Photosynthetic Electron Donor Research Articles

Quantitative proteomics reveals the Sox system's role in sulphur and arsenic metabolism of phototroph Halorhodospira halophila.

The metabolic process of purple sulphur bacteria's anoxygenic photosynthesis has been primarily studied in Allochromatium vinosum, a member of the Chromatiaceae family. However, the metabolic processes of purple sulphur bacteria from the Ectothiorhodospiraceae and Halorhodospiraceae families remain unexplored. We have analysed the proteome of Halorhodospira halophila, a member of the Halorhodospiraceae family, which was cultivated with various sulphur compounds. This analysis allowed us to reconstruct the first comprehensive sulphur-oxidative photosynthetic network for this family. Some members of the Ectothiorhodospiraceae family have been shown to use arsenite as a photosynthetic electron donor. Therefore, we analysed the proteome response of Halorhodospira halophila when grown under arsenite and sulphide conditions. Our analyses using ion chromatography-inductively coupled plasma mass spectrometry showed that thioarsenates are chemically formed under these conditions. However, they are more extensively generated and converted in the presence of bacteria, suggesting a biological process. Our quantitative proteomics revealed that the SoxAXYZB system, typically dedicated to thiosulphate oxidation, is overproduced under these growth conditions. Additionally, two electron carriers, cytochrome c551/c5 and HiPIP III, are also overproduced. Electron paramagnetic resonance spectroscopy suggested that these transporters participate in the reduction of the photosynthetic Reaction Centre. These results support the idea of a chemically and biologically formed thioarsenate being oxidized by the Sox system, with cytochrome c551/c5 and HiPIP III directing electrons towards the Reaction Centre.

Complete genome of the thermophilic purple sulfur Bacterium Thermochromatium tepidum compared to Allochromatium vinosum and other Chromatiaceae.

The complete genome sequence of the thermophilic purple sulfur bacterium Thermochromatium tepidum strain MCT (DSM 3771T) is described and contrasted with that of its mesophilic relative Allochromatium vinosum strain D (DSM 180T) and other Chromatiaceae. The Tch. tepidum genome is a single circular chromosome of 2,958,290 base pairs with no plasmids and is substantially smaller than the genome of Alc. vinosum. The Tch. tepidum genome encodes two forms of RuBisCO and contains nifHDK and several other genes encoding a molybdenum nitrogenase but lacks a gene encoding a protein that assembles the Fe-S cluster required to form a functional nitrogenase molybdenum-iron cofactor, leaving the phototroph phenotypically Nif-. Tch. tepidum contains genes necessary for oxidizing sulfide to sulfate as photosynthetic electron donor but is genetically unequipped to either oxidize thiosulfate as an electron donor or carry out assimilative sulfate reduction, both of which are physiological hallmarks of Alc. vinosum. Also unlike Alc. vinosum, Tch. tepidum is obligately phototrophic and unable to grow chemotrophically in darkness by respiration. Several genes present in the Alc. vinosum genome that are absent from the genome of Tch. tepidum likely contribute to the major physiological differences observed between these related purple sulfur bacteria that inhabit distinct ecological niches.

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

Photobiological transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using Rhodobacter sphaeroides

Pump-and-treat strategies for groundwater containing explosives may be necessary when the contaminated water approaches sensitive receptors. This project investigated bacterial photosynthesis as a strategy for ex situ treatment, using light as the primary energy source to facilitate RDX transformation. The objective was to characterize the ability of photosynthetic Rhodobacter sphaeroides (strain ATCC® 17023 ™) to transform the high-energy explosive RDX. R. sphaeroides transformed 30 μM RDX within 40 h under light conditions; RDX was not fully transformed in the dark (non-photosynthetic conditions), suggesting that photosynthetic electron transfer was the primary mechanism. Experiments with RDX demonstrated that succinate and malate were the most effective electron donors for photosynthesis, but glycerol was also utilized as a photosynthetic electron donor. RDX was transformed irrespective of the presence of carbon dioxide. The electron shuttling compound anthraquinone-2,6-disulfonate (AQDS) increased transformation kinetics in the absence of CO2, when the cells had excess NADPH that needed to be re-oxidized because there was limited CO2 for carbon fixation. When CO2 was added, the cells generated more biomass, and AQDS had no stimulatory effect. End products indicated that RDX carbon became CO2, biomass, and a soluble, uncharacterized aqueous metabolite, determined using 14C-labeled RDX. These data are the first to suggest that photobiological explosives transformation is possible and will provide a framework for which phototrophy can be used in environmental restoration of explosives contaminated water.

