Novel genes of the dsr gene cluster and evidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum

Abstract

Seven new genes designated dsrLJOPNSR were identified immediately downstream of dsrABEFHCMK, completing the dsr gene cluster of the phototrophic sulfur bacterium Allochromatium vinosum D (DSM 180(T)). Interposon mutagenesis proved an essential role of the encoded proteins for the oxidation of intracellular sulfur, an obligate intermediate during the oxidation of sulfide and thiosulfate. While dsrR and dsrS encode cytoplasmic proteins of unknown function, the other genes encode a predicted NADPH:acceptor oxidoreductase (DsrL), a triheme c-type cytochrome (DsrJ), a periplasmic iron-sulfur protein (DsrO), and an integral membrane protein (DsrP). DsrN resembles cobyrinic acid a,c-diamide synthases and is probably involved in the biosynthesis of siro(heme)amide, the prosthetic group of the dsrAB-encoded sulfite reductase. The presence of most predicted Dsr proteins in A. vinosum was verified by Western blot analysis. With the exception of the constitutively present DsrC, the formation of Dsr gene products was greatly enhanced by sulfide. DsrEFH were purified from the soluble fraction and constitute a soluble alpha(2)beta(2)gamma(2)-structured 75-kDa holoprotein. DsrKJO were purified from membranes pointing at the presence of a transmembrane electron-transporting complex consisting of DsrKMJOP. In accordance with the suggestion that related complexes from dissimilatory sulfate reducers transfer electrons to sulfite reductase, the A. vinosum Dsr complex is copurified with sulfite reductase, DsrEFH, and DsrC. We therefore now have an ideal and unique possibility to study the interaction of sulfite reductase with other proteins and to clarify the long-standing problem of electron transport from and to sulfite reductase, not only in phototrophic bacteria but also in sulfate-reducing prokaryotes.

FIG. 1.

(A) Schematic overview of the dsr locus of A. vinosum and two related genes clusters in the green sulfur bacterium C. tepidum. The same names were given to clearly related genes. Predicted functions of gene products are indicated under the genes where appropriate. The positions of insertion sites of the kanamycin Ω interposon in the A. vinosum double-crossover mutants 21D and 39D are indicated. (B) Schematic presentation of the Dsr proteins from A. vinosum. The scheme is based on sequence analysis of the encoding genes and on biochemical information where available.

FIG. 2.

Western blot analysis of proteins fractions of A. vinosum with antibodies against the indicated Dsr proteins. Lanes: 1, crude extracts; 2, soluble fraction; 3, membrane fraction. Proteins of cell extracts (10 μg per lane) were separated by SDS-PAGE.

FIG. 3.

Sulfide-dependent formation of DsrE, DsrH, DsrC, DsrK, and DsrL in A. vinosum wild type and mutants 21D and 39D. A. vinosum was cultivated photoorganoheterotrophically in complex RCV medium in completely filled, closed 100-ml bottles. After the cells reached the late exponential phase, sulfide (4.2 mM) was added. Cells (2 ml) were collected by centrifugation 5 h after sulfide addition, resuspended with 100 μl of 20 mM Tris HCl (pH 7.5) and 50 μl of SDS sample buffer, and incubated at 100°C for 5 min. A 10-μl volume of each sample was subjected to SDS-PAGE and Western blot analysis with antibodies raised against the indicated proteins.

FIG. 4.

SDS-PAGE and Western blot analyses of DsrEFH from A. vinosum. A 1.5-μg portion of purified protein was used for SDS-PAGE and Western blot analyses, respectively.

FIG. 5.

SDS-PAGE and Western blot analyses of DsrK-containing fractions (5 μg of protein) after anion-exchange and hydroxyapatite chromatography.

FIG. 6.

Immunochemical analysis of a crude extract of a DsrJ-overproducing E. coli strain (E. coli HM125) containing pEC86 and pISC-2(dsrJ) after SDS-PAGE (lane 1); heme stain of a crude extract of E. coli HM125 containing pEC86 and pISC-2 (lane 2); heme stain of a crude extract of E. coli HM125 containing pEC86 and pISC-2(dsrJ) (lane 3).