Structural And Molecular Genetic Insight Into A Widespread Sulfur Oxidation Pathway

doi:10.1016/j.jmb.2008.10.016 J Mol Biol (2008) 384, 1287–1300 Available online at www.sciencedirect.com Structural and Molecular Genetic Insight into a Widespread Sulfur Oxidation Pathway Christiane Dahl , Andrea Schulte , Yvonne Stockdreher , Connie Hong , Frauke Grimm , Johannes Sander , Rosalind Kim , Sung-Hou Kim 2,3 and Dong Hae Shin * Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany Department of Chemistry, University of California, Berkeley, CA 94720-5230, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea Received 16 July 2008; received in revised form 27 September 2008; accepted October 2008 Available online 15 October 2008 Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds Oxidation of this sulfur requires the concerted action of Dsr proteins However, individual functions and interplay of these proteins are largely unclear We proved with a ΔdsrE mutant experiment that the cytoplasmic α2β2γ2-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria Conserved Cys78 of A vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins © 2008 Elsevier Ltd All rights reserved Edited by M F Summers Keywords: DsrEFH; dissimilatory sulfur oxidation; crystal structure; anoxygenic phototrophic sulfur bacteria YchN fold; dissimilatory sulfite reductase Introduction Reduced sulfur compounds such as sulfide and thiosulfate are oxidized by a large and diverse group of prokaryotes, including the phototrophic sulfur bacteria, the thiobacilli, and other chemotrophic sulfur bacteria and some thermophilic archaea Typically, these sulfur compounds are oxidized to sulfate, but in many cases, globules of polymeric, water*Corresponding author E-mail address: dhshin55@ewha.ac.kr Abbreviations used: MTM, Methanothermobacter, Thermotoga, and Moorella; PDB, Protein Data Bank insoluble sulfur accumulate as a transient product The sulfur can be deposited outside of the cell as is the case for green sulfur bacteria On the other hand, purple sulfur bacteria of the family Chromatiaceae store sulfur globules inside the cells They have this trait in common not only with a large number of environmentally important free-living chemotrophic sulfur oxidizers such as Beggiatoa, Thioploca, or magnetotactic bacteria but also with sulfur-oxidizing bacterial symbionts of marine animals such as Riftia pachyptila or Olavius algarvensis It is very important to note that the sulfur resides in the bacterial periplasm in the purple sulfur bacterial model organism Allochromatium vinosum and in many if not all other bacteria forming intracellular sulfur globules.1,2 Bio- 0022-2836/$ - see front matter © 2008 Elsevier Ltd All rights reserved 1288 chemical data, genetic studies with A vinosum, and genome comparisons indicate that in all these organisms as well as in green sulfur bacteria and thiobacilli, a complicated pathway is at work, involving transport of sulfur carrier molecules from outside the cells or the periplasm into the cytoplasm and requiring the presence of many different enzymes including sulfite reductase (DsrAB).2,3 In A vinosum, several proteins encoded in the dsr gene cluster (Fig 1a) have been shown to be essential for further oxidation of stored sulfur to the end product sulfate.4–8 The Dsr proteins are either cytoplasmic or membrane-bound It is proposed that sulfur is transported into the cytoplasm in a persulfidic form, possibly as glutathione amide persulfide.3,6,9–11 Once in the cytoplasm, the sulfane sulfur has to be made available to sulfite reductase, which oxidizes it to sulfite The siroheme-containing sulfite reductase specifically interacts with the membrane-bound electron-transporting DsrMKJOP complex7 that may feed electrons into photosynthetic electron transport Such a pathway would be analogous to that postulated for dissimilatory sulfate-reducing bacteria,12 operating in the reverse direction DsrC, a protein with two conserved carboxy-terminal cysteine residues (Cys100 and C111), has been discussed to be involved in electron transfer between DsrAB and DsrMKJOP via thioldisulfide switches.3,6,7 Recently, it has been shown that the DsrC protein from the sulfate reducer Desulfovibrio vulgaris can be bound in a cleft between DsrA and DsrB with the cysteine corresponding to Cys111 A vinosum DsrC reaching the distal side of the active-site siroheme On this basis, it has been proposed that DsrC is involved in the catalytic reaction as a product-binding protein and that a persulfide of DsrC is a crucial intermediate in the reduction of sulfite.13 The protein DsrEFH occurs exclusively in sulfur oxidizers.9 In Escherichia coli, the DsrEFH-related protein TusBCD and the DsrC homologous protein TusE are firmly established parts of a sulfur relay system during thiouridine biosyntheses.14 On this background, the recently documented interaction of A vinosum DsrEFH and DsrC led to the suggestion of an alternative model for intracellular sulfur oxidation implying DsrEFH and DsrC as parts of sulfur trafficking between persulfidic sulfur imported into the cytoplasm and sulfite reductase.6 Structure and Role of DsrEFH DsrEFH is a soluble, cytoplasmic α2β2γ2-structured holoprotein with an apparent molecular mass of 75 kDa.7 The polypeptides DsrE, DsrF, and DsrH are homologous to each other (Fig 3) DsrE and DsrF are the prototypes of a family of conserved domains (Pfam 02635.11, COG 1553, COG 2044, COG 2923) DsrH is the prototype of yet another family of conserved proteins found in bacteria and archaea (Pfam04077.6; COG 