Christiane Dahl

Sulfur Metabolism in Phototrophic Sulfur Bacteria

Phototrophic sulfur bacteria are characterized by oxidizing various inorganic sulfur compounds for use as electron donors in carbon dioxide fixation during anoxygenic photosynthetic growth. These bacteria are divided into the purple sulfur bacteria (PSB) and the green sulfur bacteria (GSB). They utilize various combinations of sulfide, elemental sulfur, and thiosulfate and sometimes also ferrous iron and hydrogen as electron donors. This review focuses on the dissimilatory and assimilatory metabolism of inorganic sulfur compounds in these bacteria and also briefly discusses these metabolisms in other types of anoxygenic phototrophic bacteria. The biochemistry and genetics of sulfur compound oxidation in PSB and GSB are described in detail. A variety of enzymes catalyzing sulfur oxidation reactions have been isolated from GSB and PSB (especially Allochromatium vinosum, a representative of the Chromatiaceae), and many are well characterized also on a molecular genetic level. Complete genome sequence data are currently available for 10 strains of GSB and for one strain of PSB. We present here a genome-based survey of the distribution and phylogenies of genes involved in oxidation of sulfur compounds in these strains. It is evident from biochemical and genetic analyses that the dissimilatory sulfur metabolism of these organisms is very complex and incompletely understood. This metabolism is modular in the sense that individual steps in the metabolism may be performed by different enzymes in different organisms. Despite the distant evolutionary relationship between GSB and PSB, their photosynthetic nature and their dependency on oxidation of sulfur compounds resulted in similar ecological roles in the sulfur cycle as important anaerobic oxidizers of sulfur compounds.

Quantitative speciation of sulfur in bacterial sulfur globules: X-ray absorption spectroscopy reveals at least three different species of sulfur.

X-ray absorption near edge structure (XANES) spectroscopy at the sulfur K-edge was applied to probe the speciation of sulfur of metabolically different sulfur-accumulating bacteria in situ. Fitting the spectra using a least-square fitting routine XANES reveals at least three different forms of sulfur in bacterial sulfur globules. Cyclooctasulfur dominates in the sulfur globules of Beggiatoa alba and the very recently described giant bacterium Thiomargarita namibiensis. A second type of sulfur globules is present in Acidithiobacillus ferrooxidans: here the sulfur occurs as polythionates. In contrast, in purple and green sulfur bacteria the sulfur mainly consists of sulfur chains, irrespective of whether it is accumulated in globules inside or outside the cells. These results indicate that the speciation of sulfur in the sulfur globules reflects the different ecological and physiological properties of different metabolic groups of bacteria.

Thiosulphate oxidation in the phototrophic sulphur bacterium Allochromatium vinosum

Two different pathways for thiosulphate oxidation are present in the purple sulphur bacterium Allochromatium vinosum: oxidation to tetrathionate and complete oxidation to sulphate with obligatory formation of sulphur globules as intermediates. The tetrathionate:sulphate ratio is strongly pH‐dependent with tetrathionate formation being preferred under acidic conditions. Thiosulphate dehydrogenase, a constitutively expressed monomeric 30 kDa c‐type cytochrome with a pH optimum at pH 4.2 catalyses tetrathionate formation. A periplasmic thiosulphate‐oxidizing multienzyme complex (Sox) has been described to be responsible for formation of sulphate from thiosulphate in chemotrophic and phototrophic sulphur oxidizers that do not form sulphur deposits. In the sulphur‐storing A. vinosum we identified five sox genes in two independent loci (soxBXA and soxYZ). For SoxA a thiosulphate‐dependent induction of expression, above a low constitutive level, was observed. Three sox‐encoded proteins were purified: the heterodimeric c‐type cytochrome SoxXA, the monomeric SoxB and the heterodimeric SoxYZ. Gene inactivation and complementation experiments proved these proteins to be indispensable for thiosulphate oxidation to sulphate. The intermediary formation of sulphur globules in A. vinosum appears to be related to the lack of soxCD genes, the products of which are proposed to oxidize SoxY‐bound sulphane sulphur. In their absence the latter is instead transferred to growing sulphur globules.

