Unexpected extracellular and intracellular sulfur species during growth of Allochromatium vinosum with reduced sulfur compounds.

Before its uptake and oxidation by purple sulfur bacteria, elemental sulfur probably first has to be mobilized. To obtain more insight into this mobilization process in the phototrophic purple sulfur bacterium Allochromatium vinosum, we used HPLC analysis and X-ray absorption near-edge structure (XANES) spectroscopy for the detection and identification of sulfur compounds in culture supernatants and bacterial cells. We intended to identify soluble sulfur compounds that specifically occur during growth on elemental sulfur, and therefore compared spectra of cultures grown on sulfur with those of cultures grown on sulfide or thiosulfate. While various unexpected oxidized organic sulfur species (sulfones, C-SO(2)-C, and sulfonates, C-SO(3)(-)) were observed via XANES spectroscopy in the supernatants, we obtained evidence for the presence of monosulfane sulfonic acids inside the bacterial cells by HPLC analysis. The concentrations of the latter compounds showed a tight correlation with the content of intracellular sulfur, reaching their maximum when sulfur began to be oxidized. None of the detected sulfur compounds appeared to be a specific soluble intermediate or product of elemental sulfur mobilization. It therefore seems unlikely that mobilization of elemental sulfur by purple sulfur bacteria involves excretion of soluble sulfur-containing substances that would be able to act on substrate distant from the cells.

Chromatium species: an emerging bioindicator of crude oil pollution of tidal mud flats in the Niger Delta mangrove ecosystem, Nigeria.

Establishing microbiological indices for the monitoring of environmental decay by crude oil pollution in the Niger Delta region has been a major concern of our current researches. Chromatium species, a purple, Gram positive pleomorphic, motile, microaerophlic sulfur bacterium offers a good potential for use in the assessment of the short term effects of oil pollution of tidal mud flats in the Niger Delta mangrove ecosystem. Its response to the November 22, 2003 spillage at the Qua Iboe Estuary and the adjoining Cross River Estuary was investigated. Our results have revealed that the sulphur bacterium is easily identified and widely distributed in the epipellic sediment of the mangrove ecosystem but very sensitive to hydrocarbon pollution. The bacterium was readily detected in the tidal mud flats containing as much as 2.0 mg kg(-1) but not detected in sediment with THC level of 3.65 mg kg(-1) and above. It is thus, suggestive that the threshold and lethal limits of effect of hydrocarbons against the sulfur bacterium lies between 2.04 and 3.65 mg kg(-1). These imply that in any case of crude oil pollution that Chromatium is not detected during monitoring the THC levels of the sediment may have been raised to a level close to or above 3.65 mg kg(-1). Statistical analysis of the relationship between THC level and density of Chromatium in sediment revealed a significant (p < 0.05) negative relationship (r = -0.85) in Qua Iboe mangrove ecosystem as against an insignificant (p > 0.05) relationship (r = -0.41) recorded for the Cross River mangrove ecosystem which served as the control. The result indicates that oil pollution affect the homeostatic status of Chromatium in tidal mud flats despite its even distribution (R2 = 71.4%). The finding though not definitive may contribute to the hierarchical process of oil pollution assessment in the Niger Delta region of Nigeria. However, its effective utilization will require not only the development of a selective medium for enumeration and isolation of the bacterium but also the establishment of a defined dose-response relationship under controlled conditions which requires further research.

Reducing red color intensity of seafood wastewater in facultative pond.

Studies were carried out on the growth of Chromatium sp. on seafood wastewater (SFWW), which under facultative conditions and light exposure produced red pigment. The strain grew and utilized organic matter in both dark and light exposure conditions, but it produced red pigment when exposed to light. The growth was repressed by aerobic condition. The red color intensity was reduced by about 32.5+/-1.5 and 70.8+/-2.8% when kept under dark and static conditions, or aerobic and light exposure conditions, respectively. The COD of SFWW and the number of cells of Chromatium sp. were also rapidly reduced by about 78.6+/-2.7 and 92.0+/-1.0%, respectively, under aerobic and light exposure condition. KNO3 and FeCl3 also reduced red color intensity and maximum removal of organic matter and red color were 30 and 4 mg/l, respectively. Aerobic conditions increased the color removal efficiency with 30 mg/l KNO3 and 4 mg/l FeCl3 treatments up to 96.5+/-1 and 98.9+/-1%, respectively.

