole of SoxT1A in\u003c/b\u003e \u003cb\u003eH. denitrificans\u003c/b\u003e. Like SoxT1B, the related membrane protein SoxT1A could in principle act either as an importer of sulfur for further oxidation in the cytoplasm or as a means of transmitting the information that oxidizable sulfur is available externally. To decide between the two possibilities, the strain \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1A\u003c/em\u003e was constructed, phenotypically characterized and studied concerning \u003cem\u003eshdr\u003c/em\u003e and \u003cem\u003esox\u003c/em\u003e gene transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The strain proved to be thiosulfate oxidation negative, although transcript abundance for \u003cem\u003eshdrA\u003c/em\u003e and \u003cem\u003esoxXA\u003c/em\u003e increased significantly in the presence of thiosulfate.\u003c/p\u003e \u003cp\u003eFurther insights were obtained when \u003cem\u003eH. denitrificans\u003c/em\u003e strains Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1A\u003c/em\u003e Δ\u003cem\u003esoxR\u003c/em\u003e and Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1A\u003c/em\u003e Δ\u003cem\u003eshdrR\u003c/em\u003e were studied. Both strains show very high transcript abundance for \u003cem\u003esoxXA\u003c/em\u003e and \u003cem\u003eshdrA\u003c/em\u003e in the absence as well as in the presence of thiosulfate, but are unable to oxidize thiosulfate, suggesting at an essential function of SoxT1A in the overall sulfur oxidation pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we provide information on the distribution and phylogeny of YeeE-like transporters in sulfur-oxidizing prokaryotes and even more importantly, we assign fundamentally different functions to two of these proteins, SoxT1A and SoxT1B, that co-occur in the same Alphaproteobacterium, \u003cem\u003eH. denitrificans\u003c/em\u003e. The completely different regulation of the respective genes upon exposure of the organism to thiosulfate is the first milestone for functional assignment. Expression of \u003cem\u003esoxT1A\u003c/em\u003e is highly increased, while \u003cem\u003esoxT1B\u003c/em\u003e expression is hardly affected at all by the presence of the reduced sulfur compound. All of our observations are consistent with a central role of SoxT1A in sulfur oxidation. The amount of SoxT1A molecules in the cells is increased to ensure efficient import of sulfur into the cytoplasm where it is further processed by the sHdr-LbpA system (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To the best of our knowledge, \u003cem\u003eH. denitrificans\u003c/em\u003e SoxT1A is the only experimentally demonstrated sulfur importer in dissimilatory sulfur-oxidizing prokaryotes. However, it does not provide a general solution because it not even occurs in all sulfur oxidizers using the cytoplasmic sHdr pathway. SoxT transporters are completely absent genomes with type II sHdr, even though some of these organisms harbor the capacity for Sox-driven thiosulfate oxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoxT1B functions as a signal transducing module. The same function can be assumed for the SoxT proteins in Alphaproteobacteria with complete Sox systems. In these organisms, thiosulfate is completely oxidized to sulfate in the periplasm and accordingly they lack cytoplasmic sulfur-oxidizing enzymes. As a consequence, there is no need for mass import of sulfur as carried out by SoxT1A. In full agreement with these conclusions, a function of SoxT from \u003cem\u003ePseudaminobacter salicylatoxidans\u003c/em\u003e in the transport of an inducer to the cytosol to activate the transcriptional regulator SoxR has been suggested\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genetic neighborhood of the \u003cem\u003esoxT1A\u003c/em\u003e and \u003cem\u003esoxT1B\u003c/em\u003e genes provides a basis for a model of how sulfur might be presented to the transporters, transported through them, and delivered to their final targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In immediate vicinity to and in the same direction of transcription with \u003cem\u003esoxT1A\u003c/em\u003e, a gene (Hden_0679) is