Chlorobaculum tepidum growth on biogenic S(0) as the sole photosynthetic electron donor.

The green sulfur bacteria, the Chlorobi, are phototrophic bacteria that oxidize sulfide and deposit extracellular elemental sulfur globules [S(0)]. These are subsequently consumed after sulfide is exhausted. S(0) globules from a Chlorobaculum tepidum mutant strain were purified and used to show that the wild-type strain of Cba. tepidum can grow on biogenic S(0) globules as the sole photosynthetic electron donor, i.e. in medium with no other source of reducing power. Growth yields and rates on biogenic S(0) are comparable with those previously determined for Cba. tepidum grown on sulfide as the sole electron donor. Contact between cells and S(0) was required for growth. However, only a fraction of the cell population was firmly attached to S(0) globules. Microscopic examination of cultures growing on S(0) demonstrated cell-S(0) attachment and allowed for the direct observation of S(0) globule degradation. Bulk chemical analysis, scanning electron microscopy, secondary ion mass spectrometry and SDS-PAGE indicate that Cba. tepidum biogenic S(0) globules contain carbon, oxygen and nitrogen besides S and may be associated with specific proteins. These observations suggest that current models of S(0) oxidation in the Chlorobi need to be revised to take into account the role of cell-S(0) interactions in promoting S(0) degradation.

Sulfite oxidation in the purple sulfur bacterium Allochromatium vinosum: identification of SoeABC as a major player and relevance of SoxYZ in the process

In phototrophic sulfur bacteria, sulfite is a well-established intermediate during reduced sulfur compound oxidation. Sulfite is generated in the cytoplasm by the reverse-acting dissimilatory sulfite reductase DsrAB. Many purple sulfur bacteria can even use externally available sulfite as a photosynthetic electron donor. Nevertheless, the exact mode of sulfite oxidation in these organisms is a long-standing enigma. Indirect oxidation in the cytoplasm via adenosine-5'-phosphosulfate (APS) catalysed by APS reductase and ATP sulfurylase is neither generally present nor essential. The inhibition of sulfite oxidation by tungstate in the model organism Allochromatium vinosum indicated the involvement of a molybdoenzyme, but homologues of the periplasmic molybdopterin-containing SorAB or SorT sulfite dehydrogenases are not encoded in genome-sequenced purple or green sulfur bacteria. However, genes for a membrane-bound polysulfide reductase-like iron-sulfur molybdoprotein (SoeABC) are universally present. The catalytic subunit of the protein is predicted to be oriented towards the cytoplasm. We compared the sulfide- and sulfite-oxidizing capabilities of A. vinosum WT with single mutants deficient in SoeABC or APS reductase and the respective double mutant, and were thus able to prove that SoeABC is the major sulfite-oxidizing enzyme in A. vinosum and probably also in other phototrophic sulfur bacteria. The genes also occur in a large number of chemotrophs, indicating a general importance of SoeABC for sulfite oxidation in the cytoplasm. Furthermore, we showed that the periplasmic sulfur substrate-binding protein SoxYZ is needed in parallel to the cytoplasmic enzymes for effective sulfite oxidation in A. vinosum and provided a model for the interplay between these systems despite their localization in different cellular compartments.

Proton transport facilitating water-oxidation: the role of second sphere ligands surrounding the catalytic metal cluster

The ability of PSII to extract electrons from water, with molecular oxygen as a by-product, is a remarkable biochemical and evolutionary innovation. From an evolutionary perspective, the invention of PSII approximately 2.7 Ga led to the accelerated accumulation of biomass in the biosphere and the accumulation of oxygen in the atmosphere, a combination that allowed for the evolution of a much more complex and extensive biosphere than would otherwise have been possible. From the biochemical and enzymatic perspective, PSII is remarkable because of the thermodynamic and kinetic obstacles that needed to have been overcome to oxidize water as the ultimate photosynthetic electron donor. This article focuses on how proton release is an integral part of how these kinetic and thermodynamic obstacles have been overcome: the sequential removal of protons from the active site of H2O-oxidation facilitates the multistep oxidation of the substrate water at the Mn4CaOx, the catalytic heart of the H2O-oxidation reaction. As noted previously, the facilitated deprotonation of the Mn4CaOx cluster exerts a redox-leveling function preventing the accumulation of excess positive charge on the cluster, which might otherwise hinder the already energetically difficult oxidation of water. Using recent results, including the characteristics of site-directed mutants, the role of the second sphere of amino acid ligands and the associated network of water molecules surrounding the Mn4CaOx is discussed in relation to proton transport in other systems. In addition to the redox-leveling function, a trapping function is assigned to the proton release step occurring immediately prior to the dioxygen chemistry. This trapping appears to involve a yet-to-be clarified gating mechanism that facilitates to coordinated release of a proton from the neighborhood of the active site thereby insuring that the backward charge-recombination reaction does not out-compete the forward reaction of dioxygen chemistry during this final step of H2O-oxidation.