Long-term population dynamics of phototrophic sulfur bacteria in the chemocline of Lake Cadagno, Switzerland.

Population analyses in water samples obtained from the chemocline of crenogenic, meromictic Lake Cadagno, Switzerland, in October for the years 1994 to 2003 were studied using in situ hybridization with specific probes. During this 10-year period, large shifts in abundance between purple and green sulfur bacteria and among different populations were obtained. Purple sulfur bacteria were the numerically most prominent phototrophic sulfur bacteria in samples obtained from 1994 to 2001, when they represented between 70 and 95% of the phototrophic sulfur bacteria. All populations of purple sulfur bacteria showed large fluctuations in time with populations belonging to the genus Lamprocystis being numerically much more important than those of the genera Chromatium and Thiocystis. Green sulfur bacteria were initially represented by Chlorobium phaeobacteroides but were replaced by Chlorobium clathratiforme by the end of the study. C. clathratiforme was the only green sulfur bacterium detected during the last 2 years of the analysis, when a shift in dominance from purple sulfur bacteria to green sulfur bacteria was observed in the chemocline. At this time, numbers of purple sulfur bacteria had decreased and those of green sulfur bacteria increased by about 1 order of magnitude and C. clathratiforme represented about 95% of the phototrophic sulfur bacteria. This major change in community structure in the chemocline was accompanied by changes in profiles of turbidity and photosynthetically available radiation, as well as for sulfide concentrations and light intensity. Overall, these findings suggest that a disruption of the chemocline in 2000 may have altered environmental niches and populations in subsequent years.

Direct comparison of the electrocatalytic oxidation of hydrogen by an enzyme and a platinum catalyst.

It is shown that for molecules of Allochromatium vinosum [NiFe]-hydrogenase adsorbed on a pyrolytic graphite electrode the nickel-iron active site catalyzes hydrogen oxidation at a diffusion-controlled rate matching that achieved by platinum.

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Kinetics of photoacclimation in cultures of Chromatium vinosum DSM 185 during shifts in light irradiance.

Continuous cultures of Chromatium vinosum DSM 185 were shifted from a high to a low irradiance (67 to 4 microE m(-2) s(-1)) and vice versa (4 to 67 microE m(-2) s(-1)). The kinetics of photoacclimation of the cultures were analysed during these transitions until steady state was reached. When irradiance was shifted from 4 to 67 microE m(-2) s(-1), bacteriochlorophyll synthesis halted for 4 h. During this period, pigments were progressively diluted in the newly formed biomass, resulting in a lower specific pigment content. The specific growth rate of the organisms did not change immediately after the shift, but rather underwent a gradual increase during the following 10 h. This transition was accompanied by a transient increase in the levels of glycogen, indicating that CO2 fixation rates increased immediately after the shift, and that unused photosynthate was stored as glycogen. The shift from a high to a low irradiance was characterized by an immediate drop in the specific growth rate to virtually zero, and by comparatively sharp decreases in the specific rates of sulfur and sulfide oxidation and in the specific rate of glycogen accumulation. The specific content of bacteriochlorophyll a increased during the first 10 h. During the same period the specific content of glycogen decreased.

Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur.

The sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180) was determined to clarify the in vivo role of 'reverse' sirohaem sulfite reductase. The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling [4Fe-4S]-cluster-containing heterodisulfide reductase from methanogenic archaea. Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds. The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed. Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur. The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered. Photoorganoheterotrophic growth was also unaffected. 'Reverse' sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation. These results, together with the finding that the sulfur globules of C. vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium.

The cytochrome subunit structure in the photosynthetic reaction center of Chromatium minutissimum.