2168) However, DsrH also fits into the DsrE/F family Structural information on representatives of the DsrH family of proteins is available through the work of Shin et al on YchN from E coli,15 Gaspar et al on Tm0979 from Thermotoga maritima,16 Christendat et al on MTH1491 from Methanobacterium thermoautotrophicum,17 and Numata et al on E coli TusBCD.18 In contrast to DsrEFH and TusBCD, all others form homooligomers YchN is present as two rings of trimers, MTH1491 as a trimer, and Tm0979 as a dimer Except Tm0979, all of these proteins harbor conserved cysteine residues in a probable active-site region In our effort to further dissect the functions of the proteins encoded at the A vinosum dsr locus and to test the existing models for the dsr-encoded sulfur oxidation pathway, we firstly constructed an A vinosum mutant with an in-frame deletion of dsrE, complemented the dsrEFH genes in trans, and studied the resulting phenotypes regarding sulfur oxidation Secondly, we determined the threedimensional structure of DsrEFH by X-ray crystallography Furthermore, we determined the site of interaction with DsrC via site-directed mutagenesis of putative active-site cysteines in DsrE and/or DsrH Results Biological significance of DsrEFH In order to examine the importance of DsrEFH for sulfur oxidation, we first deleted the complete dsrEFH genes However, the resulting A vinosum mutant turned out to be genetically unstable, most probably due to the deletion of the promoter of the constitutively expressed dsrC present in dsrF.7,8 The dsrC gene cannot be stably deleted from A vinosum, Fig Schematic overview of the dsr locus of A vinosum Genes that have been proven to be individually essential for sulfur oxidation by in-frame deletion mutagenesis4,5 are shown in black Absolute requirement of DsrE is proven in this study DsrN (light gray), a probable siroamide synthase providing the prosthetic group for DsrAB sulfite reductase, is important though not absolutely essential.5 The dsrC gene is marked with an asterisk In-frame deletion of this gene leads to a genetically unstable mutant, indicating that dsrC is indispensable in A vinosum even in the absence of reduced sulfur compounds.6 1289 Structure and Role of DsrEFH indicating that its product is essential for central metabolic pathways in this organism.6 Therefore, we deleted solely dsrE, leaving the promoter for dsrC intact The A vinosum ΔdsrE mutant was genetically stable even after prolonged incubation In order to examine the phenotype of A vinosum ΔdsrE, we cultivated the strain photolithoautotrophically in batch culture with sulfide as electron source As expected for a classical purple sulfur bacterium,19 the sulfide concentration immediately decreased and intracellular sulfur was formed The rate of sulfide oxidation to sulfur was unaffected in the ΔdsrE mutant During the oxidation of sulfide to sulfur of oxidation state zero, two different polysulfides are formed as intermediates by A vinosum wild type.20 The formation of both polysulfides was not affected in the ΔdsrE mutant (not shown) In the wild type, stored sulfur is further oxidized to sulfate when sulfide is depleted.8 In contrast, the ΔdsrE mutant was completely unable to oxidize the accumulated sulfur (Fig 2a) Another unambiguous indicator for the inability of the mutant strain to oxidize stored sulfur was the complete lack of sulfate as the final product of sulfur oxidation (Fig 2b) Furthermore, an accumulation of intermediates en route to sulfate, for example, sulfite, was discounted by HPLC analysis Complementation of dsrEFH in trans completely restored the mutant sulfur oxidation to that of the wild type (Fig 2a) Sulfate was again formed as the final product (Fig 2b) This experiment confirmed that the observed phenotype was indeed caused by the specific loss of dsrE and consequently a lack of the DsrEFH protein Growth under photoorganoheterotrophic conditions was not influenced in the A vinosum ΔdsrE strain In summary, the phenotype of the studied single-locus dsrE mutant clearly demonstrates the vital importance of the DsrEFH protein for the oxidation of stored sulfur in A vinosum DsrEFH proteins are encoded in immediate vicinity of other dsr genes (including dsrAB encoding dissimilatory sulfite reductase) similar to the situation in A vinosum.7 From this observation, we conclude that these proteins form a homogeneous physiological group We can therefore state that the presence of one conserved putative active-site cysteine in each, DsrE and DsrH, is a common and typical property of DsrEFH proteins from sulfuroxidizing bacteria In all these organisms, the dsrEFH genes are situated close to a dsrC gene In order to investigate whether the different groups of DsrEFH-related proteins are also phylogenetically distinct, we performed neighbor-joining, parsimony, likelihood, and Bayesian analyses of concatenated DsrE, F, and H sequences The occurrence of conserved cysteine residues fits well with DsrEFH-related proteins are widespread and form distinct groups A whole array of bacteria and also some archaea contain dsrEFH homologous genes located immediately adjacent to each other The function of the encoded proteins is probably variable: An in silico alignment of DsrEFH fusion proteins revealed that they can be subdivided into distinct groups characterized by the number and position of conserved cysteine residues in putative active-site regions (Fig 3) A vinosum DsrEFH belongs to a group of deduced proteins, which contain conserved cysteine residues only in DsrE (Cys78) and DsrH (Cys20) Notably, the