Coupling dark metabolism to electricity generation using photosynthetic cocultures

We investigated the role of green sulfur bacteria inlight-responsive electricity generation in microbial electrochemical cells (MXCs). We operated MXCs containing either monocultures or defined cocultures of previously enriched phototrophic Chlorobium and anode-respiring Geobacter under anaerobic conditions in the absence of electron donor. Monoculture control MXCs containing Geobacter or Chlorobium neither responded to light nor produced current, respectively. Instead, light-responsive current generation occurred only in coculture MXCs. Current increased above background levels only in the dark and declined slowly over 96 h. This pattern suggested that Chlorobium exhausted intracellular glycogen reserves via dark fermentation to supply an electron donor, presumably acetate, to Geobacter. With medium containing sulfide as the sole photosynthetic electron donor, current generation had a similar and reproducible negative light response. To investigate whether this metabolic interaction also occurred without an electrode, we performed coculture experiments in batch serum bottles. In this setup, sulfide served as the sole electron donor, whose oxidation by Chlorobium was required to provide S(0) as the electron acceptor to Geobacter. Copies of Geobacter 16S rDNA increased approximately 14-fold in batch bottle cocultures containing sulfide compared to those lacking sulfide, and did not decline after termination of sulfide feeding. These results suggest that products of both photosynthesis and dark fermentation by Chlorobium were sufficient both to yield an electrochemical response by Geobacter biofilms, and to promote Geobacter growthin batch cocultures. Our work expands upon the fusion of MXCs with coculture techniques and reinforces the utility of microbial electrochemistry for sensitive, real-time monitoring of microbial interactions in which a metabolic intermediate can be converted to electrical current.

Photosynthetic Electron Transport in an Anoxygenic Photosynthetic Bacterium Afifella (Rhodopseudomonas) marina Measured Using PAM Fluorometry

Blue diode-based pulse amplitude modulation (PAM) technology can be used to measure the photosynthetic electron transport rate (ETR) in a purple nonsulfur anoxygenic photobacterium, Afifella (Rhodopseudomonas) marina. Rhodopseudomonads have a reaction center light harvesting antenna complex containing an RC-2 type bacteriochlorophyll a protein (BChl a RC-2-LH1) which has a blue absorption peak and variable fluorescence similar to PSII. Absorptance of cells filtered onto glass fiber disks was measured using a blue-diode-based absorptance meter (Blue-RAT) so that absolute ETR could be calculated from PAM experiments. Maximum quantum yield (Y) was ≈0.6, decreasing exponentially as irradiance increased. ETR vs irradiance (P vs E) curves fitted the waiting-in-line model (ETR=(ETRmax ×E/Eopt )×exp(1-E/Eopt )). Maximum ETR (ETRmax ) was ≈1000-2000μmole(-) mg(-1) BChl ah(-1) . Fe(2+) , bisulfite and thiosulfate act as photosynthetic electron donors. Optimum irradiance was ≈100μmolm(-2) s(-1) PPFD even in Afifella grown in sunlight. Quantum efficiencies (α) were ≈0.3-0.4mole(-) molhλ(-1) ; or ≈11.8±2.9mole(-) molhλ(-1) m(2) μg(-1) BChl a). An underlying layer of Afifella in a constructed algal/photosynthetic bacterial mat has little effect on the measured ETR of the overlying oxyphotoautotroph (Chlorella).

A tribute to Seymour Steven Brody: in memoriam (November 29, 1927 to May 25, 2010)

We honor Steve Brody, a dear friend and a mentor on what would have been his 83rd birthday (November 29, 2010). Steve was a pioneer of chlorophyll structure and function, an outstanding biophysicist, an innovator, an artist and an adventurer, a true renaissance man. We present here first his first-of-a-kind contributions on the primary processes of photosynthesis at the University of Illinois at Urbana-Champaign, and then review his research on the interactions of chlorophyll monolayers and various photosynthetic electron donors and acceptors in artificial membrane systems at New York University. We highlight significant research contributions of interest to the reader and conclude with biographical notes.