located that encodes a periplasmic DsbA-like thioredoxin with two thioredoxin-like cysteine motifs (Cys-X\u003csub\u003e2\u003c/sub\u003e-Cys), one of which resides at the very carboxy-terminal end of the protein. Thioredoxins serve as general protein disulfide oxidoreductases that interact with a broad range of proteins by a redox mechanism based on reversible oxidation of two cysteine thiol groups to a disulfide, accompanied by the transfer of two electrons and two protons (IPR013766). We consider the possibility that the \u003cem\u003eH. denitrificans\u003c/em\u003e DsbA is involved in release of sulfane sulfur from the persulfidated periplasmic sulfur carrier SoxYZ and that the sulfur is then transferred into the cytoplasm through SoxT1A. In the cytoplasm, the sulfur is further handled by cytoplasmic Rhd442 (Hden_680), a protein that we recently characterized as a rhodanese-like sulfur transferase\u003csup\u003e6\u003c/sup\u003e. From there, the sulfur is delivered to the sulfur transferase DsrE3C and finally oxidized to sulfite by the sHdr-LbpA system, possibly involving TusA\u003csup\u003e43\u003c/sup\u003e. Hden_0678 encodes short 56 aa membrane protein, lacking cysteine residues and consisting of one central transmembrane helix (aa 12 to 27) with the N-terminus predicted to reside in the cytoplasm. Functional assignment is currently not possible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe genes in the vicinity of \u003cem\u003esoxT1B\u003c/em\u003e appear to encode a second module dedicated to the transport of sulfur, albeit for a different purpose. As suggested earlier, it is conceivable that sulfur bound to the sulfur carrier protein SoxYZ is in this case presented to the transporter by the periplasmic thiol\u0026ndash;disulfide oxidoreductase SoxS\u003csup\u003e44\u003c/sup\u003e. In fact, SoxS from \u003cem\u003eP. denitrificans\u003c/em\u003e specifically binds SoxY\u003csup\u003e44\u003c/sup\u003e. Once in the cytoplasm, the sulfur transferase TusA\u003csup\u003e6,18\u003c/sup\u003e is a likely acceptor protein for the sulfur. This idea is corroborated by recent findings for the \u003cem\u003eE. coli\u003c/em\u003e thiosulfate transporter TsuA\u003csup\u003e14,16\u003c/sup\u003e. TsuA belongs to same family as the SoxT transporters and the TusA-like TsuB protein was shown to be essential for TsuA mediated thiosulfate uptake in vivo. TsuB can cleave thiosulfate resulting in persulfidation of its conserved cysteine and the release of sulfite. In \u003cem\u003eH. denitrificans\u003c/em\u003e, sulfur atoms could be passed on from TusA to either one or both or the transcriptional repressors encoded in the \u003cem\u003eshr-lbpA-sox\u003c/em\u003e genomic region. For SoxR, we showed that it forms an intramolecular sulfur bridge between two conserved cysteines\u003csup\u003e8\u003c/sup\u003e. The formation of this bridge is the trigger to detach from its target DNA and thus to enable transcription. We assume that sHdrR, which closely resembles SoxR\u003csup\u003e8\u003c/sup\u003e, functions accordingly. Whether SoxR and/or sHdrR are indeed loaded with sulfur in a reaction mediated by TusA or rather directly by the sulfur species transported through SoxT1B, cannot be answered on the current data basis.\u003c/p\u003e \u003cp\u003eThe exact chemical nature of the sulfur species transported by SoxT1A and SoxT1B requires further investigation. The substrate for TsuA (YeeE) is thiosulfate. At present, we cannot rule out the possibility that in \u003cem\u003eH. denitrificans\u003c/em\u003e a small fraction of the thiosulfate available for oxidation is itself used as a signal molecule, channeled through SoxT1B and then cleaved by TusA, as proposed for TsuA. However, thiosulfate is certainly not the substrate for SoxT1A. The periplasmic proteins of the truncated Sox system in \u003cem\u003eH. denitrificans\u003c/em\u003e effectively oxidize this substrate and release sulfate and SoxYZ-bound sulfane sulfur from it, which then has to be further processed in the cytoplasm. In fact, a transport by passing sulfur from the SoxY cysteine along the three cysteines lining the central channel of the SoxT1 proteins is