organisms containing these proteins include Thiobacillus denitrificans, the green sulfur bacteria, and the magnetotactic bacteria All of these organisms are well-known sulfur-oxidizing bacteria In addition, genome sequence data have been claimed to suggest that Methylococcus capsulatus, another member of this group, is also capable of chemolithotrophic sulfur oxidation.21 In all of the established sulfur oxidizers mentioned above, the Fig Sulfur accumulation and oxidation (a) and sulfate production (b) by A vinosum wild type (▵), A vinosum ΔdsrE ( ), and A vinosum ΔdsrE + dsrE (●) Cells were grown photolithoautotrophically in batch culture in the presence of mM sulfide Sulfide is not completely recovered as sulfate due to loss of gaseous H2S during sampling.8 Sulfide and the polysulfides formed as intermediates during the formation of sulfur globules from sulfide are not shown for clarity Sulfite and thiosulfate were not detected during degradation of sulfur globules in any of the cultures Protein concentrations at the onset and at the end of the experiments were 50 and 70 μg, respectively Representative growth experiments for each strain are shown ▪ 1290 Structure and Role of DsrEFH Fig Sequence comparison among some of YchN fold members The three-dimensional structures of the listed members are known Abbreviations are as follows: YchN, YchN from E coli; 1X9A, PDB ID of Tm0979; 1L1S, PDB ID of Mth1491 The “-” represents a gap, “⁎” denotes identical residues, “:” indicates highly conserved residues, and “.” denotes less highly conserved residues The blue character H represents a sequence belonging to α-helices, a green G for 310helices, a pink β for β-strands, and a black L for loops The conserved cysteine residues reported in the YchN family15 are marked yellow the position of the respective protein in the phylogenetic tree (Fig 4), supporting the notion that different physiological functions correlate with the presence of certain conserved cysteine residues As expected, the DsrEFH-related proteins of known sulfur oxidizers are affiliated to each other Surprisingly, this clade cannot be regarded as monophyletic since the proteins of Methanothermobacter, Thermotoga, and Moorella (MTM clade) are nested within this clade, regardless of method or data set used (Fig 4) Only neighbor-joining trees show the MTM clade as a sister group to the sulfur-oxidizing prokaryotes Since the MTM proteins are very different at the sequence level from all other proteins included in this analysis, we suppose this to be a result of a phenomenon called “long branch attraction”.22 As a control and in order to exclude independent evolution of the dsrE, dsrF, and dsrH genes, detailed phylogenies for the single genes were also calculated The resulting trees were similar but not absolutely identical Some variations were observed concerning the positions of subbranches; however, such minor differences also occurred when the different methods (Bayesian analysis, maximum parsimony, and neighbor joining) were compared In all cases, the same major groups were observed; that is, the proteins from the sulfur oxidizers always group together It has to be noted that the members of the genus Pseudomonas as well as Shewanella oneidensis, Chromohalobacter salexigens, Oceanobacter sp., M capsulatus, and Idiomarina loihiensis possess a dsrC-like gene directly adjacent to the dsrEFH-related genes This points at functional linkage between DsrEFH-like and DsrC-like proteins in these organisms Biochemical studies in E coli14 and A vinosum6,7 already provided evidence for such an interaction The presence of a conserved cysteine residue in DsrH might point at a so far unidentified specific reaction partner Quality of the model and overall structure of DsrEFH In an attempt to gain more insight into the function of A vinosum DsrEFH, its X-ray crystal structure was determined There were three α2β2γ2-structured DsrEFHs (three heterohexamers) in the asymmetric unit In the final refined models to 2.5 Å resolution, all residues of DsrE and all residues of DsrH except the first methionine are included However, the first four residues and the eight residues between 103 and 110 are undefined in the electron density map of DsrF The average B-factors for main-chain and side-chain atoms are 32.1 and 35.6 Å2, respectively Table summarizes refinement statistics All residues except Tyr21 of DsrE and Tyr21 of DsrF lie in the allowed region of the Ramachandran plot produced with PROCHECK.24 In the crystal structure of TusBCD, a structural homologue of DsrEFH, His13 of TusC, which corresponds to the tyrosines (Tyr21) of DsrE and DsrF, also lies in the disallowed region of the Ramachandran plot The Cα trace of the atomic model of the DsrEFH structure is shown in Fig 5a Each of DsrE, DsrF, and DsrH consists of a single domain with a three-layer (αβ)-sandwich architecture The α2β2γ2-structured hexamer (Fig 5b) found in the asymmetric unit is 1291 Structure and Role of DsrEFH Fig Bayesian tree of in silico fusions of DsrEFH-related proteins Node significances are given as posterior probabilities (first values) for the Bayesian analyses and as bootstrap support for the neighbor-joining (second values) and maximum parsimony analyses (third values) Names of organisms containing putative dsr operons similar to that of A vinosum are printed in bold letters In the left part of the figure, the presence of conserved cysteine residues is indicated by bars: DsrE1, residue 78; E2, residue 81; F1, residue 80; F2, residue 83; and H, residue 20 (numbering according to the