Prosthecochloris indica sp. nov., a novel green sulfur bacterium from a marine aquaculture pond, Kakinada, India

A green sulfur bacterium, strain JAGS6T was isolated from a marine aquaculture pond located near Kakinada on the east coast of India. Cells of strain JAGS6T were Gram-negative, non-motile, coccoid, 1-1.2 microm in diameter, with prosthecae. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JAGS6T clusters with members of the genus Prosthecochloris and the sequence similarity with the nearest relative, Prosthecochloris vibrioformis, is 96.7%. Cultures of strain JAGS6T are green in color and the cells contain bacteriochlorophyll c and most likely carotenoids of the chlorobactene series as photosynthetic pigments. Strain JAGS6T is mesophilic, halotolerant (up to 7% NaCl) and is obligately phototrophic, utilizing sulfide but not thiosulfate as a photosynthetic electron donor. Sulfur globules are deposited outside the cells during oxidation of sulfide. On the basis of 16S rRNA gene sequence analysis and its morphological and physiological characteristics, strain JAGS6T is distinct from described species of the genus Prosthecochloris and we propose to describe it as a new species, Prosthecochloris indica, sp. nov. The type strain is JAGS6T (=JCM 13299T=ATCC BAA1214T).

Arsenic(III) Fuels Anoxygenic Photosynthesis in Hot Spring Biofilms from Mono Lake, California

Phylogenetic analysis indicates that microbial arsenic metabolism is ancient and probably extends back to the primordial Earth. In microbial biofilms growing on the rock surfaces of anoxic brine pools fed by hot springs containing arsenite and sulfide at high concentrations, we discovered light-dependent oxidation of arsenite [As(III)] to arsenate [As(V)] occurring under anoxic conditions. The communities were composed primarily of Ectothiorhodospira-like purple bacteria or Oscillatoria-like cyanobacteria. A pure culture of a photosynthetic bacterium grew as a photoautotroph when As(III) was used as the sole photosynthetic electron donor. The strain contained genes encoding a putative As(V) reductase but no detectable homologs of the As(III) oxidase genes of aerobic chemolithotrophs, suggesting a reverse functionality for the reductase. Production of As(V) by anoxygenic photosynthesis probably opened niches for primordial Earth's first As(V)-respiring prokaryotes.

A new purple sulfur bacterium isolated from a littoral microbial mat, Thiorhodococcus drewsii sp. nov.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO(2), it is capable of assimilating C(1)-C(5) fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5-6.7, 30-35 degrees C, > or =50 micro mol quanta m(-2) s(-1), and 2.4-2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518(T), was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.

An alternate photosynthetic electron donor system for PSI supports light dependent nitrogen fixation in a non-heterocystous cyanobacterium, Plectonema boryanum

Plectonema boryanum exhibits temporal separation of photosynthesis and nitrogen fixation under diazotrophic conditions. During nitrogen fixation, the photosynthetic electron transport chain becomes impaired, which leads to the uncoupling of the PSII and PSI activities. A 30-40% increase in PSI activity and continuous generation of ATP through light-dependent processes seem to support the nitrogen fixation. The use of an artificial electron carrier that shuttles electrons between the plastoquinone pool and plastocyanin, bypassing cytochrome b/f complex, enhanced the photosynthetic electron transport activity five to six fold during nitrogen fixation. Measuring of full photosynthetic electron transport activity using methyl voilogen as a terminal acceptor revealed that the photosynthetic electron transport components beyond plastocyanin might be functional. Further, glycolate can act as a source of electrons for PSI for the nitrogen fixing cells, which have residual PSII activity. Under conditions when PSI becomes largely independent of PSII and glycolate provides electrons for PSI activity, the light-dependent nitrogen fixation also was stimulated by glycolate. These results suggest that during nitrogen fixation, when the photosynthetic electron transport from PSII is inhibited at the level of cytochrome b/f complex, an alternate electron donor system for PSI may be required for the cells to carry out light dependent nitrogen fixation.

Phototrophic consortia: model systems for symbiotic interrelations between prokaryotes.