Gel-electrophoretic assay revealed that the photosynthetic reaction center (RC) of Chromatium minutissimum, in contrast to the well-known RC Rhodopseudomonas viridis, consists of five rather than four subunits with molecular masses of 37, 34, 25, 19, and 17 kDa. The 37- and 19-kDa subunits are stained with tetramethylbenzidine for the cytochrome c hemes. Absorption spectra show that the concentration of reduced cytochromes in the C. minutissimum RC poised at redox potential of -150 mV (fully reduced pool of hemes) is about three times more than in the C. minutissimum RC poised at redox potential of +260 mV (only high-potential hemes are reduced). The results of redox titration of absorption changes at the cytochrome c alpha-band are most appropriately approximated by a six-component theoretical curve with the midpoint potentials of Em1 = 390 mV, Em2 = 320 mV, Em3 = 210 mV, Em4 = 100 mV, Em5 = 20 mV, and Em6 = -50 mV. Possible functions of the cytochromes with the midpoint potentials 210 and 100 mV, which have not been found in purple bacteria before, are discussed.

Insertional gene inactivation in a phototrophic sulphur bacterium: APS-reductase-deficient mutants of Chromatium vinosum.

In purple sulphur bacteria of the family Chromatiaceae sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process. In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite:acceptor oxidoreductase has not been resolved. This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5'-phosphosulphate (APS) reductase from Chromatium vinosum strain D (DSM 180'), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin omega cartridge. The mutated genes were transferred to C. vinosum on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination. Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria. Enzymological studies demonstrated the absence of APS reductase from the mutants. Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions. In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite:acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in C. vinosum. Specific inactivation of selected genes as developed for C. vinosum in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria.

Glutathione amide and its perthiol in anaerobic sulfur bacteria.

Chromatium species produced the novel biological thiol glutathione amide, gamma-L-glutamyl-L-cysteinylglycine amide (GASH), when grown photoheterotrophically. GASH was largely converted to the corresponding perthiol during photoautotrophic growth on sulfide, suggesting that GASH may have a function in anaerobic sulfide metabolism. This unprecedented form of glutathione metabolism was probably present in anaerobic ancestors of modern cyanobacteria and purple bacteria.

Isolation and characterization of soluble electron transfer proteins from Chromatium purpuratum.

Several soluble electron transfer proteins were isolated and characterized from the marine purple-sulfur bacterium Chromatium purpuratum. The C. purpuratum flavocytochrome c is similar in molecular mass (68 kDa) and isoelectric point (6.5) to flavocytochromes isolated from other phototrophs. Redox titrations of the flavocytochrome c hemes show two components with midpoint potential values of +15 and -120 mV, behavior similar to that observed with the flavocytochrome isolated from the thermophilic Chromatium tepidum. Moreover, N-terminal amino acid sequence analysis of both the flavin and the cytochrome subunit indicates substantial homology to the primary structure of the flavocytochrome c of Chromatium vinosum. In contrast, the C. purpuratum high-potential iron-sulfur protein (HiPIP) differs from those isolated from other photosynthetic bacteria in its relatively high midpoint potential (+390 mV) and the possibility that it exists as a dimer in solution. Two low molecular mass c-type cytochromes were also characterized. One appears to be a high-potential (+310 mV) c8-type cytochrome. Amino acid sequencing suggests that the second cytochrome may be a homologue of the low-potential cytochrome c-551, previously described in two species of Ectothiorhodospirillaceae.

Picosecond dynamics of excitations in light-harvesting complex B800-850 from Chromatium minutissimum studied using fluorescence spectrochronography.

The picosecond dynamics of excitations in the isolated B800-850 light-harvesting complex of the purple sulfur bacterium Chromatium minutissimum has been studied using picosecond fluorescence spectrochronography. A short-lived component of about 20 ps lifetime has been found at 77K at the short wavelength part of the B850 fluorescence spectrum similar to that previously described for the core antenna bacteriochlorophyll band B880 of Rhodospirillum rubrum. Evidence has been presented indicating that this component is likely to reflect excitation energy relaxation step(s) involving both photoexcited bacteriochlorophyll and the protein environment. A new kinetic scheme of excitation transfer from the peripheral antenna to the photoreceptor units in purple bacteria is suggested which takes into account these findings.

Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: evidence for autotrophic growth in natural populations.

Purple phototrophic bacteria of the genus Chromatium can grow as either photoautotrophs or photoheterotrophs. To determine the growth mode of the thermophilic Chromatium species, Chromatium tepidum, under in situ conditions, we have examined the carbon isotope fractionation patterns in laboratory cultures of this organism and in mats of C. tepidum which develop in sulfide thermal springs in Yellowstone National Park. Isotopic analysis (13C/12C) of total carbon, carotenoid pigments, and bacteriochlorophyll from photoautotrophically grown cultures of C. tepidum yielded 13C fractionation factors near -20%. Cells of C. tepidum grown on excess acetate, wherein synthesis of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase ribulose bisphosphate carboxylase) was greatly repressed, were isotopically heavier, fractionation factors of ca. -7% being observed. Fractionation factors determined by isotopic analyses of cells and pigment fractions of natural populations of C. tepidum growing in three different sulfide thermal springs in Yellowstone National Park were approximately -20%, indicating that this purple sulfur bacterium grows as a photoautotroph in nature.

13C-NMR evidence of bacteriochlorophyll a formation by the C5 pathway in Chromatium.

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of L-[1-13C]glutamate and [2-13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.

Competition for sulfide among colorless and purple sulfur bacteria in cyanobacterial mats.

The vertical zonation of light, O2, H2S, pH, and sulfur bacteria was studied in two benthic cyanobacterial mats from hypersaline ponds at Guerrero Negro, Baja California, Mexico. The physical-chemical gradients were analyzed in the upper few mm at < or = 100 micrometers spatial resolution by microelectrodes and by a fiber optic microprobe. In mats, where oxygen produced by photosynthesis diffused far below the depth of the photic zone, colorless sulfur bacteria (Beggiatoa sp.) were the dominant sulfide oxidizing organisms. In a mat, where the O2-H2S interface was close to the photic zone, but yet received no significant visible light, purple sulfur bacteria (Chromatium sp.) were the dominant sulfide oxidizers. Analysis of the spectral light distribution here showed that the penetration of only 1% of the incident near-IR light (800-900 nm) into the sulfide zone was sufficient for the mass development of Chromatium in a narrow band of 300 micromoles thickness. The balance between O2 and light penetration down into the sulfide zone thus determined in micro-scale which type of sulfur bacteria became dominant.

Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum.

Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. Both protein components were purified to homogeneity, and some of their properties were determined. Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties. As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid). Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m). The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.

In vivo 31P and 13C nuclear magnetic resonance studies of acetate metabolism in Chromatium vinosum.

31P and 13C nuclear magnetic resonance (NMR) experiments were performed on suspensions of the phototrophic bacterium Chromatium vinosum incubated anaerobically in the dark. 31P NMR spectra revealed that during prolonged dark incubation high ATP levels are maintained. This phenomenon was independent of the presence of the energy reserves polyglucose and polyphosphate. 13C NMR experiments revealed that the amino acids glutamate, aspartate, and alanine are the major products of acetate incorporation in the dark. Apart from these amino acids, poly-beta-hydroxybutyrate was also formed. Acetate metabolism was markedly stimulated by the presence of polyglucose. The specific 13C activity of glutamate C-2 was approximately 50% that of glutamate C-4. The idea is discussed that this difference is the consequence of the maintenance of redox balance during entry of acetate into cell metabolism.

[Effect of light intensity on the content of bacteriochlorophyll and on the growth of phototrophic marine sulfur bacteria]. / Influence de l'intensité lumineuse sur la teneur en bactériochlorophylle et sur la croissance de sulfobactéries phototrophes marines.

Eight strains of Chromatiaceae isolated from marine sediments are cultivated under light intensities of 50-5000 lx. A decreased in the light intensity brings about an increase in the specific bacteriochlorophyll content and also in the length of development. In certain strains, the increase in pigment contents partly compensates for the loss in light intensity, up to the maximum concentration of bacteriochlorophyll. This mechanism is only a physiological compatibility which ensures the survival of these organisms under feeble light intensities.