respective A vinosum protein) In the outer left lane, the presence of a bar indicates that dsrC and dsrEFH genes are located in vicinity to each other in the respective organism's genome Accession numbers of the DsrE-like proteins are given after the organism names Genes for DsrF and DsrH are generally found in the immediate vicinity of dsrE genes The accession numbers for the DsrE-like proteins of Moorella thermoacetica, T maritima, and M thermoautotrophicum are ZP_00330986, NP_228789, and AAB 85834, respectively a biological form confirmed by analytical sizeexclusion column and dynamic light-scattering experiments.27 The heterohexamer has approximate dimensions of 75 Å × 60 Å × 55 Å Oligomeric forms of DsrEFH Interestingly, all the subunits of DsrEFH have a YchN fold first found in E coli YchN.15 One of the 1292 Structure and Role of DsrEFH Table Refinement parameters Crystal parameters and refinement statistics Space group Cell dimensions Volume fraction of solvent (%) Vm (Å3/Da) Total number of residues Total non-H atoms Number of Se atoms Number of water molecules Average temperature factors (Å2) Protein Solvent Resolution range of reflections used (Å) Amplitude cutoffa (σ) R-factor (%) Free R-factor (%) Stereochemical ideality Bond (Å) Angle (°) Improper (°) Dihedral (°) 34.0 39.1 20.0–2.5 Identification and mutational analysis of putative active sites in DsrEFH 0.0 20.9 25.6 Considering the E coli YchN structure, there should be a putative active site at the beginning of the H3 helix of each subunit.15 DsrE has a conserved cysteine (Cys78) corresponding to the active cysteines of E coli YchN at this position (Fig 6a and b) However, nonconserved residues, Asp83 and Gly63, are found in this position in the case of DsrF and DsrH, respectively Therefore, only DsrE has the putative active cysteine similar to that of E coli YchN The putative active site is formed between interfaces of DsrE and DsrF with a depth of ∼ 11 Å and a width of ∼ Å × 14 Å where the highly conserved Tyr40 is also present There is a large cleft on the equatorial interface lined up by two DsrF and two DsrH (Figs 5b and 7a) Generally, a long L3 loop of the YchN fold contributes L3–L3 loop interactions during hexamerization on the outer equatorial surface 15 However, DsrH has a shortened L3 loop (Fig 5b), which results in the formation of a big cleft with a depth of ∼ 11 Å and a width of about 10 Å × 30 Å on the surface of DsrFH (Fig 7a) The bottom of the cleft is lined up by two L1 loops (Fig 5b) Interestingly, highly conserved residues, His5 and Trp101 of DsrH, are constellated around Cys20 of DsrH in this pocket As shown in Fig 4, Cys20 of DsrH is a conserved cysteine Therefore, these features strongly support that this big cleft may be another putative active site In summary, the DsrEFH structure reveals two different types of putative active sites, which is different from the case of the E coli YchN structure where the six interfaces among the adjoining subunits contain the same recessed cavities along the equatorial surface of the hexamer Recently, it has been shown that DsrEFH interacts with DsrC from A vinosum and that this interaction is strictly dependent on the presence of the penultimate conserved cysteine residue of DsrC.6 We now set out to identify the site of interaction in the DsrEFH protein and exchanged either one or both putative active-site cysteine residues (Cys78 of DsrE and Cys20 of DsrH) to serine Interaction of the proteins was assessed using a band-shift technique under nondenaturing conditions (Fig 8) It clearly appeared that Cys78 of DsrE is absolutely required for the interaction, as interacting bands are not formed when Cys78 of DsrE alone or both Cys78 of DsrE and Cys20 of DsrH are mutated to Ser The 0.006 1.25 0.74 23.67 Ramachandran plot (%) Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regions a P21 56.6 Å × 183.1 Å × 107.8 Å, β = 99.6° 42.6 2.23 2124 17,203 30 418 DsrH) form an ion pair network Since these residues are close to each other, a closed cavity is not formed at the core of the hexamer unlike the case of E coli YchN.15 The dimensions of the hexamer are ∼ 75 Å along the pseudo-triad axis and ∼ 60 Å across the 2-fold axis The lack of hydrophobic interactions between the two trimers may result in disassembly of the hemispheres depending on surrounding conditions influencing these ionic interactions, such as pH, salt, or protein concentration 91.8 7.0 0.7 0.5 23 Sigma cutoff in CNS during refinement structural characteristics of the YchN fold is that a large portion of the fold is involved in oligomerization Oligomerization is accompanied by an increase in the buried surface areas from ∼550 to ∼ 1800 Å2 per monomer during hexamerization (Table 2) Various charged interactions contribute to stabilization of each subunit and various oligomeric forms (Table 2) The trimeric form (Fig 5a) is stabilized mainly by hydrophobic interactions between subunits Interestingly, many aromatic side chains are involved in these interactions: (1) Phe3, Phe23, and Phe128 of DsrE and Phe33, Tyr85, and Phe88 of DsrH at the interfaces of DsrEH; (2) Tyr40 of DsrE and Phe108, Phe235, Phe134, and Phe136 of DsrF at the interfaces of DsrEF The interactions among the last β-strands (β5s) of each subunit in the center of the trimer also play a role to stabilize the trimeric form through a water-mediated interaction as first shown in the E coli YchN structure.15 Therefore, the trimeric DsrEFH forms a very stable structure In the hexameric form, there are two different types of contacts at the interfaces of