Journal of Molecular Biology
Volume 384, Issue 5, 31 December 2008, Pages 1287-1300
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Structural and Molecular Genetic Insight into a Widespread Sulfur Oxidation Pathway

https://doi.org/10.1016/j.jmb.2008.10.016Get rights and content

Abstract

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.

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-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 Biochemical 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, 5, 6, 7, 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, 10, 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 thiol-disulfide 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

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 three-dimensional 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.

Section snippets

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, indicating that its product is essential for central metabolic pathways in this organism.6 Therefore, we deleted solely dsrE, leaving the

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 sulfur-oxidizing bacteria.

Our structural characterization clearly places DsrEFH into the YchN family. Based on the structure of E. coli YchN,15 several broad

Bacterial strains, media, and growth conditions

E. coli strains DH5α [Fϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 (rKmK+) supE44 λthi-1 gyrA relA1]30 and S17-1 [294 (recA pro res mod+) Tpr Smr (pRP4-2-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

Acknowledgements

Skillful technical assistance by Hisao Yokota, Jaru Jancarik, and Birgitt Hüttig is gratefully acknowledged. 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, KRF-2007-313-C00618), by Grant

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