Rhodanese-Mediated sulfur transfer to succinate dehydrogenase.

The interaction of the sulfurtransferase rhodanese (EC 2.8.1.1) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied. Succinate dehydrogenase incorporates the sulfane sulfur of [35S]rhodanese and, in the presence of unlabelled rhodanese, also incorporates that of [35S]thiosulfate. Rhodanese releases most of its transferable sulfur and is re-loaded in the presence of thiosulfate. Rhodanese undergoes similar modifications with yeast alcohol dehydrogenase but this latter does not bind 35S in amounts comparable to those incorporated in succinate dehydrogenase: nearly all the 35S released by [35S]rhodanese is with low-molecular-weight compounds. Bovine serum albumin also binds very little sulfur and [35S]rhodanese present in the reaction mixture does not discharge its radioactive sulfur nor does it take up sulfur from thiosulfate. Sulfur release from rhodanese appears to depend on the presence of - SH groups in the acceptor protein. Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavorprotein. The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation. The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Q_y absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. One-color and two-color pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.     

Cytoplasmic sulfurtransferases in the purple sulfur bacterium Allochromatium vinosum: evidence for sulfur transfer from DsrEFH to DsrC

While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium Allochromatium vinosum. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the "Dsr" (dissimilatory sulfite reductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from E. coli. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the A. vinosum Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 in vitro. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC in vitro. The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation in vivo supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.     

Excitation energy transfer kinetics and efficiency in phototrophic green sulfur bacteria

A series of spectroscopic measurements were performed on membrane fractions and detergent-solubilized complexes from the green sulfur bacterium (GSB) Chlorobaculum (Cba.) tepidum. The excitation migration through the entire GSB photosynthetic apparatus cannot be observed upon excitation of membranes in the chlorosome region at 77?K. In order to observe energy transfer from the Fenna-Matthews-Olson (FMO) protein to the reaction center (RC), FMO was directly excited at ~800?nm in transient absorption experiments. However, interpretation of the results is complicated by the spectral overlap between FMO and the RC. The availability of the Y16F FMO mutant, whose absorption spectrum is drastically different from that of the WT, has enabled the selection of spectral regions where either only FMO or the RC contributes. The application of a directed kinetic modeling approach, or target analysis, revealed the various decay and energy transfer pathways within the pigment-protein complexes. The calculated FMO-to-RC excitation energy transfer efficiencies are approximately 25% and 48% for the Y16F and WT samples, respectively.     

Synthesis of oligodeoxyribonucleoside phosphorothioates using Lawesson's reagent for the sulfur transfer step

2,4-Bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent) efficiently converts the triphosphite intermediate used in the solid-phase synthesis of oligonucleotides into an oligodeoxyribonucleoside phosphorothioate.     

Role of conserved cysteines in mediating sulfur transfer from IscS to IscU

The role of the three conserved cysteine residues on Azotobacter vinelandii IscU in accepting sulfane sulfur and forming a covalent complex with IscS has been evaluated using electrospray-ionization mass spectrometry studies of variants involving individual cysteine-to-alanine substitutions. The results reveal that IscS can transfer sulfur to each of the three alanine-substituted forms of IscU to yield persulfide or polysulfide species, and formation of a heterodisulfide covalent complex between IscS and Cys(37) on IscU. It is concluded that S transfer from IscS to IscU does not involve a specific cysteine on IscU or the formation of an IscS-IscU heterodisulfide complex.     

Biosynthesis of the thioquinolobactin siderophore: an interesting variation on sulfur transfer

The thioquinolobactin siderophore from Pseudomonas fluorescens ATCC 17400 utilizes a variation of the sulfur transfer chemistry found in thiamine and molydobterin biosynthesis. A JAMM motif protein cleaves the C-terminal amino acid residues following a diglycine moiety on a small sulfur carrier protein, and the modified C terminus is activated and sulfurylated, forming a thiocarboxylate.     

Oxygen transfer and consumption in a thiosulfate oxidizing bioreactor with sulfur production

AIMS: To evaluate the contribution of oxygen transfer and consumption in a sulfoxidizing system to increase the elemental sulfur yield from thiosulfate oxidation. METHODS AND RESULTS: A 10 l thiosulfate oxidizing bioreactor with suspended cells operating under microaerophilic conditions and a separated aerator with a variable volume of 0.8--1.7 l were operated with a consortium containing mainly Thiobacillus sp. that oxidizes several sulfide species to elemental sulfur and sulfate. From the gas-liquid oxygen balance, the k(L)a was estimated under different operation conditions. A k(L)a of around 200 h(-1) favoured elemental sulfur production and can serve as scale-up criterion. It was further shown that more than 50% of the oxygen fed to the system was consumed in the aerator. CONCLUSIONS: The performance of the sulfoxidizing system can be improved by controlling oxygen transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method for the k(L)a determination was based on the oxygen balance, which incorporates the oxygen concentrations measured in the liquid in steady state, reducing the interference of the response time in the traditional non-steady state methods. This approach can be used to optimize reactors where microaerophilic conditions are desirable.     