trimers (Fig 5b): (1) DsrE–DsrE′ interaction and (2) DsrF–DsrH′ or DsrH–DsrF′ interactions These types of contacts result in unique subunit symmetry also found in the TusBCD structure.18 Most of the contacts between hemispheres are governed by the hydrophilic interactions Therefore, many water molecules are found between the interfaces of trimers At the core of the hexamer, several charged residues (His14 and Asp130 of DsrE and Lys9, Arg14, and Glu34 of Structure and Role of DsrEFH 1293 Fig Crystal structure of DsrEFH (a) A Cα trace of DsrEFH DsrE, DsrF, and DsrH are represented by green, magenta, and yellow, respectively Every 20th residue is numbered and represented by a dot The N-terminus (Met1 of DsrE, Val5 of DsrF, and Ser2 of DsrH) and C-terminus (Asp130 of DsrE, F136 of DsrF, and Leu102 of DsrH) are labeled The figure was generated by MOLSCRIPT.25 (b) Loop interactions in the second putative active pocket of hexameric DsrEFH L1 and L3 loops are labeled to indicate L1–L1 and L3–L3 interactions (see the text) The figures were generated using the program RIBBONS.26 interaction is not prevented by the exchange of Cys20 of DsrH to Ser Structural differences between DsrEFH and TusBCD coincide with divergent cellular functions Apart from the described similarities, striking structural differences between DsrEFH and TusBCD are also apparent In the primary structure, DsrEFH has a much lower aliphatic index (89.02) than TusBCD (105.64) The difference is caused by the higher alanine content in TusBCD (13.5%) compared with DsrEFH (9.2%) The high aliphatic index also results from the reduced content of charged residues in TusBCD Actually, the total number of charged residues (aspartate, glutamate, lysine, and arginine) is 96 in DsrEFH and 65 in TusBCD Therefore, the contribution of charged residues to structure and function may be severely decreased in the case of TusBCD The tertiary structure difference is prominent between DsrF and TusC, though the core structure of both subunits is conserved DsrF has a large insertion on L5 (the undefined loop including residues 103 and 110), which is not present in the TusC 1294 Structure and Role of DsrEFH Table Comparison between DsrEFH and TusBCD Oligomeric state Subunit interaction Total surface area (Å2) Monomer DsrE DsrF DsrH DsrEF DsrEH DsrFH DsrEFH (DsrEFH)2 5698 5922 4361 10,383 8875 9372 12,930 21,156 Dimer Trimer Hexamer a b Area buried per monomer (Å2) No of salt bridgea Subunit interaction Total surface area (Å2) 577 559 525 1026 1874 3 15 11 19 44 TusD TusC TusB TusCD TusBD TusBC TusBCD (TusBCD) 5716 5359 4254 10,109 8737 8897 12,714 21,032 Area buried per monomer (Å2) No of salt bridgea Sequence identity (%) and rmsd (Å)b 44.5/0.8 26.1/1.4 35.8/1.2 483 537 438 872 1604 8 11 16 37 The number of salt bridges within 3.2 Å distance Structural comparison with the combinatorial extension method [http://cl.sdsc.edu/ce.html] sequence (Fig 3) The quaternary structure difference is also visible in the second putative active site on the surface of DsrFH The size of the pocket of DsrFH (Fig 7a) is much larger than that of TusBC (Fig 7b) In this pocket, TusBC has a glutamate (Glu85 of TusC) and a tyrosine (Tyr45 of TusB) instead of an alanine (Ala89 in the case of DsrF) and a valine (Val46 of DsrH) These bigger substitutions narrow down the size of the pocket of TusBC In addition, the conserved cysteine residue (Cys20 of DsrH) found in the pocket of DsrFH is not present in the sequence of TusB Instead, TusC has a conserved cysteine (Cys79) in the corresponding pocket of TusBC.18 Interestingly, Cys79 of TusC is part of a CXXC motif similar to that of YchN Therefore, the molecular function of the second putative active site of DsrEFH is thought to be quite different from that of TusBCD Discussion Here, we have shown via in-frame deletion mutagenesis and complementation that DsrEFH is an essential and central player in oxidative sulfur metabolism More specifically, it is an absolutely essential component for the oxidation of stored sulfur via the Dsr system, a pathway occurring not only in phototrophic but also in many chemotrophic sulfuroxidizing bacteria Our structural characterization clearly places DsrEFH into the YchN family Based on the structure of E coli YchN,15 several broad putative molecular functions such as peroxiredoxin, oxidoreductase, and hydrolase have originally been proposed for members of that family In addition, an involvement in sulfur metabolism has been suggested for MTH1491 from M thermoautotrophicum mainly on the basis of gene arrangement.17 As outlined above, our comparison of primary to quaternary structures of DsrEFH and E coli TusBCD revealed a number of common characteristics (Fig 6) and the sulfur transferase activity of TusBCD14 finally provided a more direct clue to understand the possible molecular function of DsrEFH TusBCD is involved in 2-thiouridine biosynthesis in bacterial tRNAs In a first step, the cysteine desulfurase IscS obtains sulfur from L-cysteine and this sulfur is then transferred to the protein TusA (former gene name in the E coli genome: yhhP) Sulfur-activated TusA then binds to TusBCD (encoded by the former genes yheLMN), in which TusD accepts sulfur from the TusA persulfide TusD transfers persulfide sulfur to TusE (encoded by the former yccK), which finishes 2-thiouridine biosynthesis with the aid of MnmA The residues comprising the putative active site of DsrE are exactly identical with those of TusD (Fig 6b) It is therefore well possible that the principal molecular function of A vinosum DsrE and E coli TusD