Dissimilatory sulphite reductase from Archaeoglobus fulgidus: physico-chemical properties of the enzyme and cloning, sequencing and analysis of the reductase genes

A dissimilatory sulphite reductase was isolated from the extremely thermophilic dissimilatory sulphate-reducing archaeon Archaeoglobus fulgidus. In common with other dissimilatory sulphite reductases thus far characterized, the enzyme has an alpha 2 beta 2-structure and contains sirohaem, non-haem iron atoms and acid labile sulphide. The oxidized enzyme exhibited absorption maxima at 281, 394, 545 and 593 nm with a weak band around 715 nm. We have cloned and sequenced the genes for the alpha and beta subunits of this enzyme, which we designate dsrA and dsrB, respectively. They are contiguous in the order dsrA dsrB and probably comprise an operon, since dsrA is preceded by sequences characteristic of promoters in methanogenic archaea, and dsrB is followed by a sequence resembling termination signals in extremely thermophilic sulphur-dependent archaea. dsrA and dsrB encode 47.4 kDa and 41.7 kDa peptides, which have 25.6% amino acid sequence identity, indicating that they may have arisen by duplication of an ancestral gene. Each deduced peptide contains cysteine clusters resembling those postulated to bind sirohaem-[Fe4S4] complexes in sulphite reductases and nitrite reductases from other species. The dsrB encoded peptide lacks a single cysteine residue in one of the two clusters, suggesting that only the alpha subunit binds a sirohaem-[Fe4S4] complex, and chemical analyses showed the presence of only two sirohaems per alpha 2 beta 2 enzyme molecule. Both deduced peptides also contain an arrangement of cysteine residues characteristic of [Fe4S4] ferredoxins, and chemical analyses were consistent with the presence of six [Fe4S4] clusters per alpha 2 beta 2 enzyme molecule, two of which would be expected to be associated with sirohaem while the other four could bind to the ferredoxin-like sites.

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Abstract Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum.

Reverse dissimilatory sulfite reductase as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes.

Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of dsrAB that encode the reversely operating sirohaem dissimilatory sulfite reductase (rDSR) and are present in many but not all published genomes of SOP. The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.