Theoretical characterization of excitation energy transfer in chlorosome light-harvesting antennae from green sulfur bacteria

We present a theoretical study of excitation dynamics in the chlorosome antenna complex of green photosynthetic bacteria based on a recently proposed model for the molecular assembly. Our model for the excitation energy transfer (EET) throughout the antenna combines a stochastic time propagation of the excitonic wave function with molecular dynamics simulations of the supramolecular structure and electronic structure calculations of the excited states. We characterized the optical properties of the chlorosome with absorption, circular dichroism and fluorescence polarization anisotropy decay spectra. The simulation results for the excitation dynamics reveal a detailed picture of the EET in the chlorosome. Coherent energy transfer is significant only for the first 50 fs after the initial excitation, and the wavelike motion of the exciton is completely damped at 100 fs. Characteristic time constants of incoherent energy transfer, subsequently, vary from 1 ps to several tens of ps. We assign the time scales of the EET to specific physical processes by comparing our results with the data obtained from time-resolved spectroscopy experiments.     

How proteins trigger excitation energy transfer in the FMO complex of green sulfur bacteria

A simple electrostatic method for the calculation of optical transition energies of pigments in protein environments is presented and applied to the Fenna-Matthews-Olson (FMO) complex of Prosthecochloris aestuarii and Chlorobium tepidum. The method, for the first time, allows us to reach agreement between experimental optical spectra and calculations based on transition energies of pigments that are calculated in large part independently, rather than fitted to the spectra. In this way it becomes possible to understand the molecular mechanism allowing the protein to trigger excitation energy transfer reactions. The relative shift in excitation energies of the seven bacteriochlorophyll-a pigments of the FMO complex of P. aestuarii and C. tepidum are obtained from calculations of electrochromic shifts due to charged amino acids, assuming a standard protonation pattern of the protein, and by taking into account the three different ligand types of the pigments. The calculations provide an explanation of some of the earlier results for the transition energies obtained from fits of optical spectra. In addition, those earlier fits are verified here by using a more advanced theory of optical spectra, a genetic algorithm, and excitonic couplings obtained from electrostatic calculations that take into account the influence of the dielectric protein environment. The two independent calculations of site energies strongly favor one of the two possible orientations of the FMO trimer relative to the photosynthetic membrane, which were identified by electron microscopic studies and linear dichroism experiments. Efficient transfer of excitation energy to the reaction center requires bacteriochlorophylls 3 and 4 to be the linker pigments. The temporal and spatial transfer of excitation energy through the FMO complex is calculated to proceed along two branches, with transfer times that differ by an order of magnitude.     

Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum.

The broad-host-range IncQ group plasmids pDSK519 and pGSS33 were transferred by conjugation from Escherichia coli into the thermophilic green sulfur bacterium Chlorobium tepidum. C. tepidum exconjugants expressed the kanamycin and ampicillin-chloramphenicol resistances encoded by pDSK519 and pGSS33, respectively. Ampicillin resistance was a particularly good marker for selection in C. tepidum. Both pDSK519 and pGSS33 were stably maintained in C. tepidum at temperatures below 42 degrees C and could be transferred between C. tepidum and E. coli without modifications. Conjugation frequencies ranged from 10(-1) to 10(-4) exconjugants per donor cell, and frequencies of 10(-2) to 10(-3) were consistently obtained when ampicillin resistance was used as a selectable marker. Methods for growth of C. tepidum on agar, isolation of plating strains and antibiotic-resistant mutants of wild-type C. tepidum cells, and optimum conditions for conjugation were also investigated.     