is conserved and a sulfur transferase activity of DsrEFH through the conserved cysteine located on the primary active site appears likely This assumption is strongly supported by our finding that the active-site cysteine of DsrE is absolutely essential for interaction in vitro with DsrC (Fig 8) As mentioned above, TusD in TusBCD forms persulfide and then transfers persulfide sulfur to TusE Not surprisingly, TusE is a homologue of DsrC.8,14 Since TusE and DsrC also share a conserved carboxy-terminal cysteine, their molecular role may be similar too A persulfide of the penultimate conserved cysteine of DsrC has been proposed to be an important intermediate not only during the production of sulfite by the reverseacting sulfite reductase of A vinosum6 but also during the reduction of sulfite in a sulfate reducer.13 Besides the many common characteristics, important differences between DsrEFH and TusBDC are also apparent Structural as well as phylogenetic analyses of DsrEFH revealed two different types of putative active sites, one involving Cys78 of DsrE and the other involving Cys20 of DsrH The presence of these two sites clearly differentiates DsrEFH from the enterobacterial TusBCD Furthermore, the large insertion on loop L5 and the different surface structure of DsrF may have an influence on selecting partner molecules of DsrEFH, which is known not only to interact with DsrC6 but also to form a supercomplex with other Dsr proteins.7 In summary, the observed differences may contribute to the divergent cellular functions of DsrEFH and TusBCD, that is, sulfur oxidation versus thiouridine biosynthesis, of the two proteins though their subunits, and overall structures are conserved Structure and Role of DsrEFH 1295 Fig Structural superposition of DsrEFH (green) with TusBCD (red) (a) An asterisk indicates a location of a putative active site of DsrE and TusD The figure is created by the program PyMOL (red, negative; blue, positive).28 (b) A structural superposition of residues comprising the putative active sites of DsrEFH and TusBCD The residues' numbers are the same for both structures In conclusion, the results presented here indicate an important and so far largely neglected role of sulfur transfer reactions during sulfur oxidation via the Dsr proteins The structural characteristics of DsrEFH support the recently suggested model that suggests DsrEFH and DsrC to be agents transferring sulfur to dsrAB-encoded sulfite reductase.6 DsrE of DsrEFH could be the acceptor for persulfide sulfur originating from sulfur stored in the periplasm In analogy to the related E coli protein and according to our mutational analysis, DsrC may accept a sulfur atom from Cys78 of DsrE As pointed out before,6 1296 Structure and Role of DsrEFH Fig Diagrams of the electronic surface potential of DsrEFH and TusBCD The molecular surface around the second putative active pocket is drawn The figure is created by the program GRASP (red, negative; blue, positive; white, uncharged).29 (a) DsrEFH, (b) TusBCD 1297 Structure and Role of DsrEFH Fig Band-shift assay of DsrC with wild-type and mutant DsrC proteins DsrC (200 pmol) was incubated with 100 pmol of the respective DsrEFH protein as described in Materials and Methods Shifted bands are clearly visible for wild-type DsrEFH and DsrEFH20 (carrying the DsrH-Cys20Ser mutation) but not present in samples with DsrE78FH (carrying the DsrE-Cys78Ser mutation) or DsrE78FH20 (carrying both mutations) The proteins carrying the DsrE-Cys78Ser mutation show a slight but reproducible decrease of electrophoretic mobility (compare lanes 1, 2, 5, and with lanes 3, 4, 7, and 8) the persulfide sulfur could be reductively released as a substrate for sulfite reductase, possibly by formation of a disulfide between the two conserved cysteine residues of DsrC (in contrast to DsrC, TusE has only one conserved carboxy-terminal cysteine) Another possibility is that a DsrC persulfide acts as an even more direct substrate-donating molecule for sulfite reductase by immediately accessing the active-site siroheme just as has been documented for the D vulgaris protein.13 Although the structural characterization of DsrC6 and DsrEFH already allowed refinement of the models for the widespread Dsr pathway of sulfur oxidation, more work is clearly necessary to exactly follow the fate of sulfur from the periplasmic sulfur globules into the cytoplasm and further until it is finally excreted as sulfate Materials and Methods Bacterial strains, media, and growth conditions E coli strains DH5α [F−ϕ80dlacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (r−Km+K) supE44 λ−thi-1 gyrA relA1]30 and S17-1 [294 (recA pro res mod+) Tpr Smr (pRP42-Tc∷Mu-Km∷Tn7)]31 were cultivated on LB medium.32 For identification of recombinant plasmids containing inserts in the α portion of lacZ, IPTG and X-Gal were added to the medium Growth conditions for A vinosum strains were set as described earlier.33 A vinosum Rif50,5 a spontaneous rifampicin-resistant mutant of A vinosum DSM 180T, as well as A vinosum ΔdsrEFH (Rifr, 1071 bp deletion of dsrEFH), ΔdsrE (Rifr, 403 bp deletion of dsrE), and A vinosum ΔdsrE+ dsrEFH (strain ΔdsrE carrying complementation plasmid pBBR1MCS2-EFH) were grown photoorganoheterotrophically on malate (RCV medium34), using trace element solution SL12.35 For solidification of the medium, 1% (w/v) Phytagel (P8169, Sigma) was added, as well as 0.5% NaCl to aid gelling and 0.02% (w/v) Na2S2O3 × H2O, mM sodium acetate, and 2.6 ml feeding solution (for 100 ml: 3.1 g NaSH × H2O, 5.0 g NaHCO3) for growth enhancement The