Sulfur metabolism in phototrophic organisms

Editorial.-Contents.-Preface-Author Index.-Colour Plates.-I. Sulfate Activation And Reduction, Biosynthesis Of Sulfur Containing Amino Acids.- 1.Introduction To Sulfur Metabolism In Phototrophic Organisms Christiane Dahl, Rudiger Hell,David Knaff, Thomas Leustek.-2.Uptake, Allocation And Subcellular Transport Of Sulfate Malcolm J. Hawkesford .-3.Phylogenetic Analysis Of Sulfate Assimilation And Cysteine Biosynthesis In Phototrophic Organisms Stanislav Kopriva , Nicola Patron, Tom Leustek, Patrick Keeling.-4.Metabolism Of Cysteine In Plants And Phototrophic Bacteria Rudiger Hell, Markus Wirtz .-5.Metabolism Of Methionine In Plants And Phototrophic Bacteria Rainer Hoefgen, Holger Hesse.-6.Sulfotransferases From Plants, Algae And Phototrophic Bacteria Cinta Hernandez-Sebastia, Frederic Marsolais, Luc Varin.-7.Cysteine Desulfurase-Mediated Sulfur Donation Pathways In Plants And Phototrophic Bacteria Lolla Padmavathi, Hong Ye, Elizabeth AH Pilon-Smits, Marinus Pilon .-II Sulfur In Plants And Algae:8.Molecular Biology And Functional Genomics For Identification Of Regulatory Networks Of Plant Sulfate Uptake And Assimilatory Metabolism Hideki Takahashi, Kazuki Saito.-9.Biosynthesis, Compartmentation And Cellular Functions Of Glutathione In Plant Cells Andreas Meyer, Thomas Rausch .-10.Sulfolipid Biosynthesis And Function In Plants Christoph Benning, R. Michael Garavito, Mie Shimojia.- 11.Sulfur-Containing Secondary Metabolites And Their Role In Plant Defense Meike Burow, Jonathan Gershenzon, Ute Wittstock.-12.Sulfite Oxidation In Plants Robert Hansch, Ralf R. Mendel.-13.The State Of Sulfur Metabolism In Algae: From Ecology To Genomics Nakako Shibagaki, Arthur Grossman.- III. Sulfur In Phototrophic Prokaryotes:14.Systematics Of Anoxygenic Phototrophic Bacteria Johannes Imhoff.-15.InorganicSulfur Compounds As Electron Donors In Purple Sulfur Bacteria Christiane Dahl.-16.Sulfide Oxidation From Cyanobacteria To Humans: Sulfide-Quinone Oxidoreductase Yosepha Shahak, Gunther Hauska.-17.Genomic Insights Into The Sulfur Metabolism Of Phototrophic Green Sulfur Bacteria Niels-Ulrik Frigaard, Don Bryant.-18.Genetic And Proteomic Studies Of Sulfur Oxidation In Chlorobium Tepdium Chlorobium Tepidum Leong-Keat Chan, Rachael Morgan-Kiss, Thomas E. Hanson.-IV. Ecology And Biotechnology: 19.Ecology Of Phototrophic Sulfur Bacteria Jorg Overmann.-20.Role Of Sulfur For Algae: Acquisition, Metabolism, Ecology And Evolution Mario Giordano, Alessandra Norici, Simona Ratti, John A. Raven.-21.Role Of Sulfur For Plant Production In Agricultural And Natural Ecosystems Fangjie Zhao, Michael Tausz , Luit De Kok.-22. Using Anoxygenic Photosynthetic Bacteria For The Removal Of Sulfide From Wastewater Timothy J. Hurse, Ulrike Kappler, Jurg Keller.-V. Specific Methods: 23.X-Ray Absorption Spectroscopy As Tool For The Detection And Identification Of Sulfur Compounds In Phototrophic Organisms Alexander Prange, Josef Hormes, Hartwig Modrow.-24.Imaging Thiol-Based Redox Processes In Live Cells Andreas Meyer, Mark D. Fricker.-Index

Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus)

Abstract. The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.

A dissimilatory sirohaem-sulfite-reductase-type protein from the hyperthermophilic archaeon Pyrobaculum islandicum

A sulfite-reductase-type protein was purified from the hyperthermophilic crenarchaeote Pyrobaculum islandicum grown chemoorganoheterotrophically with thiosulfate as terminal electron acceptor. In common with dissimilatory sulfite reductases the protein has an alpha 2 beta 2 structure and contains high-spin sirohaem, non-haem iron and acid-labile sulfide. The oxidized protein exhibits absorption maxima at 280, 392, 578 and 710 nm with shoulders at 430 and 610 nm. The isoelectric point of pH 8.4 sets the protein apart from all dissimilatory sulfite reductases characterized thus far. The genes for the alpha- and beta-subunits (dsrA and dsrB) are contiguous in the order dsrAdsrB and most probably comprise an operon with the directly following dsrG and dsrC genes. dsrG and dsrC encode products which are homologous to eukaryotic glutathione S-transferases and the proposed gamma-subunit of Desulfovibrio vulgaris sulfite reductase, respectively. dsrA and dsrB encode 44.2 kDa and 41.2 kDa peptides which show significant similarity to the two homologous subunits DsrA and DsrB of dissimilatory sulfite reductases. Phylogenetic analyses indicate a common protogenotic origin of the P. islandicum protein and the dissimilatory sulfite reductases from sulfate-reducing and sulfide-oxidizing prokaryotes. However, the protein from P. islandicum and the sulfite reductases from sulfate-reducers and from sulfur-oxidizers most probably evolved into three independent lineages prior to divergence of archaea and bacteria.

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum

Abstract Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180T). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling σ70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which – when compared to the isolated proteins – carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Δp that drives ATP synthesis and reverse electron flow in Chr. vinosum.