Thio-modification of yeast cytosolic tRNA requires a ubiquitin-related system that resembles bacterial sulfur transfer systems

The wobble uridine in yeast cytosolic tRNA(Lys2)(UUU) and tRNA(Glu3)(UUC) undergoes a thio-modification at the second position (s(2) modification) and a methoxycarbonylmethyl modification at the fifth position (mcm(5) modification). We previously demonstrated that the cytosolic and mitochondrial iron-sulfur (Fe/S) cluster assembly machineries termed CIA and ISC, including a cysteine desulfurase called Nfs1, were essential for the s(2) modification. However, the cytosolic component that directly participates in this process remains unclear. We found that ubiquitin-like protein Urm1 and ubiquitin-activating enzyme-like protein Uba4, as well as Tuc1 and Tuc2, were strictly required for the s(2) modification. The carboxyl-terminal glycine residue of Urm1 was critical for the s(2) modification, indicating direct involvement of the unique ubiquitin-related system in this process. We also demonstrated that the s(2) and mcm(5) modifications in cytosolic tRNAs influence each other's efficiency. Taken together, our data indicate that the s(2) modification of cytosolic tRNAs is a more complex process that requires additional unidentified components.     

C-type cytochromes in the photosynthetic electron transfer pathways in green sulfur bacteria and heliobacteria

Green sulfur bacteria and heliobacteria are strictly anaerobic phototrophs that have homodimeric type 1 reaction center complexes. Within these complexes, highly reducing substances are produced through an initial charge separation followed by electron transfer reactions driven by light energy absorption. In order to attain efficient energy conversion, it is important for the photooxidized reaction center to be rapidly rereduced. Green sulfur bacteria utilize reduced inorganic sulfur compounds (sulfide, thiosulfate, and/or sulfur) as electron sources for their anoxygenic photosynthetic growth. Membrane-bound and soluble cytochromes c play essential roles in the supply of electrons from sulfur oxidation pathways to the P840 reaction center. In the case of gram-positive heliobacteria, the photooxidized P800 reaction center is rereduced by cytochrome c-553 (PetJ) whose N-terminal cysteine residue is modified with fatty acid chains anchored to the cytoplasmic membrane.     

Modelling of sulfur oxidation from elemental sulfur

An elemental S oxidation model has been developed which combines a maximum S release rate with modifiers for temperature and soil moisture conditions. This model has been combined with a pasture growth and CNSP nutrient cycling model to match S oxidation rate to pasture S demand. In two Southern Australian enviroments, 100m elemental S was superior to 200m particles whilst in Northern Australia the 200m particles were superior. These models can be used to match S release to plant demand.     

Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE

SufS is a cysteine desulfurase of the suf operon shown to be involved in iron-sulfur cluster biosynthesis under iron limitation and oxidative stress conditions. The enzyme catalyzes the conversion of L-cysteine to L-alanine and sulfide through the intermediate formation of a protein-bound cysteine persulfide in the active site. SufE, another component of the suf operon, has been previously shown to bind tightly to SufS and to drastically stimulate its cysteine desulfurase activity. Working with Escherichia coli proteins, we here demonstrate that a conserved cysteine residue in SufE at position 51 is essential for the SufS/SufE cysteine desulfurase activity. Mass spectrometry has been used to demonstrate (i). the ability of SufE to bind sulfur atoms on its cysteine 51 and (ii). the direct transfer of the sulfur atom from the cysteine persulfide of SufS to SufE. A reaction mechanism is proposed for this novel two-component cysteine desulfurase.     

Development of a genetic system for a purple sulfur bacterium: Conjugative plasmid transfer inChromatium vinosum

Establishment of a system for manipulative genetics in phototrophic sulfur bacteria of the family Chromatiaceae has mainly been hampered by the lack of reliable methods for growth of these organisms on agar surfaces, techniques for streaking, growth on selective media, screening for antibiotic resistance markers, and most importantly by the lack of a system for DNA transfer. We, therefore, developed minimal and complex agar media for Chromatium vinosum strain D (DSM 180^T), a representative of the purple sulfur bacteria. Sensitivity of C. vinosum towards a broad range of antibiotics was tested in liquid cultures and solidified media, allowing us to select appropriate antibiotic resistance markers. Furthermore, a system for conjugative transfer of IncP-mobilizable plasmids from Escherichia coli to C. vinosum was established. Broad-host-range IncQ vectors were mobilized to C. vinosum with the aid of plasmid RP4 either present extrachromosomally or integrated in the chromosome of E. coli S17-1. Conjugation efficiencies of up to 1 were observed. Agarose gel electrophoretic analysis showed that transconjugants contained the transferred plasmids in addition to the two detectable plasmids of wild-type C. vinosum. All genetic markers tested (kanamycin, gentamicin, ampicillin, amikacin, tetracycline) were expressed in C. vinosum. Furthermore, high-frequency transfer of plasmid RP4 from C. vinosum to E. coli and to Rhodospirillum rubrum K100 was demonstrated. Received: 3 March 1995 / Accepted: 22 May 1995