conceivable. On the other hand, Ikei and coworkers suggest that interaction of thiosulfate with the cysteine residues occurs via S─H─S hydrogen bonds\u003csup\u003e16\u003c/sup\u003e. The three cysteine residues in TsuA (YeeE) are linearly located at intervals of ~\u0026thinsp;7 \u0026Aring;, while disulfide bonds are usually about 2.05 \u0026Aring; in length, and 3.0 \u0026Aring; is taken as the cutoff for disulfides in the PDB database. It is therefore questionable whether sulfur atoms can be directly transferred from one cysteine sulfur to the next. Free HS\u003csup\u003e\u0026minus;\u003c/sup\u003e ions or short polysulfides (\u003csup\u003e\u0026minus;\u003c/sup\u003eS-S\u003csub\u003en\u003c/sub\u003e-S\u003csup\u003e\u0026minus;\u003c/sup\u003e), that are possibly formed by the action of the periplasmic protein disulfide oxidoreductases DsbA and SoxS, are alternatives and conceivable substrates for cytoplasmic sulfur transferases such as Rhd442 or TusA. Even a direct reaction of polysulfides with the transcriptional repressors, as occurs in vitro\u003csup\u003e8\u003c/sup\u003e, is conceivable.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eBacterial strains, plasmids, primers, and growth conditions.\u003c/b\u003e Supplementary Table\u0026nbsp;4 lists the bacterial strains, and plasmids that were used for this study. \u003cem\u003eEscherichia coli\u003c/em\u003e strains were grown on complex lysogeny broth (LB) medium \u003csup\u003e45\u003c/sup\u003e. \u003cem\u003eE. coli\u003c/em\u003e 10β was used for molecular cloning. \u003cem\u003eH. denitrificans\u003c/em\u003e strains were cultured in minimal medium kept at pH 7.2 with 100 mM 3-(\u003cem\u003eN\u003c/em\u003e-Morpholino)propanesulfonic acid (MOPS) buffer as previously described\u003csup\u003e28\u003c/sup\u003e. Media contained 24.4 mM methanol. Antibiotics for \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eH. denitrificans\u003c/em\u003e were used at the following concentrations (in \u0026micro;g ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): ampicillin, 100; kanamycin, 50; streptomycin, 200; chloramphenicol, 25.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRecombinant DNA techniques.\u003c/b\u003e Standard techniques for DNA manipulation and cloning were used unless otherwise indicated \u003csup\u003e46\u003c/sup\u003e. Restriction enzymes, T4 ligase and Q5 polymerase were obtained from New England Biolabs (Ipswich, UK) and used according to the manusfacturer\u0026rsquo;s instructions. Oligonucleotides were obtained from Eurofins Genomics Germany GmbH (Ebersberg, Germany). Plasmid DNA from \u003cem\u003eE. coli\u003c/em\u003e was purified using the GenJET Plasmid Miniprep kit (Thermo Scientific, Waltham, USA). Chromosomal DNA from \u003cem\u003eH. denitrificans\u003c/em\u003e strains was prepared using the Simplex Easy DNA Extract Kit (GEN-IAL GmbH, Troisdorf, Germany). DNA fragments were extracted from agarose gels using the GeneJET Gel Extraction Kit (Thermo Scientific, Waltham, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of\u003c/b\u003e \u003cb\u003eH. denitrificans\u003c/b\u003e \u003cb\u003emutant strains.\u003c/b\u003e Plasmids for reverse genetics in \u003cem\u003eH. denitrificans\u003c/em\u003e were constructed using the suicide plasmid pk18\u003cem\u003emobsacB\u003c/em\u003e \u003csup\u003e47\u003c/sup\u003e and the tetracycline cassette from pHP45Ω-Tc\u003csup\u003e48\u003c/sup\u003e on the basis of previously published procedures\u003csup\u003e28,29\u003c/sup\u003e. For markerless \u003cem\u003ein frame\u003c/em\u003e deletion of the individual \u003cem\u003eH. denitrificans soxT1A\u003c/em\u003e and \u003cem\u003esoxT1B\u003c/em\u003e genes by splicing overlap extension (SOE)\u003csup\u003e49\u003c/sup\u003e, PCR fragments were constructed using the primers listed in Supplementary Table\u0026nbsp;4. The \u003cem\u003esoxT1A\u003c/em\u003e or \u003cem\u003esoxT1B\u003c/em\u003e fragments were inserted into pk18\u003cem\u003emobsacB\u003c/em\u003e using XbaI and SaII or XbaI and PstI restriction sites, respectively. The SmaI-excised tetracycline cassette from pHP45Ω-Tc \u003csup\u003e48\u003c/sup\u003e was