plates were used directly after solidification and were always prepared freshly Photolithoautotrophic growth was achieved by using a sulfide-containing medium based on Pfennig's medium36 prepared as follows: for 10 l of medium, 8000 ml solution (3.3 g KCl, 3.3 g MgCl2·6 H2O, 4.3 g CaCl2·2 H2O, 3.3 g NH4Cl, and 10 ml trace element solution SL12), 1000 ml solution containing 3.3 g KH2PO4, and 1000 ml solution containing 15 g NaHCO3 were autoclaved separately After cooling, solutions and were added to solution under stirring and nitrogen atmosphere The medium was saturated with CO2 by stirring under CO2 atmosphere until the medium turned clear The pH was adjusted to 6.6– 6.7 The medium was distributed aseptically into 1000-ml screw-capped bottles.36 Sulfide was added with the inoculum Antibiotics were used at the following concentrations (in micrograms per milliliter): for E coli, kanamycin, 50; for A vinosum, kanamycin, 10; rifampicin, 50 Recombinant DNA techniques Standard methods were used for molecular biological techniques.32,37 Chromosomal DNA of A vinosum strains was obtained as described previously.20 Southern hybridizations were performed overnight at 68 °C as described earlier.38 DNA probes for Southern experiments were digoxigenin-labeled by polymerase chain reaction (PCR).39 PCRs with Taq DNA polymerase were done essentially as described previously.40 The GC-RICH PCR System (12140306001, Roche) was used, when the amplicon was designated for the construction of deletion plasmids GC-RICH PCR System reactions were set up according to the protocol supplied by Roche Construction of in-frame deletions and complementations in A vinosum The in-frame deletion of dsrEFH or dsrE alone was achieved by utilizing the gene splicing by overlap extension PCR41 using the following primers for the dsrEFH deletion: EXbaf1 (5′-aatgcgtgtctagatcgagtccggctgtc-3′), erev1 (5′-ctcggtcgtcagatcgttgatctgaagcgc-3′), Efor1 (5′-gcgcttcagatcaaggatctgacgaccgag-3′), and eXbar1 (5′-gcgctcgatctagacggctcatgactgctcg-3′) and the following primers for the dsrE deletion: EXbaf1, erev2 (5′-tccgacatgttggcgcctcgtggctatcca-3′), Efor2 (5′-tggatagccacgaggcgccaacatgtc-3′), and eXbar1 An XbaI restriction site was introduced to the 5′ and 3′ ends of the final deletion framing PCR fragment The PCR amplicons were cloned into the XbaI site of the mobilizable suicide vector pK18mobsacB (Kmr, lacZ`, sacB, Mob+).42 The resulting plasmids were pK18mobsacBΔdsrEFH (Kmr, XbaI fragment of PCR-amplified genome region surrounding a 1071-bp deletion of dsrEFH) and pK18mobsacBΔdsrE (Kmr, XbaI fragment of PCR-amplified genome region surrounding a 403-bp deletion of dsrE) Conjugative transfer of plasmids from E coli into A vinosum Rif50, procedures for achieving gene exchange, and verification of genotypes were performed as described earlier.5 Site-directed mutagenesis Point mutations were introduced into dsrE and/or dsrH by gene splicing by overlap extension41 using standard PCR with Pfu DNA polymerase (Fermentas, St Leon-Rot) and pETEFH as the template In pETEFH, the A vinosum dsrEFH genes are cloned between the NdeI and BamHI sites of pET15b (Novagen).27 For the DsrECys78Ser 1298 exchange, two fragments were amplified with the following primers: for the first fragment, dsrE78for (5′-CGTGCAGGGCCAGACTGG-3′) and dsrE78Crev⁎ (5′-GGCGACTGAGACGACCATG-3′); for the second fragment, dsrE78Cfor⁎ (5′-ATGGTCGTCTCAGTCGCCG-3′) and T7Term (5′-GCTAGTTATTGCTCAGCGG-3′) Both fragments were used as templates for amplification of the complete dsrEFH genes, with dsrE carrying the desired point mutation In this step, dsrE78for and T7Term served as primers The resulting fragment was restricted with NdeI and BamHI and cloned into pET15b, resulting in plasmid pETE78FH The DsrHCys20Ser exchange was done accordingly using primers T7Prom (5′-TAATACGACTCACTATAGGG-3′) and dsrH20Crev⁎ (5′-AAACTTCAGGGAGGATTCTAAC-3′) for the first fragment, dsrH20Cfor⁎ (5′-TTAGAATCCTCCCTGAAGTTTG-3′) and dsrH20rev (5′-GGAAAACGTTCTTCGGGG-3′) for the second fragment, and a combination of T7Prom and dsrH20rev for the third fragment The resulting plasmid was termed pETEFH20 For the construction of the double mutation, plasmid pETE78FH was used as the template for the introduction of the DsrHCys20Ser mutation using primers dsrH20rev⁎ and dsrH20for⁎, resulting in plasmid pETE78FH20 All mutations were verified by nucleotide sequencing Wild-type and mutated proteins were overproduced with an amino-terminal His tag in E coli BL21 (DE3) and purified as described earlier.27 Construction of complementation plasmids The conjugative broad host range vector pBBR1-MCS243 was used to reintroduce the dsrEFH genes under the control of the original dsr promoter into the ΔdsrEFH mutant The dsrEFH-containing fragment of the plasmid used for production of DsrEFH in E coli27 was retrieved by digestion with NdeI and BamHI and cloned between NdeI and BamHI of pBBR1MCS2-L.5 Conjugative transfer of plasmids from E coli into A vinosum Rif50, procedures for achieving gene exchange, and verification of genotypes were performed as described earlier.5 Phenotypic characterization of A vinosum in-frame deletion and complementation strains Photolithoautotrophic growth of A vinosum wild-type and mutant strains was examined in batch culture under continuous illumination essentially as described by Prange et al.20 One liter of a photoheterotrophically grown stationary-phase culture was harvested (5900g, 10 min), and the cell material was used to inoculate l of modified Pfennig's medium in a thermostated fermenter The experiments were