Most symbiotic prokaryotes known to date have been found in association with eukaryotes, whereas only few (3.5%) bacteria or archaea are known that have established interactions with other prokaryotes. As revealed by direct microscopic investigations, however, multiple morphotypes of highly structured associations of different prokaryotes exist in nature. These so-called consortia appear to represent the most developed type of bacterial interaction. Phototrophic consortia are associations of green sulfur bacteria that surround a central chemotrophic bacterium. In some natural environments, almost all cells of green sulfur bacteria occur in the associated state, i.e. as epibionts of phototrophic consortia. In contrast to earlier speculations, the central bacterium belongs to the beta-Proteobacteria. Within the consortia, the green sulfur bacterial epibionts grow photolithoautotrophically, utilizing exogenous sulfide as photosynthetic electron donor. The entire consortium does not appear to be independent of organic carbon compounds, since it exhibits chemotaxis towards 2-oxoglutarate and, as demonstrated by microautoradiography, can also incorporate this compound. Intact consortia exhibit a scotophobic response in which the bacteriochlorophylls of the epibionts function as light sensors, whereas the chemotrophic central bacterium confers motility upon the association. Hence, a signal exchange must occur between the different bacteria. Based on their 16S rRNA gene sequences, the epibionts represent distinct phylotypes that are often only distantly related to known species of green sulfur bacteria. Since phototrophic consortia have recently become available in enrichment cultures, they can now serve as suitable model systems for the investigation of the molecular mechanisms of cell-cell recognition and signal exchange, and for studies of the coevolution of nonrelated prokaryotes.

Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems II and I.

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin-thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.

Thioalkalicoccus limnaeus gen. nov., sp. nov., a new alkaliphilic purple sulfur bacterium with bacteriochlorophyll b.

Four strains of purple sulfur bacteria containing bacteriochlorophyll b were isolated from cyanobacterial mats of soda lakes in the steppe of south-east Siberia, Russia. Cells of all strains were cocci without gas vesicles. Eventually, cells with flagella were seen in the electron microscope, but motile cells were observed very rarely in cultures. Internal photosynthetic membranes were of the tubular type. Photosynthetic pigments were bacteriochlorophyll b and carotenoids with spectral characteristics similar to 3,4,3',4'-tetrahydrospirilloxanthin. The bacteria were obligately phototrophic and strictly anaerobic. Hydrogen sulfide and elemental sulfur were used as photosynthetic electron donors. Thiosulfate was not used. During growth on sulfide, sulfur globules were formed as intermediate oxidation products, deposited inside the cells and centrally located. In the presence of sulfide and sodium bicarbonate, acetate, malate, propionate, pyruvate, succinate, fumarate and yeast extract were photoassimilated. Growth factors were not required. The new bacterium is an obligate alkaliphile growing at pH 8-10 with an optimum at pH 9. It showed good growth up to 6.0% sodium chloride and up to 8.5% sodium carbonates. Phenotypically, it is similar to Thiococcus pfennigii, but different by virtue of its alkaliphily and salt tolerance. The DNA G+C content was 63.6-64.8 mol %, compared to 69.4-69.9 mol % for Thiococcus pfennigii. The 16S rDNA sequence of strain A26T was approximately 92% similar to that of Thiococcus pfennigii DSM 226 and therefore a new genus and species name, Thioalkalicoccus limnaeus gen. nov. and sp. nov., are proposed for the new bacterium.

Thiorhodospira sibirica gen. nov., sp. nov., a new alkaliphilic purple sulfur bacterium from a Siberian soda lake.

A new purple sulfur bacterium was isolated from microbial films on decaying plant mass in the near-shore area of the soda lake Malyi Kasytui (pH 9.5, 0.2% salinity) located in the steppe of the Chita region of south-east Siberia. Single cells were vibrioid- or spiral-shaped (3-4 microns wide and 7-20 microns long) and motile by means of a polar tuft of flagella. Internal photosynthetic membranes were of the lamellar type. Lamellae almost filled the whole cell, forming strands and coils. Photosynthetic pigments were bacteriochlorophyll a and carotenoids of the spirilloxanthin group. The new bacterium was strictly anaerobic. Under anoxic conditions, hydrogen sulfide and elemental sulfur were used as photosynthetic electron donors. During growth on sulfide, sulfur globules were formed as intermediate oxidation products. They were deposited outside the cytoplasm of the cells, in the peripheral periplasmic space and extracellularly. Thiosulfate was not used. Carbon dioxide, acetate, pyruvate, propionate, succinate, fumarate and malate were utilized as carbon sources. Optimum growth rates were obtained at pH 9.0 and optimum temperature was 30 degrees C. Good growth was observed in a mineral salts medium containing 5 g sodium bicarbonate l-1 without sodium chloride. The new bacterium tolerated up to 60 g sodium chloride l-1 and up to 80 g sodium carbonates l-1. Growth factors were not required. The DNA G + C composition was 56.0-57.4 mol%. Based on physiological, biochemical and genetic characteristics, the newly isolated bacterium is recognized as a new species of a new genus with the proposed name Thiorhodospira sibirica.

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.