inserted into the SmaI site, resulting in plasmids pK18\u003cem\u003emobsacB-\u003c/em\u003eΔ\u003cem\u003esoxT1A-\u003c/em\u003eTc and pK18\u003cem\u003emobsacB-\u003c/em\u003eΔ\u003cem\u003esoxT1B-\u003c/em\u003eTc. Another plasmid was constructed for concomitant deletion of \u003cem\u003esoxR\u003c/em\u003e and \u003cem\u003esoxT1B\u003c/em\u003e by SOE PCR with primers P1 fwd up hden_0700, P5 fwd down hden_soxR/soxT1B, P6 rev down hden_ soxR/soxT1B and P7 rev up hden_ soxR/soxT1B (Supplementary Table\u0026nbsp;4). The PCR fragment was cloned into the XbaI and PstI sites of pk18\u003cem\u003emobsacB\u003c/em\u003e-Tc\u003csup\u003e7\u003c/sup\u003e. For chromosomal complementation of the \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e strain, the s\u003cem\u003eoxT1B\u003c/em\u003e gene was amplified together with upstream and downstream regions using primers SoxT1B_Del_Up_Fw and SoxT1B_Del_Down_Rev and cloned into the XbaI/PstI sites of pk18\u003cem\u003emobsacB\u003c/em\u003e-Tc. For chromosomal integration of the genes encoding SoxT1B Cys\u003csup\u003e24\u003c/sup\u003eSer, SoxT1B Cys\u003csup\u003e98\u003c/sup\u003eSer and SoxT1B Cys\u003csup\u003e304\u003c/sup\u003eSer, the modified genes and upstream and downstream sequences were amplified by SOE PCR using the appropriate primers listed in Supplementary Table\u0026nbsp;4.\u003c/p\u003e \u003cp\u003eAll final constructs were electroporated into the desired \u003cem\u003eH. denitrificans\u003c/em\u003e strains and transformants were selected using previously published procedures\u003csup\u003e28,29\u003c/sup\u003e. \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e served as acceptor for plasmids pK18\u003cem\u003emobsacB-\u003c/em\u003eΔ\u003cem\u003esoxT1B-\u003c/em\u003eTc, pK18\u003cem\u003emobsacB-\u003c/em\u003eΔ\u003cem\u003esoxT1A-\u003c/em\u003eTc and pk18\u003cem\u003emobsacB\u003c/em\u003e_Tc_Δ\u003cem\u003esoxR/soxT1B\u003c/em\u003e. \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003eshdrR\u003c/em\u003e and \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxR\u003c/em\u003e served as strain backgrounds for deletion of \u003cem\u003esoxT1A\u003c/em\u003e. The \u003cem\u003esoxT1B\u003c/em\u003e deletion was also established in the \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003eshdrR\u003c/em\u003e strain. The plasmids for complementation and cysteine exchanges of SoxT1B were transferred into \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e Δ\u003cem\u003esoxT1B\u003c/em\u003e in all cases, single crossover recombinants were Cm\u003csup\u003er\u003c/sup\u003e and Tc\u003csup\u003er\u003c/sup\u003e. Double crossover recombinants were Tc\u003csup\u003es\u003c/sup\u003e and survived in the presence of sucrose due to loss of both, the vector-encoded levansucrase (SacB) and the tetracyclin resistance gene. The genotype of the \u003cem\u003eH. denitrificans\u003c/em\u003e strains generated in this study were confirmed by PCR.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of phenotypes, quantification of sulfur compounds and biomass content.\u003c/b\u003e Growth experiments with \u003cem\u003eH. denitrificans\u003c/em\u003e were run in medium with 24.4 mM methanol in Erlenmeyer flasks or in 96-well microtiter plates as described earlier\u003csup\u003e7\u003c/sup\u003e. 2 mM thiosulfate were added when needed. Biomass content, thiosulfate and sulfite concentrations were determined by previously described methods\u003csup\u003e7,50\u003c/sup\u003e. All growth experiments were repeated three to five times. Representative experiments with two biological replicates for each strain are shown. All quantifications are based on at least three technical replicates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression studies based on RT-qPCR\u003c/b\u003e. Total RNA of the relevant \u003cem\u003eH. denitrificans\u003c/em\u003e strains was isolated from cells harvested in mid-log phase according to an established procedure\u003csup\u003e8\u003c/sup\u003e. RNA samples of 100 ng were used for RT-qPCR analysis which was performed with the primers listed in Supplementary Table\u0026nbsp;4 following the method described in Li et al 2023\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome-wide transcriptomic analysis of\u003c/b\u003e \u003cb\u003eH. denitrificans\u003c/b\u003e \u003cb\u003eΔ\u003c/b\u003e\u003cb\u003etsdA\u003c/b\u003e \u003cb\u003ein the absence and presence of thiosulfate.