started by the addition of mM sulfide from a sterile stock solution (1 M) Sulfur compounds (sulfide, polysulfides, sulfite, thiosulfate, and sulfate) were detected and determined by HPLC (Thermo Separation Products) using the methods of Rethmeier et al.44 Elemental sulfur was determined by cyanolysis.45 Phylogenetic analysis Nucleotide sequences were compiled with the Clone Manager (SEC central) software Similarity searches were conducted using the BLAST algorithm.46 Global multiple sequence alignments were generated using ClustalX.47 Phylogenetic analysis was performed in PAUP⁎ 4.0 bl0.48 Bootstrap support49 was estimated with 10,000 replicates Unweighted maximum parsimony trees were determined Structure and Role of DsrEFH by the branch-and-bound algorithm or by heuristic search using 100 replicates Since PAUP⁎ does not provide an algorithm to calculate maximum likelihood trees based on protein sequences, protein maximum likelihood trees were calculated using the PROML program50 included in the sequence alignment editor Bioedit.51 The Bayesian analysis was done with MrBayes52 version 3.0b4 (four chains, 50,000 generations, a sample frequency of 100, and a burn-in set to 100 trees, rates = invgamma) Trees were drawn with TreeView version 1.6.5.53 Structure determination and refinement The cloning, purification, crystallization, and preliminary X-ray studies have been published elsewhere.27 Out of 48 possible Se atom positions, 24 were located using a new substructure searching procedure, Hybrid Substructure Search, included in the PHENIX software.54 The initial single-wavelength anomalous dispersion phases were obtained using the program SOLVE55 with these sites having a figure of merit of 0.47 at 2.8 Å resolution The single-wavelength anomalous dispersion phases were further improved by solvent flattening using the program RESOLVE.55 The initial electron density map was not interpretable However, noncrystallographic symmetry matrices were manually obtained using strong selenium atom peaks and were again applied in the program RESOLVE The best interpretable map was calculated using data between 20.0 and 2.8 Å resolution with figure of merit increased to 0.71 There were three heterohexameric molecules in the asymmetric unit The preliminary model was built and refined using the program CNS56 with a stepwise addition of residues using the O program23 The noncrystallographic symmetry restraint has been applied during refinement and released at the final round of refinement The reflection data between 20.0 and 2.5 Å were included throughout the refinement calculations Ten percent of the data were randomly chosen for free R-factor cross validation The refinement statistics are shown in Table Isotropic B-factors for individual atoms were initially fixed to 20 Å2 and were refined in the last stages The 2Fo − Fc and Fo − Fc maps were used for the manual rebuilding between refinement cycles and for the location of solvent molecules In the final model, some flexible N-terminal residues, the first methionine of DsrH and the first four residues of DsrF, are not included In addition, the eight residues between 103 and 110 of DsrF, which not have any contact with neighboring molecules, have high B-factors Therefore, these residues were undefined in the electron density map and were not included in the final model too Tyr21 of DsrE and Tyr21 of DsrF lie in the disallowed region of the Ramachandran plot produced with PROCHECK.24 Therefore, a total of 12 residues in the asymmetric unit lie in the disallowed region Accession code Atomic coordinates have been deposited in the Protein Data Bank (PDB accession code: 2HYB) Acknowledgements Skillful technical assistance by Hisao Yokota, Jaru Jancarik, and Birgitt Hüttig is gratefully acknowl- 1299 Structure and Role of DsrEFH edged The biochemical and genetic parts of this research were supported by the Deutsche Forschungsgemeinschaft (grants Da 351/3-3, 3-4, and 3-5 to C.D.) The crystallographic part described here was supported by the Korea Research Foundation Grant funded by the Korean Government (Ministry of Education and Human Resource Development, Basic Research Promotion Fund, KRF2007-313-C00618), by Grant No R15-2006-020 from the National Core Research Center program of the Ministry of Science and Technology and Korea Science and Engineering Foundation through the Center for Cell Signaling and Drug Discovery Research at Ewha Womans University, and by the Protein Structure Initiative grant from National Institutes of Health GM 62412 References Pattaragulwanit, K., Brune, D C., Trüper, H G & Dahl, C (1998) Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum Arch Microbiol 169, 434–444 Dahl, C & Prange, A (2006) Bacterial sulfur globules: occurrence, structure and metabolism In Inclusions in Prokaryotes (Shively, J M., ed), pp 21–51, Springer, Berlin, Germany Frigaard, N.-U & Dahl, C (2008) Sulfur 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EXbaf1 (5′-aatgcgtgtctagatcgagtccggctgtc-3′), erev1 (5′-ctcggtcgtcagatcgttgatctgaagcgc-3′), Efor1 (5′-gcgcttcagatcaaggatctgacgaccgag-3′), and eXbar1 (5′-gcgctcgatctagacggctcatgactgctcg-3′) and. .. (5′-TAATACGACTCACTATAGGG-3′) and dsrH20Crev⁎ (5′-AAACTTCAGGGAGGATTCTAAC-3′) for the first fragment, dsrH20Cfor⁎ (5′-TTAGAATCCTCCCTGAAGTTTG-3′) and dsrH20rev (5′-GGAAAACGTTCTTCGGGG-3′) for the second fragment, and a combination... chemical data, genetic studies with A vinosum, and genome comparisons indicate that in all these organisms as well as in green sulfur bacteria and thiobacilli, a complicated pathway is at work,