\u003c/b\u003e For transcriptome sequencing (RNA-Seq), \u003cem\u003eH. denitrificans\u003c/em\u003e Δ\u003cem\u003etsdA\u003c/em\u003e was cultured in 50 ml minimal medium containing either 24.4 mM methanol or 24.4 mM methanol plus 2 mM thiosulfate in 200 ml Erlenmeyer flasks at 30\u0026deg;C with shaking at 200 rpm to early log phase. Cells from 20 ml culture were harvested and flash frozen in liquid N\u003csub\u003e2\u003c/sub\u003e and stored at -70\u0026deg;C. From the frozen pellets, the RNA was purified with the FastGene RNA Premium Kit (NIPPON Genetics EUROPE, D\u0026uuml;ren, Germany) according to the manufacturer\u0026rsquo;s instructions. A modification was introduced regarding the cell lysis step. After addition of the lysis buffer that contained 1% (v/v) 2-mercaptoethanol, cells were disrupted by bead beating (Bead Ruptor 12 Bead Mill Homogenizer, Omni International, Kennesaw, GA, USA) for three cycles of 30 s at maximum speed and incubation on ice for 1 min. RNA quality was checked on 1% agarose gels and its concentration was measured using NanoPhotometer NP80 (IMPLEN, Munich, Germany). The RNA was shipped on dry ice to Eurofins Genomics GmbH (Ebersberg, Germany). The subsequent analysis pipeline included rRNA depletion, library preparation (mRNA fragmentation, strand specific cDNA synthesis), Illumina paired end sequencing (2 x 150 bp, minimum 10 MB reads)), and bioinformatic analysis (mapping against the reference genome, identification and quantification of transcripts, pairwise comparison of expression levels and determination of significant fold differences) and was conducted by the company.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of datasets for phylogenetic analyses.\u003c/b\u003e Archaeal and bacterial genomes were downloaded from Genome Taxonomy Database (GTDB, release R207). In GTDB, all genomes are sorted according to validly published taxonomies, they are pre-validated and have high quality (completeness minus 5*contamination must be higher than 50%). One representative of each of the current 65,703 species clusters was analyzed. Open reading frames were determined using Prodigal\u003csup\u003e51\u003c/sup\u003e and subsequently annotated for sulfur related proteins via HMSS2\u003csup\u003e37\u003c/sup\u003e. Annotation was extended by HMMs from TIGRFAMs\u003csup\u003e52\u003c/sup\u003e and Pfam\u003csup\u003e53\u003c/sup\u003e databases representing the 16 syntenic ribosomal proteins RpL2, 3, 4, 5, 6, 14, 15, 16, 18, 22, and 24, and RpS3, 8, 10, 17, and 19. A type I sHdr system was considered to be present if the core genes \u003cem\u003eshdrC1B1AHC2B2\u003c/em\u003e were present in a syntenic gene cluster. For a type II sHdr system gene cluster \u003cem\u003eshdrC1B1AHB3\u003c/em\u003e and \u003cem\u003eetfAB\u003c/em\u003e had to be present in a single syntenic gene cluster\u003csup\u003e29,54\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic tree inference.\u003c/b\u003e For species tree inference, results for each ribosomal protein were individually aligned, trimmed and subsequently concatenated before they were used for phylogenetic tree construction. Proteins were aligned using MAFFT\u003csup\u003e55\u003c/sup\u003e and trimmed with BMGE\u003csup\u003e56\u003c/sup\u003e (entropy threshold\u0026thinsp;=\u0026thinsp;0.95, minimum length\u0026thinsp;=\u0026thinsp;1, matrix\u0026thinsp;=\u0026thinsp;BLOSUM30). Alignments were then used for maximum likelihood phylogeny inference using IQ-TREE v1.6.12\u003csup\u003e57\u003c/sup\u003e implemented on the \u0026ldquo;bonna\u0026rdquo; high performance clusters of the University of Bonn. The best-fitting model of sequence evolution was selected using ModelFinder\u003csup\u003e58\u003c/sup\u003e. Branch support was then calculated by SH-aLRT (2000 replicates),\u003csup\u003e59\u003c/sup\u003e aBayes (2000 replicates)\u003csup\u003e60\u003c/sup\u003e and ultrafast bootstrap (2000 replicates)\u003csup\u003e34\u003c/sup\u003e. Finally, trees were displayed using iTol\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistics and reproducibility.\u003c/b\u003e Experimental data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of the mean (SEM) of the number of tests stated for each experiment. All analysis was reproduced in at least three independent experiments. The significant difference between the two groups was analyzed using an independent student\u0026rsquo;s t-test; the p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated statistical significance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReporting summary.\u003c/b\u003e Further information on research design will be available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the article (and its supplementary information files).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Deutsche Forschungsgemeinschaft (Grants Da351/8-2, Da 351/13-1 and Da 351/14-1).\u0026nbsp;Jingjing Li was financed by a Scholarship of the Chinese Scholarship Council and Tomohisa Sebastian Tanabe received a scholarship from the Studienstiftung des Deutschen Volkes. We thank Stefania de Benedetti for help with RNA isolation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional\u0026nbsp;\u003c/strong\u003einformation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for material should be addressed to Christiane Dahl.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRabus, R.\u003cem\u003e et al.\u003c/em\u003e A post-genomic view of the ecophysiology, catabolism and biotechnological relevance of sulphate-reducing prokaryotes. \u003cem\u003eAdv. 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Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, W293-W296 (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Hyphomicrobium denitrificans, sulfur oxidation, thiosulfate, Sox pathway, sulfur transport, regulation","lastPublishedDoi":"10.21203/rs.3.rs-4461547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4461547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany sulfur-oxidizing prokaryotes oxidize sulfur compounds through a combination of initial extracytoplasmic and downstream cytoplasmic reactions. Facultative sulfur oxidizers adjust transcription to sulfur availability. While sulfur-oxidizing enzymes and transcriptional repressors have been extensively studied, sulfur import into the cytoplasm and how regulators sense external sulfur are poorly understood. Addressing this gap, we show that SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters and are encoded alongside sulfur oxidation and transcriptional regulation genes, fulfill these roles in the Alphaproteobacterium \u003cem\u003eHyphomicrobium denitrificans\u003c/em\u003e. SoxT1A mutants are sulfur oxidation-negative despite high transcription levels of sulfur oxidation genes, showing that SoxT1A delivers sulfur to the cytoplasm for its further oxidation. SoxT1B serves as a signal transduction unit for the transcriptional repressor SoxR, as SoxT1B mutants are sulfur oxidation-negative due to low transcription unless SoxR is also absent. Thus, SoxT1A and SoxT1B play essential but distinct roles in oxidative sulfur metabolism and its regulation.\u003c/p\u003e","manuscriptTitle":"YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 16:35:09","doi":"10.21203/rs.3.rs-4461547/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e2444986-9051-4eea-9ebb-5f7001712b3d","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-22T08:09:23+00:00","versionOfRecord":{"articleIdentity":"rs-4461547","link":"https://doi.org/10.1038/s42003-024-07270-7","journal":{"identity":"communications-biology","isVorOnly":false,"title":"Communications Biology"},"publishedOn":"2024-11-21 05:00:00","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-06-18 16:35:09","video":"","vorDoi":"10.1038/s42003-024-07270-7","vorDoiUrl":"https://doi.org/10.1038/s42003-024-07270-7","workflowStages":[]},"version":"v1","identity":"rs-4461547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4461547","identity":"rs-4461547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}